Microorganism

Toxicol Lett, 1996 Aug, 86(2-3), 155 - 62
Environmental chemicals relevant for respiratory hypersensitivity: the indoor environment; Becher R et al.; The allergenic constituents of non-industrial indoor environments are predominantly found in the biologic fraction . Several reports have related biological particles such as mites and their excreta, dander from pets and other furred animals, fungi and bacteria to allergic manifestations including respiratory hypersensitivity among the occupants of buildings . Also, bacterial cell-wall components and the spores of toxin-producing moulds may contribute to the induction of hypersensitivity, but the relevance for human health is not yet determined . The knowledge regarding hypersensitivity and asthmatic reactions after exposure to chemical agents is primarily based on data from occupational settings with much higher exposure levels than usually found in non-industrial indoor environments . However, there is evidence that indoor exposure to tobacco smoke, some volatile organic compounds (VOC) and various combustion products (either by using unvented stoves or from outdoor sources) can be related to asthmatic symptoms . In some susceptible individuals, the development of respiratory hypersensitivity or elicitation of asthmatic symptoms may also be related to the indiscriminate use of different household products followed by exposure to compounds such as diisocyanates, organic acid anhydrides, formaldehyde, styrene and hydroquinone . At present, the contribution of the indoor environment both to the development of respiratory hypersensitivity and for triggering asthmatic symptoms is far from elucidated.

Clin Exp Immunol, 1996 Aug, 105(2), 213 - 9
Chronic mucocutaneous candidiasis . II . Class and subclass of specific antibody responses in vivo and in vitro; Lilic D et al.; Patients with chronic mucocutaneous candidiasis (CMC) succumb to persistent infections with the opportunistic yeast Candida . Impaired cell-mediated responses to Candida have been repeatedly reported while antibody responses were mostly found to be normal . The underlying defect remains poorly understood . It has recently been shown that CMC patients are also susceptible to infections with encapsulated bacteria, and may have associated IgG2 and IgG4 deficiency . Our previous studies demonstrated altered cytokine production in CMC patients . As cytokines can influence production and isotype of specific antibody, in 10 patients with CMC we measured the levels and isotype distribution of serum antibodies to Candida antigens (CAg), pneumococcal polysaccharide (PPS) and tetanus toxoid (TT) antigens . Peripheral blood lymphocytes were also stimulated in culture and the antibodies made in vitro were measured . Our data demonstrated that in vivo, CMC patients had very high levels of IgG and IgA CAg-specific antibodies . CAg-specific and PPS-specific IgG1 was markedly higher than in controls . Children but not adults with CMC had significantly lower levels of IgG2-specific antibody to CAg and PPS compared with age-matched controls . Patients had significantly higher levels of IgG3-specific antibody to all three antigens tested . These findings were in accordance with increased total IgG and IgG3 levels seen in CMC patients . In vitro, CMC patients, particularly children, did not respond as frequently to antigen stimulation as did their healthy controls . The level of specific antibody produced was also lower to all antigens tested, as was the amount of total immunoglobulins following antigenic and particularly mitogenic stimulation . Addition of interferon-alpha (IFN-alpha) or IFN-gamma to cultures had variable, sometimes marked, effects . Our results demonstrate that CMC patients manifest subtle alterations in specific antibody responses to CAg, PPS and TT, which are most pronounced in children . This may relate to altered cytokine production also seen in these patients.

Am J Respir Cell Mol Biol, 1996 Aug, 15(2), 207 - 15
Characterization of a neutrophil inhibitor peptide harvested from human bronchial lavage: homology to influenza A nucleoprotein; Cooper JA Jr et al.; Bronchi are exposed to particulate matter, including bacteria, fungi and dusts, that should trigger release of molecules which attract polymorphonuclear neutrophils (PMN) . However, normal bronchi are relatively devoid of PMN, suggesting that there exists a mechanism to dampen acute inflammation in the lung . We have previously reported that bronchial lavage from normal humans contains a nonpolar peptide that inhibits PMN chemotaxis and oxidant production . In the present study we devised preparative methods to obtain sufficient quantities of a similar inhibitor molecule for partial amino acid sequencing and allow production of truncated analogues . Amino acid sequencing demonstrated that the peptide includes a 10-amino-acid sequence that is completely homologous to a sequence of amino acids contained in the influenza A nucleoprotein . Synthesized peptides containing this 10-amino-acid sequence inhibited PMN chemotaxis and oxidant production . In addition, PMN lysates actively phosphorylated peptides containing the 10-amino-acid sequence or a partial sequence containing an apparent phosphorylation site . U937 cells were noted to be one source of this inhibitor, as a similarly sized nonpolar inhibitor peptide was purified from U937 culture supernatants . In addition, U937 and monocyte cellular lysates contained proteins recognized by an antiserum directed at the influenza A nucleoprotein . Further characterization of the molecule described in this study or related molecules may lead to significantly new antiinflammatory strategies.

Appl Environ Microbiol, 1996 Aug, 62(8), 3034 - 6
Analysis of partial sequences of genes coding for 16S rRNA of actinomycetes isolated from Casuarina equisetifolia nodules in Mexico; Niner BM et al.; Filamentous bacteria isolated from surface-sterilized nodules of Casuarina equisetifolia trees in Mexico were capable of reducing acetylene, a diagnostic test for nitrogenase, but were unable to nodulate their host . Analysis of partial 16S rRNA gene sequences suggests that the Mexican isolates are not Frankia strains but members of a novel clade.

Appl Environ Microbiol, 1996 Aug, 62(8), 2904 - 9
Amplification of 16S rRNA genes from Frankia strains in root nodules of Ceanothus griseus, Coriaria arborea, Coriaria plumosa, Discaria toumatou, and Purshia tridentata; Benson DR et al.; To study the global diversity of plant-symbiotic nitrogen-fixing Frankia strains, a rapid method was used to isolate DNA from these actinomycetes in root nodules . The procedure used involved dissecting the symbiont from nodule lobes; ascorbic acid was used to maintain plant phenolic compounds in the reduced state . Genes for the small-subunit rRNA (16S ribosomal DNA) were amplified by the PCR, and the amplicons were cycle sequenced . Less than 1 mg (fresh weight) of nodule tissue and fewer than 10 vesicle clusters could serve as the starting material for template preparation . Partial sequences were obtained from symbionts residing in nodules from Ceanothus griseus, Coriaria arborea, Coriaria plumosa, Discaria toumatou, and Purshia tridentata . The sequences obtained from Ceonothus griseus and P . tridentata nodules were identical to the sequence previously reported for the endophyte of Dryas drummondii . The sequences from Frankia strains in Coriaria arborea and Coriaria plumosa nodules were identical to one another and indicate a separate lineage for these strains . The Frankia strains in Discaria toumatou nodules yielded a unique sequence that places them in a lineage close to bacteria that infect members of the Elaeagnaceae.

Appl Environ Microbiol, 1996 Aug, 62(8), 2710 - 5
Characterization of active recombinant 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase from Comamonas testosteroni B-356 and sequence of the encoding gene (bphB); Sylvestre M et al.; 2,3-Dihydro-2,3-dihydroxybiphenyl-2,3-dehydrogenase (B2,3D) catalyzes the second step in the biphenyl degradation pathway . The nucleotide sequence of Comamonas testosteroni B-356 bphB, which encodes B2,3D, was determined . Structural analysis showed that the dehydrogenases involved in the bacterial degradation of aromatic compounds are related to each other and that their phylogenetic relationships are very similar to the relationships observed for dioxygenases that catalyze the initial reaction in the degradation pathway . The bphB sequence was used to produce recombinant active His-tagged B2,3D, which allowed us to describe for the first time some of the main features of a B2,3D . This enzyme requires NAD+, its optimal pH is 9.5, and its native M(r) was found to be 123,000, which makes it a tetramer . These characteristics are very similar to those reported for the related enzyme cis-toluene dihydrodiol dehydrogenase . The Km value and maximum rate of metabolism for 2,3-dihydro-2,3-dihydroxybiphenyl were 73 +/- 16 microM and 46 +/- 4 nmol min-1 microgram-1, respectively . Compared with the cis-toluene dihydrodiol dehydrogenase, B2,3D appeared to be more substrate specific since it was unable to attack cis-1,2-dihydroxy-cyclohexa-3,5-diene.

Gastroenterology, 1996 Aug, 111(2), 419 - 25
Helicobacter pylori urease is a potent stimulus of mononuclear phagocyte activation and inflammatory cytokine production; Harris PR et al.; BACKGROUND & AIMS: Helicobacter pylori surface proteins induce the production of proinflammatory mediators by mononuclear phagocytes, but the protein responsible for this stimulation has not been identified . This study determined whether urease, the major component of the soluble proteins extracted from H . pylori grown in culture, activates mononuclear phagocytes and stimulates them to produce proinflammatory cytokines . METHODS: Primary human blood monocytes were incubated with column-purified H . pylori urease and assayed by flow cytometry, Immunoassay, and reverse-transcription polymerase chain reaction for phenotypic, functional, and molecular evidence of activation . RESULTS: H . pylori urease induced monocyte expression of surface interleukin 2 receptors and increased expression of HLA-DR, phenotypic changes consistent with activation . Urease also stimulated dose-dependent production of interleukin 1 beta, interleukin 6, interleukin 8, and tumor necrosis factor alpha peptides and messenger RNA . These urease-induced phenotypic and functional changes were inhibited by preincubation of the urease with antisera to H . pylori whole bacteria, purified urease, or the 31-kilodalton subunit of urease . CONCLUSIONS: Among the soluble proteins released by H . pylori, urease is capable of activating monocytes for proinflammatory cytokines production . The local production of cytokines by urease-stimulated mononuclear phagocytes may play a central role in the development of H . pylori gastroduodenal inflammation.

Nature, 1996 Aug 1, 382(6590), 471 - 3
Visualization of ordered genomic RNA and localization of transcriptional complexes in rotavirus; Prasad BV et al.; In double-stranded-RNA (dsRNA) viruses found in animals, bacteria and yeast, the genome is transcribed within the structurally intact core of the virion with extraordinary efficiency . The structural organization of the genome and the enzymes involved in the transcription inside any of these viruses, critical for understanding this process, is not known . Here we report what we believe is the first three-dimensional characterization of the viral genome and the transcription complex in a prototypical dsRNA virus . Rotavirus is a large (diameter 1,000 A) icosahedral virus composed of three capsid protein layers and 11 dsRNA segments . It is the most important cause of gastroenteritis in children, accounting for over a million deaths annually . We show that viral dsRNA forms a dodecahedral structure in which the RNA double helices, interacting closely with the inner capsid layer, are packed around the enzyme complex located at the icosahedral 5-fold axes . The ordered RNA accounts for about 4,500 out of a total 18,525 base pairs in the genome, the largest amount of icosahedrally ordered RNA observed in any virus structure to date . We propose that the observed organization of the dsRNA is conducive for an orchestrated movement of the RNA relative to the enzyme complex during transcription.

Biochim Biophys Acta, 1996 Jul 31, 1275(3), 161 - 203
The ferritins: molecular properties, iron storage function and cellular regulation; Harrison PM et al.; The iron storage protein, ferritin, plays a key role in iron metabolism . Its ability to sequester the element gives ferritin the dual functions of iron detoxification and iron reserve . The importance of these functions is emphasised by ferritin's ubiquitous distribution among living species . Ferritin's three-dimensional structure is highly conserved . All ferritins have 24 protein subunits arranged in 432 symmetry to give a hollow shell with an 80 A diameter cavity capable of storing up to 4500 Fe(III) atoms as an inorganic complex . Subunits are folded as 4-helix bundles each having a fifth short helix at roughly 60 degrees to the bundle axis . Structural features of ferritins from humans, horse, bullfrog and bacteria are described: all have essentially the same architecture in spite of large variations in primary structure (amino acid sequence identities can be as low as 14%) and the presence in some bacterial ferritins of haem groups . Ferritin molecules isolated from vertebrates are composed of two types of subunit (H and L), whereas those from plants and bacteria contain only H-type chains, where 'H-type' is associated with the presence of centres catalysing the oxidation of two Fe(II) atoms . The similarity between the dinuclear iron centres of ferritin H-chains and those of ribonucleotide reductase and other proteins suggests a possible wider evolutionary linkage . A great deal of research effort is now concentrated on two aspects of ferritin: its functional mechanisms and its regulation . These form the major part of the review . Steps in iron storage within ferritin molecules consist of Fe(II) oxidation, Fe(III) migration and the nucleation and growth of the iron core mineral . H-chains are important for Fe(II) oxidation and L-chains assist in core formation . Iron mobilisation, relevant to ferritin's role as iron reserve, is also discussed . Translational regulation of mammalian ferritin synthesis in response to iron and the apparent links between iron and citrate metabolism through a single molecule with dual function are described . The molecule, when binding a {4Fe-4S} cluster, is a functioning (cytoplasmic) aconitase . When cellular iron is low, loss of the {4Fe-4S} cluster allows the molecule to bind to the 5'-untranslated region (5'-UTR) of the ferritin m-RNA and thus to repress translation . In this form it is known as the iron regulatory protein (IRP) and the stem-loop RNA structure to which it binds is the iron regulatory element (IRE) . IREs are found in the 3'-UTR of the transferrin receptor and in the 5'-UTR of erythroid aminolaevulinic acid synthase, enabling tight co-ordination between cellular iron uptake and the synthesis of ferritin and haem . Degradation of ferritin could potentially lead to an increase in toxicity due to uncontrolled release of iron . Degradation within membrane-encapsulated "secondary lysosomes' may avoid this problem and this seems to be the origin of another form of storage iron known as haemosiderin . However, in certain pathological states, massive deposits of "haemosiderin' are found which do not arise directly from ferritin breakdown . Understanding the numerous inter-relationships between the various intracellular iron complexes presents a major challenge.

Biochim Biophys Acta, 1996 Jul 31, 1275(3), 145 - 50
Molecular cloning, DNA sequence and transcriptional analysis of the Rhodospirillum molischianum B800/850 light-harvesting genes; Germeroth L et al.; The amino acid sequences of the B800/850 light-harvesting proteins from Rhodospirillum molischianum were determined by Edman degradation . On the basis of these amino acid sequences, two degenerated oligonucleotides were synthesized and used for PCR of genomic DNA . The resulting 150 bp DNA fragment was cloned, sequenced and used for subsequent Southern blot analysis of digested genomic DNA . A 2.3 kbp EcoRI fragment strongly hybridized to the probe and a size selected genomic library from genomic DNA was constructed . One clone scored positive during screening of the library with the PCR-fragment and subsequent DNA sequence analysis of the clone revealed the presence of three A-genes (A1A2A3) encoding alpha-polypeptides and of two B-genes (B1B2) encoding beta-polypeptides of the B800/850 complex . The arrangement of the different genes are B1A1, B2A2 and A3 where only B1 and B2 are preceded by typical Shine-Dalgarno sequences . In addition, typical nucleotide sequences for a rho-independent termination of transcription are located downstream of the genes A1 and A2 . The deduced amino acid sequences revealed that the alpha-genes encoded for identical polypeptides, whereas the deduced beta-polypeptides differed in their amino acid sequence at four positions . Transcriptional operon analysis revealed that the genes A1B1 and A2B2 are both dicistronically transcribed, whereas the gene A3 is not.

Biochemistry, 1996 Jul 30, 35(30), 9925 - 34
Energy and electron transfer upon selective femtosecond excitation of pigments in membranes of Heliobacillus mobilis; Liebl U et al.; Excitation energy transfer steps in membranes of Heliobacillus mobilis were directly monitored by transient absorption spectroscopy with a time resolution of 30 fs under selective excitation within the inhomogeneously broadened bacteriochlorophyll g QY band . The initial anisotropy was found to be > 0.4, indicating that the pigments are excitonically coupled . After initial decay of this anisotropy in < 50 fs, major sub-picosecond components associated with spectral equilibration were identified, corresponding to uphill energy transfer with a 300 fs time constant (812 nm excitation) and downhill energy transfer with 100 and 500 fs components (770 nm excitation) . These equilibrations are ascribed predominantly to single excitation transfer steps, as anisotropy measurements showed that equilibration within spectrally similar pigments occurs on the same time scale as spectral equilibration, a situation which contrasts with that in photosystem I . Downhill energy transfer occurs to a significant extent directly to an energetically heterogeneous population of excited states as well as in a sequential way via gradually lower-lying pools of bacteriochlorophyll g . This finding supports a description in which all pigments, including the bluemost absorbing, are spatially organized in a random way rather than in clusters of spectrally similar species . Spectral equilibration is not entirely completed prior to formation of the primary radical pair P798 + A0-, which was found to proceed in a multiexponential way (time constants of 5 and 30 ps) . No indication for the formation of radical species other than P798 + A0- on the time scale up to 100 ps was found.

Neuroreport, 1996 Jul 29, 7(11), 1730 - 2
Metabolism of agmatine into urea but not into nitric oxide in rat brain; Gilad GM et al.; Agmatine is a guanidino compound abundant in bacteria and plants where it serves as a precursor for polyamine synthesis . It can interfere with several neurotransmission-related functions and can exert neuroprotective effects after brain injury . Agmatine was recently identified in mammalian brain and its synthesis by arginine decarboxylation was characterized . Its metabolism by the brain is, however, unknown . Here we report evidence indicating that agmatine can be selectively metabolized in the rat brain (cerebellum) into urea and thus, may lead to formation of putrescine, the precursor of polyamine synthesis . In addition, while agmatine can inhibit brain nitric oxide synthase, it did not serve as a substrate for nitric oxide formation.

J Biol Chem, 1996 Jul 26, 271(30), 17609 - 12
Developmental and tissue-specific expression of mouse pelle-like protein kinase; Trofimova M et al.; The NF-kappaB/c-Rel proteins are a family of evolutionarily conserved transcription factors activated during development that in the adult, mediate many processes including the immune response . A high degree of sequence similarity is shared between the NF-kappaB/c-Rel family of transcription factors and the Drosophila Dorsal protein as well as between its cytoplasmic inhibitor, IkappaBalpha, and the Drosophila Cactus protein . Genetic analyses of Dorsal have defined components of a signaling pathway for Dorsal activation, including a serine/threonine kinase, Pelle, placed upstream of Dorsal and Cactus . We demonstrate that this pathway is likely to be conserved in mammals by the isolation of a cDNA that encodes a novel mouse protein highly related to Pelle, mPLK (mouse Pelle-like protein kinase) . Expression of mPLK mRNA is developmentally regulated in the mouse and in adult tissue mPLK expression is greatest in the liver, a tissue that expresses a high level of NF-kappaB . Recombinant mPLK produced in bacteria is a protein kinase capable of autophosphorylating and phosphorylating IkappaBalpha.

Biochem Biophys Res Commun, 1996 Jul 25, 224(3), 611 - 8
Nitrite reductase from Desulfovibrio desulfuricans (ATCC 27774)--a heterooligomer heme protein with sulfite reductase activity; Pereira IC et al.; The membrane bound cytochrome c nitrite reductase from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774) was found to have a high specific activity in the reduction of sulfite, producing stoichiometric amounts of sulfide . The K(m) for sulfite in the MV+.:sulfite oxidoreductase assay is 0.75 mM, and the specific activity 2.06 mumolH2/min/mg . Visible and EPR spectroscopies studies indicate that the enzyme high-spin heme reacts with sulfite in the oxidised state, and that sulfide partially reduces the enzyme . The redoxcycled enzyme, using H2/Hydrogenase/MV+ . as a reductant, is identical to the resting enzyme . This is the first time that a c-type nitrite reductase has been shown to reduce sulfite . These findings, besides revealing a new function for the nitrite reductase, raise a major question regarding the sulfur metabolism in the sulfate reducing bacteria as well as the cellular localization of the enzymatic activities involved in the dissimilatory reduction of sulfate . The purified nitrite reductase is a heterooligomer, containing two types of subunits of 62 kDa (+/- 5 kDa) and 18.8 kDa (+/- 1 kDa), and forms a complex or aggregate with a molecular mass of approximately 750 kDa.

Biochemistry, 1996 Jul 23, 35(29), 9584 - 93
Functional interaction of the c-Myc transactivation domain with the TATA binding protein: evidence for an induced fit model of transactivation domain folding; McEwan IJ et al.; c-Myc is a member of a family of sequence specific-DNA binding proteins that are thought to regulate the transcription of genes involved in normal cell growth, differentiation, and apoptosis . In order to understand how human c-myc functions as a transcription factor, we have studied the mechanism of action and structure of the N-terminal transactivation domain, amino acids 1-143 . In a protein interaction assay, c-myc1-143 bound selectively to two basal transcription factors, the TATA binding protein (TBP) and the RAP74 subunit of TFIIF . Furthermore, the isolated c-myc transactivation domain competed for limiting factors required for the assembly of a functional preinitiation complex . This squelching of basal transcription was reversed in a dose-dependent manner by recombinant TBP . Taken together, these results identify TBP as an important target for the c-myc transactivation domain, during transcriptional initiation . Similar to other transactivation domains, the c-myc1-143 polypeptide showed little or no evidence of secondary structure, when measured by circular dichroism spectroscopy (CD) in aqueous solution . However, significant alpha-helical conformation was observed in the presence of the hydrophobic solvent trifluoroethanol . Strikingly, addition of TBP caused changes in the CD spectra consistent with induction of protein conformation in c-myc1-143 during interaction with the target factor . This change was specific for TBP as a similar effect was not observed in the presence of TFIIB . These data support a model in which target factors induce or stabilize a structural conformation in activator proteins during transcriptional transactivation.

FEBS Lett, 1996 Jul 22, 390(2), 119 - 23
The emergence of major cellular processes in evolution; Ouzounis C et al.; The phylogenetic distribution of divergently related protein families into the three domains of life (archaea, bacteria and eukaryotes) can signify the presence or absence of entire cellular processes in these domains and their ancestors . We can thus study the emergence of the major transitions during cellular evolution, and resolve some of the controversies surrounding the evolutionary status of archaea and the origins of the eukaryotic cell . In view of the ongoing projects that sequence the complete genomes of several Archaea, this work forms a testable prediction when the genome sequences become available . Using the presence of the protein families as taxonomic traits, and linking them to biochemical pathways, we are able to reason about the presence of the corresponding cellular processes in the last universal ancestor of contemporary cells . The analysis shows that metabolism was already a complex network of reactions which included amino acid, nucleotide, fatty acid, sugar and coenzyme metabolism . In addition, genetic processes such as translation are conserved and close to the original form . However, other processes such as DNA replication and repair or transcription are exceptional and seem to be associated with the structural changes that drove eukaryotes and bacteria away from their common ancestor . There are two major hypotheses in the present work: first, that archaea are probably closer to the last universal ancestor than any other extant life form, and second, that the major cellular processes were in place before the major splitting . The last universal ancestor had metabolism and translation very similar to the contemporary ones, while having an operonic genome organization and archaean-like transcription . Evidently, all cells today contain remnants of the primordial genome of the last universal ancestor.

J Biol Chem, 1996 Jul 19, 271(29), 17330 - 4
Molecular cloning of human phosphomevalonate kinase and identification of a consensus peroxisomal targeting sequence; Chambliss KL et al.; Two overlapping cDNAs which encode human liver phosphomevalonate kinase (PMKase) were isolated . The human PMKase cDNAs predict a 191-amino acid protein with a molecular weight of 21,862, consistent with previous reports for mammalian PMKase (Mr = 21,000-22,500) . Further verification of the clones was obtained by expression of PMKase activity in bacteria using a composite 1024-base pair cDNA clone . Northern blot analysis of several human tissues revealed a doublet of transcripts at approximately 1 kilobase (kb) in heart, liver, skeletal muscle, kidney, and pancreas and lower but detectable transcript levels in brain, placenta, and lung . Analysis of transcripts from human lymphoblasts subcultured in lipid-depleted sera (LDS) and LDS supplemented with lovastatin indicated that PMKase gene expression is subject to regulation by sterol at the level of transcription . Southern blotting indicated that PMKase is a single copy gene covering less than 15 kb in the human genome . The human PMKase amino acid sequence contains a consensus peroxisomal targeting sequence (PTS-1), Ser-Arg-Leu, at the C terminus of the protein . This is the first report of a cholesterol biosynthetic protein which contains a consensus PTS-1, providing further evidence for the concept that early cholesterol and nonsterol isoprenoid biosynthesis may occur in the peroxisome.

Biochim Biophys Acta, 1996 Jul 18, 1275(1-2), 16 - 20
Respiratory chains of archaea and extremophiles; Schafer G et al.; Extremophilic organisms are adapted to harsh environmental conditions like high temperature, extremely acidic or alkaline pH, high salt, or a combination of those . With a few exceptions extremophilic bacteria are colonizing only moderately hot biotopes, whereas hyperthermophiles are found specifically among archaea (formerly 'archaebacteria') which can thrive at temperatures close to or even above the boiling point of water . It has been a challenging question whether the special properties of their proteins and membranes have been acquired by adaptation, or whether they might reflect early evolutionary states as suggested by their phylogenetic position at the lowest branches of the universal tree of life.

Biochem Biophys Res Commun, 1996 Jul 16, 224(2), 555 - 63
Interferon-gamma-dependent expression of inducible nitric oxide synthase, interleukin-12, and interferon-gamma-inducing factor in macrophages elicited by allografted tumor cells; Sanchez-Bueno A et al.; We have examined the mechanisms of activation of macrophages (Mos) induced by i.p . allografted Meth A tumor cells (Meth A-Mos) during the rejection of the cells by C57BL/6 mice . Inducible nitric oxide (NO) synthase (iNOS), interleukin-12 (IL-12), and interferon-gamma (IFN-gamma)-inducing factor (IGIF) were transiently expressed in Meth A-Mos during the rejection . The expression was impaired in mice in which the gene encoding IFN-gamma had been disrupted (IFN-gamma-/-) . In vitro studies showed that Meth A-Mos from IFN-gamma +/+ mice induced an apoptotic type of cell death in P815 cells, without cell-to-cell contact, in an NO-dependent manner, whereas Meth A-Mos from IFN-gamma-/- mice could not lyse these cells . The iNOS, IL-12, and IGIF expression was also impaired in bacteria-activated Mos from IFN-gamma-/-mice, indicating that IFN-gamma, but not IGIF, would be the initial signal that leads to the activation of Mos in vivo.

J Immunol, 1996 Jul 15, 157(2), 650 - 5
Introduction of exogenous antigens into the MHC class I processing and presentation pathway by Drosophila antennapedia homeodomain primes cytotoxic T cells in vivo; Schutze-Redelmeier MP et al.; The homeodomain of the Antennapedia molecule (AntpHD) spontaneously crosses cellular membranes and can be used to deliver up to 50 additional amino acids to the cytoplasm . We exploited this approach to deliver antigenic peptides to the MHC class I processing and presentation pathway . AntpHD-based fusion peptides expressing the 170-179 HLA-Cw3 CTL epitope (pCw3) were produced in bacteria . Incubation of these fusion peptides with H-2d target cells resulted in efficient delivery to the cytosol as indicated by protease resistance and confocal microscopy . Moreover, this introduction of an exogenous Ag resulted in sensitization of the cell to lysis by a CTL clone specific for the 170-179 HLA-Cw3-derived peptide . Sensitivity of the Ag processing to brefeldin A but not to chloroquine is consistent with the delivery of AntpHD fusion peptides to the conventional class I-associated processing pathway . Immunization of DBA/2 (H-2d) mice with AntpHD pCw3 fusion peptide in the presence of SDS primed H-2Kd-restricted HLA-Cw3-specific CTL . Similar results were obtained with AntpHD fusion peptides expressing the 147-156 influenza nucleoprotein peptide . The strategy outlined in this paper provides a new approach for introducing molecules into the MHC class I Ag-presenting pathway . This approach has clear relevance to the design of synthetic peptide-based vaccines.

Biochem Pharmacol, 1996 Jul 12, 52(1), 35 - 42
Differential effects of tenidap on the zymosan- and lipopolysaccharide-induced expression of mRNA for proinflammatory cytokines in macrophages; Bondeson J et al.; Tenidap is a novel antirheumatic drug that combines cyclooxygenase inhibition with cytokine modulating qualities . We demonstrate here that tenidap inhibits the zymosan-induced expression of both interleukin 1 and tumor necrosis factor alpha in macrophages, at the mRNA and protein levels . The concentration-dependence of the tenidap-induced inhibition of the expression of mRNA for these proinflammatory cytokines agrees with that of its inhibitory effects on zymosan-induced arachidonate mobilization and changes in phosphoprotein pattern . The effects of tenidap on the lipopolysaccharide-induced expression of these cytokines are more complex . Tenidap inhibits the induction of interleukin 1 by lipopolysaccharide or bacteria, but less potently than the interleukin 1-response induced by zymosan . In contrast, the drug markedly potentiates the lipopolysaccharide-induced expression of tumor necrosis factor alpha at both the mRNA and protein levels . The latter effect is demonstrated to be due to cyclooxygenase inhibition and is reversed by prostaglandin E2.

Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7131 - 6
Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication; Malkova A et al.; In wild-type diploid cells of Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) at the MAT locus can be efficiently repaired by gene conversion using the homologous chromosome sequences . Repair of the broken chromosome was nearly eliminated in rad52delta diploids; 99% lost the broken chromosome . However, in rad51delta diploids, the broken chromosomes were repaired approximately 35% of the time . None of these repair events were simple gene conversions or gene conversions with an associated crossover, instead, they created diploids homozygous for the MAT locus and all markers in the 100-kb region distal to the site of the DSB . In rad51delta diploids, the broken chromosome can apparently be inherited for several generations, as many of these repair events are found as sectored colonies, with one part being repaired and the other part being lost the broken chromosome . Similar events occur in about 2% of wild-type cells . We propose that a broken chromosome end can invade a homologous template in the absence of RAD51 and initiate DNA replication that may extend to the telomere, 100 or more kb away . Such break-induced replication appears to be similar to recombination-initiated replication in bacteria.

Res Microbiol, 1996 Jul-Sep, 147(6-7), 489 - 93
Cra and the control of carbon flux via metabolic pathways; Ramseier TM; The catabolite repressor-activator (Cra) protein controls the direction of carbon flux through metabolic pathways in enteric bacteria . Cra binds to the control regions of target genes and exerts a negative effect on the expression of genes encoding glycolytic and Entner-Doudoroff enzymes, while exerting a positive effect on genes encoding Krebs cycle, glyoxylate shunt and gluconeogenic enzymes . Cra mediates cyclic AMP-independent catabolite repression of positively Cra-regulated genes and catabolite activation of negatively Cra-controlled genes.

Res Microbiol, 1996 Jul-Sep, 147(6-7), 448 - 55
Evolution of carbohydrate metabolic pathways; Romano AH et al.; Current studies of hyperthermophilic archaea and bacteria, the phylogenetically deepest-rooted and slowest-evolving extant organisms known, are allowing new insights into the nature of presumably ancient metabolic pathways . The apparent common occurrence of modified non-phosphorylated Entner-Doudoroff (ED) pathways among saccharolytic archaea and the absence of the conventional Embden-Meyerhof-Parnas (EMP) mode of glycolysis indicate that the ED pathway is the older route of carbohydrate dissimilation . However, gluconeogenesis via the "reversed" EMP route has been found in archaea . Thus, the EMP pathway was probably an anabolic pathway to begin with; its catabolic role came later, with the evolution of fructose phosphate kinases, using ATP, ADP or pyrophosphate as phosphate donors . Similarly, the presence of reductive reactions of the citric acid cycle in anaerobic archaea and the most deeply rooted bacteria, including autotrophs, indicates that the citric acid cycle was originally a reductive biosynthetic pathway.

Lijec Vjesn, 1996 Jul-Aug, 118(7-8), 178 - 83
{Sphingolipids--a new group of lipid second messengers?}; Mesaric M; Sphingolipids have the potential to regulate cell behavior at essentially all levels of signal transduction . They serve as cell surface receptors for cytoskeletal proteins, immunoglobulins, and some bacteria; as modifiers of the properties of cell receptors for growth factors (and perhaps other agents); and as activators and inhibitors of protein kinases, ion transporters, and other proteins . the biological activity of these compounds resides not only in the more complex molecules, but also in their turnover products . Since sphingolipids change with cell growth, differentiation, and neoplastic transformation, they could be vital participants in the regulation of these processes.

Indian J Lepr, 1996 Jul-Sep, 68(3), 217 - 22
Cellularity of macrophage granuloma and morphological index; Porichha D et al.; In the present study, morphological index (MI) and average macrophage count per microscopic field in skin sections of 94 lepromatous (LL) patients is correlated . The subjects included 14 cases with some histoid features . The MI in the lepromatous cases varied from less than one to 40 and the corresponding macrophage counts ranged from 40 to 156 . In cases with histoid changes the MI varied from 30 to 60 and the cell count ranged from 215 to 360 . The histoid cases showed a higher MI and cell count compared to the other lepromatous cases . There was a positive correlation between MI and macrophage count and the hypercellular state appears to depend on living and multiplying bacteria.

Hepatogastroenterology, 1996 Jul-Aug, 43(10), 796 - 9
Sequential changes of bile contents in patients with obstructive jaundice from different etiologies; Chen CY et al.; BACKGROUND/AIMS: The decreasing of serum concentration of bilirubin and the ability of hepatocytes to excrete various biliary contents after release of obstructive jaundice are good indicators of recovery of liver function . We conducted this study to clarify whether different causes of obstructive jaundice have different effects on the biliary excretion and how they are different when obstruction is released . PATIENTS AND METHODS: Fifteen patients with obstructive jaundice undergoing percutaneous transhepatic catheter drainage or endoscopic nasobiliary drainage were classified into two groups, depending on the cause of obstruction: common bile duct stones (n = 7) and biliary tract tumors (n = 8) . All patients in the gallstone group presented with acute cholangitis while only three patients in the tumor group had a positive bacteria culture in bile . Fasting biles were collected on the day of catheter placement and the 2nd, 4th, 6th, 8th, 10th day thereafter . The sequential changes of biliary concentration of bilirubin, phospholipid, cholesterol, bile salts and serum bilirubin were checked and compared between the two groups . RESULTS: The difference in the improvement of jaundice between the stone and tumor group (p > 0.05) were not significant, nor were the excretion of biliary contents after relief of obstruction . The reduction of serum bilirubin paralleled with the increased excretion of biliary bile salts and bilirubin (gamma = -0.51, p < 0.01 and gamma = -0.4, p < 0.05) in tumor group, but not in the stone group . CONCLUSIONS: The decrease of serum bilirubin and the sequential changes of bile contents after relief of obstruction are quite similar in stone and tumor induced obstructive jaundice.

Avian Dis, 1996 Jul-Sep, 40(3), 572 - 5
Respiratory coccidiosis (Cryptosporidium baileyi) among northern Georgia broilers in one company; Goodwin MA et al.; Cryptosporidium baileyi causes respiratory disease in chickens . The purposes of this prospective study were to determine the incidence of C . baileyi tracheitis among broilers in a commercial setting, and the relationship between C . baileyi tracheitis and production performance parameters . All samples came from 56 farms that grow broilers for one company in northern Georgia . Tracheas were collected and examined with a light microscope and cultured for viruses and bacteria . Overall, 23 of 56 (41%) broiler flocks had C . baileyi tracheitis . Parasitism rates among C . baileyi-infected flocks ranged from a low of 10% to a high of 60% . Cryptosporidium baileyi tracheitis was very highly correlated (rho = 0.81, n = 56, P < 0.00001) to severity of tracheitis, negatively correlated (rho = -0.27, n = 56, P < 0.04) with average body weight, and correlated with airsacculitis (rho = 0.30, n = 56, P < 0.03) and condemnations (rho = 0.27, n = 56, P < 0.05) . The present study indicates that C . baileyi infection rates are high, and the role that this parasite plays in the pathogenesis of respiratory disease and production losses could be unexpectedly large.

Nutrition, 1996 Jul-Aug, 12(7-8), 529 - 33
Oral arginine supplementation in acute liver injury; Adawi D et al.; Acute liver failure is accompanied by a high rate of bacterial and septic complications . Arginine has a potent effect on the immune system and modulates bacterial clearance in septic models . We studied the effect of oral arginine supplementation on the extent of liver injury and the associated bacterial translocation in an acute liver injury model in rats . Sprague-Dawley rats were divided into normal, liver injury, and arginine supplemented groups . In the arginine group, 2% arginine was supplemented daily through a nasogastric tube for 8 d . Acute liver injury was induced on the eighth day by intraperitoneal injection of D-galactosamine (1.1 g/kg body wt) . Samples were collected 24 h after the liver injury . In the arginine-supplemented group, alkaline phosphatase, bilirubin, and aspartate aminotransferase were reduced significantly compared with the acute liver injury control group . The results of bacterial translocation in the arginine-supplemented group showed a significantly reduced number of translocated bacteria to the liver and mesenteric lymph nodes than occurred in the acute liver injury group . The histological study of the liver in arginine-supplemented group showed scattered areas of hepatocellular necrosis and inflammatory cell infiltration, and in the acute liver injury group there were more and widespread hepatocellular necrosis and inflammatory cell infiltration . Oral supplementation of arginine in an acute liver injury model improves significantly the state of the liver injury and reduces bacterial translocation to the liver and mesenteric lymph nodes.

Immunobiology, 1996 Jul, 195(2), 199 - 208
The proliferation of human T lymphocytes stimulated by Helicobacter pylori antigens; Chmiela M et al.; Fractionated mononuclear cells (MNCs) were obtained from peripheral blood of healthy human volunteers, seronegative for H . pylori antibodies . The MNCs were stimulated in culture with whole live or heat-killed H . pylori cells or with bacterial cell surface (SA) or cytoplasmic (CA) antigens . There was a marked proliferative response of T cells in cultures stimulated with 10(5) cells/well of live H . pylori, 5 micrograms/well of CA or 5-20 micrograms/well of SA . However, no proliferation was observed in MNC cultures containing higher "doses" of live H . pylori organisms (10(7)/well) or CA (20 micrograms/well) . Moreover, higher "doses" of the bacteria or CA entirely inhibited the response of T cells to PHA.

Glycobiology, 1996 Jul, 6(5), 543 - 50
Characterization of two mannose-binding protein cDNAs from rhesus monkey (Macaca mulatta): structure and evolutionary implications; Mogues T et al.; Mannose-binding proteins (MBPs), members of the collectin family, have been implicated as lectin opsonins for various viruses and bacteria . Two distinct but related MBPs, MBP-A and MBP-C, with approximately 55% identity at the amino acid level, have been previously characterized from rodents . In humans, however, only one form of MBP has been characterized . In this paper we report studies elucidating the evolution of primate MBPs . ELISA and Western blot analyses indicated that rhesus and cynomolgus monkeys have two forms of MBP in their sera, while chimpanzees have only one form, similar to humans . Two distinct MBP cDNA clones were isolated and characterized from a rhesus monkey liver cDNA library . Rhesus MBP-A is closely related to the mouse and rat MBP-A, showing 77% and 75% identity at the amino acid level, respectively . Rhesus MBP-A also has three cysteines at the N-terminus, similar to mouse and rat MBP-A and human MBP . Rhesus MBP-C shares 90% identity with the human MBP at the amino acid level and has three cysteines at the N-terminus, in contrast to two cysteine residues found in rodent MBP-C . A stretch of nine amino acids close to the N-terminus, absent in both mouse and rat MBP-A, but present in rodent MBP-C, chicken and human MBPs, is also found in the rhesus MBP-A . The phylogenetic analysis of rhesus and other mammalian MBPs, coupled with the serological data suggest that at least two distinct MBP genes existed prior to mammalian radiation and the hominoid ancestor apparently lost one of these genes or failed to express it.

Curr Opin Rheumatol, 1996 Jul, 8(4), 296 - 308
HLA-B27 structure, function, and disease association; Lopez-Larrea C et al.; The polymorphism of HLA-B27 alleles is located in the peptide-anchoring motif . In recent years, fundamental insights have been made into the molecular aspects of HLA-B27-restricted presentation . Subtle differences in peptide binding fine specificity are especially interesting for closely related HLA-B27 alleles that have differential association with ankylosing spondylitis . Bacterial infection has been suggested to play a role in the pathogenesis of HLA-B27-associated disease . Remarkable progress has been made in identifying peptides derived from bacteria that can be presented by HLA-B27 . Despite the mechanisms proposed to explain B27-associated diseases, there are no clear correlations between peptide sequence, differential binding to B27 subtypes, and recognition by peptide-specific T cell receptors . Furthermore, new transgenic models have now been developed that we hope will allow a clearer view of the function of B27 and the mechanisms involved in the pathogenesis of spondyloarthropathies.

Curr Opin Rheumatol, 1996 Jul, 8(4), 275 - 87
The sacroiliac joint in the spondyloarthropathies; Braun J et al.; The term spondyloarthropathy (SpA) describes and defines a group of related inflammatory joint disease that share characteristic clinical features and a unique association with the major histocompatibility complex class I molecule HLA-B27 . Five subgroups can be differentiated: ankylosing spondylitis, reactive arthritis, psoriatic arthritis, arthritis associated with inflammatory bowel disease, and undifferentiated SpA . The sacroiliac joints are centrally involved in the SpA, most clearly and pathognomonic in ankylosing spondylitis, in which most patients are affected early in the disease . Overcoming some of the diagnostic difficulties of early sacroiliitis, dynamic magnetic resonance imaging was shown to visualize both acute and chronic changes in the sacroiliac joints . The inflammation in the sacroiliac joints in patients with SpA was recently examined in more detail; using immunohistology and in situ hybridrization, T cells, macrophages, and various cytokines were found in infiltrates . Biopsy specimens were obtained under guided computed tomography, and in the same study, intra-articular corticosteroid treatment was successfully undertaken . Further investigation of such biopsy specimens showed the absence of DNA of reactive arthritis-associated bacteria . The pathogenesis of the SpA and the reason for the tropism for the sacroiliac joints is still obscure . The nature of the relation of the genetic background of SpA to initially triggering bacterial infections remains to be established . In chronic disease, autoimmune mechanisms might be more important.

Gen Pharmacol, 1996 Jul, 27(5), 761 - 71
Salivary mucins in oral mucosal defense; Slomiany BL et al.; 1 . Salivary mucins are well recognized as an important factor in the preservation of the health of the oral cavity . These large glycoproteins play a major role in the formation of protective coatings covering tooth enamel and oral mucosa, which act as a dynamic functional barrier capable of modulating the untoward effects of oral environment, and are of significance to the processes occurring within the epithelial perimeter of mucosal defense . 2 . Based on macromolecular characteristics, the mucins in saliva fall into high (> 1000 kDa) and low (200-300 kDa) molecular weight forms . The two forms, although differ with respect to bacterial clearance ability, display virtually identical carbohydrate chain make-up, ranging in size from 3 to 16 sugar units . 3 . Of the two mucin forms, the low molecular weight form more efficient in bacterial aggregation, predominates in saliva and oral mucosal mucus coat of caries-resistant individuals, while the level of the high molecular weight form is higher in caries-susceptible subjects . The saliva of caries-resistant individuals also exhibits greater activity of protease capable of conversion of the high molecular weight mucin to the low molecular weight form . 4 . The bacterial aggregating activity of salivary mucins appears to be associated with sulfomucins rather than sialomucins . While the removal of sialic acid causes only partial loss in mucin aggregating capacity, a complete loss in the bacterial aggregating activity occurs following mucin desulfation . 5 . The mucins in oral mucosal mucus coat interact with the epithelial surfaces through specific membrane receptors . This interaction apparently involves the carbohydrate moiety of mucin molecule and may be rendered vulnerable to disruption by opportunistic bacteria colonizing the oral mucosa . 6 . Salivary sulfo- and sialomucins actively participate in the modulation of the oral mucosal calcium channel activity through the inhibition of EGF-stimulated channel protein tyrosine phosphorylation . This function of salivary mucins is of paramount importance to mucosal calcium homeostasis.

J Clin Gastroenterol, 1996 Jul, 23(1), 11 - 4
Uncertain clinical significance of duodenal mucosal abnormalities in HIV-infected individuals . Results of a case-control study; Rabeneck L et al.; Previous research has described abnormalities of duodenal mucosal morphology in human immunodeficiency virus (HIV)-infected individuals . We wanted to determine the frequency of disturbed villus architecture and investigate its relationship to HIV-related chronic diarrhea . We conducted a case-control study of 120 HIV-infected men, 63 with and 57 without chronic diarrhea . Stools were cultured for bacteria and examined for ova and parasites; esophagogastroduodenoscopy and flexible sigmoidoscopy with mucosal biopsies were performed . Biopsy tissue was examined using light and electron microscopy to detect enteric pathogens and to evaluate mucosal morphology . The mean CD4+ cell count was 143/min3, and enteric pathogens were detected in 56 of 120 men (47%) . In approximately half the study sample (57%), duodenal villus architecture was normal; complete villus flattening was not observed . We detected no association between chronic diarrhea and altered villus architecture . Although further study is needed to clarify the pathogenesis of altered duodenal mucosal morphology, our results suggest that the clinical significance of the abnormalities may be small.

Trends Microbiol, 1996 Jul, 4(7), 286 - 90
The molecular ecology of legionellae; Fields BS; Legionella pneumophila is the most highly characterized member of a genus of bacteria that survive as intracellular parasites of freshwater protozoa . These bacteria can also multiply intracellularly in human phagocytic cells and cause respiratory disease in humans . Comparison of the invasive strategies of L . pneumophila in mammalian and protozoan cells and study of the interactions between Legionella and protozoa should prove useful in development of strategies for the prevention of legionellosis.

Vet Microbiol, 1996 Jul, 51(1-2), 69 - 76
Detection of Mycoplasma mycoides subspecies mycoides by monoclonal antibody-based sandwich ELISA; Rodriguez F et al.; Monoclonal antibodies (MAbs) were produced from a mouse immunised with Mycoplasma mycoides subsp . mycoides small colony (MmmSC) antigen and their use to detect and differentiate strains within the Mycoplasma mycoides cluster investigated in an antigen capture ELISA format . The MAbs produced could not distinguish between MmmSC and M . mycoides subsp . mycoides large colony (MmmLC) strains . However, the sandwich ELISAs developed were able to specifically distinguish these two biotypes from the other four members of the M . mycoides cluster, and from all other mycoplasma or bacteria species examined . The most sensitive application of the test was a combination of enrichment and capture by overnight or 48-h incubations of samples inoculated into mycoplasma broth in antibody-coated microtiter wells.

Scand J Gastroenterol, 1996 Jul, 31(7), 671 - 7
Gastric emptying and first-pass metabolism of ethanol in elderly subjects with and without atrophic gastritis; Pedrosa MC et al.; BACKGROUND: Oral ethanol intake results in lower blood ethanol concentrations than intravenous administration of the same dose of ethanol . This first-pass metabolism is thought to be due to gastric metabolism of ethanol via alcohol dehydrogenase and also to hepatic first-pass metabolism . METHODS: Since a loss of gastric mucosa may decrease first-pass metabolism of ethanol, this metabolism was studied in 10 elderly subjects (6 women and 4 men) with atrophic gastritis and bacterial overgrowth and in 17 control subjects with normal gastric secretory function . Atrophic gastritis was verified by means of the serum pepsinogen I to pepsinogen II ratio and the hypochlorhydria occurring after pentagastrin stimulation . Bacterial overgrowth was assessed by bacteria . In addition, gastric emptying rates of ethanol solution with technetium-99m sulfur colloid were calculated from scintigraphic images . Furthermore, gastric biopsy specimens were taken from 12 female patients with atrophic gastritis and from 12 controls for determination of alcohol dehydrogenase activity . RESULTS: Neither gender (female versus male, 28 +/- 5% versus 42 +/- 5%), atrophic gastritis (normal versus atrophic gastritis, 35 +/- 4% versus 32 +/- 6%), nor tetracycline treatment in atrophic gastritis subjects (before versus after, 32 +/- 6% versus 41 +/- 5%) had a statistically significant effect on the first-pass metabolism of ethanol in the elderly . Gastric alcohol dehydrogenase activity was significantly lower in atrophic gastritis subjects than in controls (p < 0.01) . A significant correlation was found between the first-pass metabolism of ethanol in healthy controls and gastric half-emptying time (p = 0.032) . CONCLUSIONS: We conclude from these data that the rate of gastric emptying modulates first-pass metabolism of ethanol in elderly individuals.

Protein Sci, 1996 Jul, 5(7), 1342 - 54
Cytochrome c3 from Desulfovibrio gigas: crystal structure at 1.8 A resolution and evidence for a specific calcium-binding site; Matias PM et al.; Crystals of the tetraheme cytochrome c3 from sulfate-reducing bacteria Desulfovibrio gigas (Dg) (MW 13 kDa, 111 residues, four heme groups) were obtained and X-ray diffraction data collected to 1.8 A resolution . The structure was solved by the method of molecular replacement and the resulting model refined to a conventional R-factor of 14.9% . The three-dimensional structure shows many similarities to other known crystal structures of tetraheme c3 cytochromes, but it also shows some remarkable differences . In particular, the location of the aromatic residues around the heme groups, which may play a fundamental role in the electron transfer processes of the molecule, are well conserved in the cases of hemes I, III, and IV . However, heme II has an aromatic environment that is completely different to that found in other related cytochromes c3 . Another unusual feature is the presence of a Ca2+ ion coordinated by oxygen atoms supplied by the protein within a loop near the N-terminus . It is speculated that this loop may be stabilized by the presence of this Ca2+ ion, may contribute to heme-redox perturbation, and might even be involved in the specificity of recognition with its redox partner.

Biotechniques, 1996 Jul, 21(1), 82 - 6
Application of 5-bromo-2'deoxyuridine as a label for in situ hybridization in chromosome microdissection and painting, and 3' OH DNA end labeling for apoptosis; Muhlmann-Diaz MC et al.; We have utilized 5-bromo-2'deoxyuridine (BrdU) substituted DNA as a probe for a number of applications including, principally, for chromosome painting by fluorescence in situ hybridization (FISH) but also for DNA end-labeling to detect apoptotic cell death and for filter hybridization . Br-dUTP was used as a substitute for biotin or digoxigenin-dUTP in probe labeling techniques, such as random priming, nick translation, end-labeling or PCR . An especially useful application is that it may be incorporated into probe DNA while cells or plasmids in bacteria are growing in the presence of BrdU . This can be particularly advantageous when large quantities of probe are needed, since the cost per mole of digoxigenin-dUTP or biotin-dUTP is nearly 1000 times that of Br-dUTP . Also, if probe is prepared by growth in BrdU, the difference in cost to prepare equal quantities of labeled DNA is more than 10,000 times greater for biotin-dUTP.

Z Naturforsch {C}, 1996 Jul-Aug, 51(7-8), 493 - 9
Lactarane type sesquiterpenoids as inhibitors of leukotriene biosynthesis and other, new metabolites from submerged cultures of Lentinellus cochleatus (Pers . ex Fr.) Karst; Wunder A et al.; Three known sesquiterpenoids of the lactarane and secolactarane type, deoxylactarorufin A (1), blennin A (2) and blennin C (3), have been obtained from cultures of Lentinellus cochleatus (Basidiomycetes) together with the new metabolites (Z)-2-chloro-3-(4-methoxyphenyl)-2-propen-1-ol (4) and lentinellone (5), a protoilludane derivative . The structures were determined by spectroscopic investigations . 1, 2 and 3 are potent inhibitors of leukotriene biosynthesis in rat basophilic leukemia (RBL-1) cells and human peripheral blood leukocytes (PBL).

Clin Microbiol Rev, 1996 Jul, 9(3), 382 - 404
Detection of infection or infectious agents by use of cytologic and histologic stains; Woods GL et al.; A wide variety of stains are useful for detection of different organisms or, for viruses, the cytopathologic changes they induce, in smears prepared directly from clinical specimens and in tissue sections . Other types of stains, such as hematoxylin and eosin, are used routinely to stain tissue sections and are most valuable for assessing the immunologic response of the host to the invading pathogen . In many cases, the pattern of inflammation provides important clues to diagnosis and helps to guide the selection of additional "special" stains used predominantly for diagnosis of infectious diseases . A stain may be nonspecific, allowing detection of a spectrum of organisms, as do the Papanicolaou stain and silver impregnation methods, or detection of only a limited group of organisms, as do the different acid-fast techniques . Some nonspecific stains, such as the Gram stain, are differential and provide valuable preliminary information concerning identification . Immunohistochemical stains, on the other hand, are specific for a particular organism, although in some cases cross-reactions with other organisms occur . Despite the wealth of information that can be gleaned from a stained smear or section of tissue, however, the specific etiology of an infection often cannot be determined on the basis of only the morphology of the organisms seen; culture data are essential and must be considered in the final diagnosis.

Chem Biol, 1996 Jul, 3(7), 519 - 24
Accommodating structurally diverse peptides in proteins; Wilkinson AJ; Many peptide-binding proteins must bind numerous ligands that differ in size, sequence and sometimes orientation . A variety of strategies for coping with structurally diverse peptide ligands have been revealed by biochemical and structural studies of proteins with roles in immunity, transport and signal transduction.

Clin Diagn Lab Immunol, 1996 Jul, 3(4), 423 - 8
High-dose catecholamine treatment decreases polymorphonuclear leukocyte phagocytic capacity and reactive oxygen production; Wenisch C et al.; Flow cytometry was used to study phagocytic function (uptake of fluorescein isothiocyanate-labeled bacteria) and release of reactive oxygen products (dihydrorhodamine 123 converted to rhodamine 123) following phagocytosis by neutrophil granulocytes of heparinized whole blood treated with adrenaline, noradrenaline, dopamine, dobutamine, or orciprenaline . Reduced neutrophil phagocytosis and reactive oxygen production were seen at 12 micrograms of adrenaline per liter (72% each compared with control values); at 120 micrograms of noradrenaline (72% each), dobutamine (83 and 80%, respectively), and orciprenaline (81 and 80%, respectively) per liter; and at 100 micrograms of dopamine per liter (66 and 70%) (P < 0.05 for all) . At these dosages, neutrophil chemotaxis was reduced to < 50% of control values for all catecholamines . Treatment with catecholamines at lower dosages had no significant effect on phagocytosis or generation of reactive oxygen products or chemotaxis . The phagocytic capacity of granulocytes was related to the generation of reactive oxygen products (r = 0.789; P < 0.05) . The results demonstrate that catecholamines have a suppressive effect on the response of phagocytic cells to bacterial pathogens at high therapeutic levels in blood.

Plant Mol Biol, 1996 Jul, 31(4), 721 - 30
The cyanobacterium Synechococcus sp . PCC 7942 possesses a close homologue to the chloroplast ClpC protein of higher plants; Clarke AK et al.; The Clp family consists of large, ubiquitous proteins that function as molecular chaperones and/or regulators of ATP-dependent proteolysis . A single copy gene coding for one of these proteins, ClpC, was cloned from the unicellular cyanobacterium Synechococcus sp . PCC 7942 . The predicted polypeptide is most similar (ca . 88%) to the chloroplast-localized ClpC protein from higher plants . Using degenerate PCR primers specific for the two distinct ATP-binding domains characteristic of all ClpA-C proteins, partial sequences homologous to clpC from Synechococcus were also identified in five other cyanobacterial strains . The Synechococcus clpC gene is transcribed under standard growth conditions as a monocistronic message of around 2.7 kb . The level of this message, however, decreases slightly after a shift from 37 to 47.5 degrees C for 2 h, similar to expression previously observed for clpC mRNA from heat-shocked higher plants . At the protein level, the amount of ClpC remains relatively unchanged during the high temperature shift, while that of the known heat shock protein GroEL rises considerably . In contrast, the constitutive level of ClpC in Synechococcus increases considerably under conditions of rapid growth, both with increasing light intensities or CO2 concentrations . This, and the fact that attempts to inactivate clpC expression fail to produce a viable phenotype, suggest that ClpC activity is essential for growth in this obligate photoautotrophic cyanobacterium.

Biomaterials, 1996 Jul, 17(13), 1273 - 7
Microwave plasmas for low-temperature dry sterilization; Chau TT et al.; The use of microwave plasmas for dry sterilization has been investigated . The dry-sterilization process is a process similar to plasma etching . Bacteria and viruses can be killed by chemical reactions which disintegrate their bodies and remove them from the surface to be sterilized . The removal of bacteria or viruses from material surfaces is caused by the reaction of activated oxygen species in the plasma with hydrocarbon bonds of the cell wall of the bacteria or the capsid of the viruses . Preliminary experiments indicate that the low-temperature dry sterilization method is easy to use, requires much less time than other methods for sterilization, and is also non-toxic . It is feasible for use in the field of sterilization in dental and medical clinics.

FASEB J, 1996 Jul, 10(9), 979 - 85
Vitamin A and retinoids in antiviral responses; Ross AC et al.; Vitamin A deficiency results in multiple derangements that impair the response to infection . This review focuses on experimental models of specific virus infections and on cytokines and cells with cytolytic activity important to antiviral defenses . Altered specific antibody responses and greater epithelial damage in vitamin A-deficient hosts are consistent findings . The cytolytic activity of natural killer cells and various cytokine responses are altered . The inflammatory response to infection may also result in derangements in the transport and metabolism of retinol . We speculate that interaction of several factors may combine to explain the greater severity of infection seen in vitamin A-deficient animals and children . In addition to a preexisting lack of tissue vitamin A, these factors may include reduced mobilization and increased excretion of retinol during the acute phase response to infection, poor innate and specific immune response to virus, and delayed repair of damaged epithelia . Foci of vitamin A-deficient epithelia may be sites of penetration of bacteria and other agents, leading to secondary infections and contributing to an increased severity of infections and poor outcome in vitamin A-deficient animals and humans.

Kansenshogaku Zasshi, 1996 Jul, 70(7), 673 - 80
{Subserogrouping of 49 Legionella pneumophila serogroup 1 strains with monoclonal antibodies by slide latex agglutination method and its usefulness for epidemiologic study}; Gondaira F et al.; Legionella pneumophila serogroup (SG) 1 has been known as a most frequent and important causative bacteria of Legionella pneumonia . It was reported that antigenicity of the serogroup was composed by numerous antigenic factors . To study antigenic factor formula of Japanese isolates from both clinical and environmental sources together with Philadelphia 1 strain, we examined slide latex agglutination system with monoclonal antibodies for subsergorouping . Philadelphia 1 and GIFU10102 strain (environmental isolate) were used as immune strains based on a cross absorption test result by rabbit antisera . Five anti L . pneumophia SG1 monoclonal antibody-producing clones were established . All monoclonal antibodies were SG1 specific but showed different reactivities with strain to strain . We prepared monoclonal antibody sensitized latex and used for subserogrouping of isolated strains . Twenty-two clinical isolates from Legionella pneumonia patients and 26 environmental isolates were examined . Each strain was reacted with at least one of the 5 sensitized latex and its antigenic formula was designed by sensitized latex reaction pattern . As a total of 6 patterns demonstrated in clinical isolates and 7 patterns in environmental isolates, 48 L . pneumophila SG1 isolates were divided into 11 subserogroup patterns . The antigenic formula of Philadelphia 1 was same as one of clinical isolate patterns . It was considered that this subserogrouping system would be a useful tool for epidemiologic study.

Arch Dis Child Fetal Neonatal Ed, 1996 Jul, 75(1), F49 - 52
CRIB (clinical risk index for babies) in relation to nosocomial bacteraemia in very low birthweight or preterm infants; Fowlie PW et al.; Positive blood cultures in very low birthweight or preterm infants usually reflect bacteraemia, septicaemia, or failure of asepsis during sampling and lead to increased costs and length of stay . Rates of nosocomial, or hospital acquired, bacteraemia may therefore be important indicators of neonatal unit performance, if comparisons are adjusted for differences in initial risk . In a preliminary study the risk of nosocomial bacteraemia was related to initial clinical risk and illness severity measured by the clinical risk index for babies (CRIB) . Nosocomial bacteraemia was defined as clinically suspected infection with culture of bacteria in blood more than 48 hours after birth . One or more episodes of nosocomial bacteraemia were identified retrospectively in 36 of 143 (25%) infants in a regional neonatal unit between 1992 and 1994 . Biologically plausible models were developed using regression analysis techniques . After correcting for period at risk, nosocomial bacteraemia was independently associated with gestation at birth and CRIB . Death was independently associated with CRIB, but not with nosocomial bacteraemia . CRIB may contribute, with other explanatory variables, to more comprehensive predictive models of death and nosocomial infection . These may facilitate future risk adjusted comparative studies between groups of neonatal units.

Int J Syst Bacteriol, 1996 Jul, 46(3), 811 - 3
Sanguibacter inulinus sp . nov; Pascual C et al.; Six strains of coryneform bacteria were isolated from blood samples obtained from healthy cows . Phenotypic and molecular genetic studies showed that these isolates represent a new species of the genus Sanguibacter, for which the name Sanguibacter inulinus is proposed . The type strain of S . inulinus is strain ST-50 (= NCFB 3024).

Appl Environ Microbiol, 1996 Jul, 62(7), 2201 - 11
Full-scale studies of factors related to coliform regrowth in drinking water; LeChevallier MW et al.; An 18-month survey of 31 water systems in North America was conducted to determine the factors that contribute to the occurrence of coliform bacteria in drinking water . The survey included analysis of assimilable organic carbon (AOC), coliforms, disinfectant residuals, and operational parameters . Coliform bacteria were detected in 27.8% of the 2-week sampling periods and were associated with the following factors: filtration, temperature, disinfectant type and disinfectant level, AOC level, corrosion control, and operational characteristics . Four systems in the study that used unfiltered surface water accounted for 26.6% of the total number of bacterial samples collected but 64.3% (1,013 of 1,576) of the positive coliform samples . The occurrence of coliform bacteria was significantly higher when water temperatures were > 15 degrees C . For filtered systems that used free chlorine, 0.97% of 33,196 samples contained coliform bacteria, while 0.51% of 35,159 samples from chloraminated systems contained coliform bacteria . The average density of coliform bacteria was 35 times higher in free-chlorinated systems than in chloraminated water (0.60 CFU/100 ml for free-chlorinated water compared with 0.017 CFU/100 ml for chloraminated water) . Systems that maintained dead-end free chlorine levels of < 0.2 mg/liter or monochloramine levels of < 0.5 mg/liter had substantially more coliform occurrences than systems that maintained higher disinfectant residuals . Free-chlorinated systems with AOC levels greater than 100 micrograms/liter had 82% more coliform-positive samples and 19 times higher coliform levels than free-chlorinated systems with average AOC levels less than 99 micrograms/liter . Systems that maintained a phosphate-based corrosion inhibitor and limited the amount of unlined cast iron pipe had fewer coliform bacteria . Several operational characteristics of the treatment process or the distribution system were also associated with increased rates of coliform occurrence . The study concludes that the occurrence of coliform bacteria within a distribution system is dependent upon a complex interaction of chemical, physical, operational, and engineering parameters . No one factor could account for all of the coliform occurrences, and one must consider all of the parameters described above in devising a solution to the regrowth problem.

Plant Cell, 1996 Jul, 8(7), 1121 - 35
Identification of the major starch synthase in the soluble fraction of potato tubers; Marshall J et al.; The major isoform of starch synthase from the soluble fraction of developing potato tubers has been purified and used to prepare an antibody and isolate a cDNA . The protein is 140 kD, and it is distinctly different in predicted primary amino acid sequence from other isoforms of the enzyme thus far described . Immunoinhibition and immunoblotting experiments and analysis of tubers in which activity of the isoform was reduced through expression of antisense mRNA revealed that the isoform accounts for approximately 80% of the activity in the soluble fraction of the tuber and that it is also bound to starch granules . Severe reductions in activity had no discernible effect on starch content or amylose-to-amylopectin ratio of starch in tubers . However, they caused a profound change in the morphology of starch granules, indicative of important underlying changes in the structure of starch polymers within the granule.

FEMS Microbiol Lett, 1996 Jul 1, 140(2-3), 171 - 8
Attempts to characterize the mechanisms involved in the growth inhibition of Mycobacterium microti in interferon-gamma or tumor necrosis factor-alpha activated J774A.1 cells; Gupta R et al.; The growth of Mycobacterium microti was inhibited within J774A.1 macrophage cells activated with either interferon-gamma or tumor necrosis factor-alpha . Activation with interferon-gamma or tumor necrosis factor-alpha alone did not stimulate the production of nitrite in J774A.1 cells . Interferon-gamma but not tumor necrosis factor-alpha increased the production of hydrogen peroxide in a concentration dependent manner but scavengers of reactive oxygen species did not influence the growth inhibiting effect of interferon-gamma within J774A.1 cells . Both interferon-gamma and tumor necrosis factor-alpha enhanced the fusion of M . microti containing phagosomes with lysosomes and the ultimate degradation of bacteria . Our results showed that growth inhibition of M . microti within interferon-gamma or tumor necrosis factor-alpha stimulated J774A.1 cells was independent of reactive oxygen intermediate and reactive nitrogen intermediate production.

J Bacteriol, 1996 Jul, 178(14), 4143 - 9
Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense; de Zamaroczy M et al.; The coexistence of two different PII, proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding PII) adjacent to glnA . Strains were grown under conditions of nitrogen limitation or nitrogen excess . The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by {32P}phosphate or {3H}uracil labeling or by cross-reaction with an anti-A . brasilense PII-antiserum . After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains . The second PII protein, named Pz, was uridylylated under conditions of nitrogen limitation . The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of PII . Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation . No glnZ mutant is yet available, but it is clear that PII and Pz are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters . The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A . brasilense.

Physiol Rev, 1996 Jul, 76(3), 839 - 85
Oxygen sensing and molecular adaptation to hypoxia; Bunn HF et al.; This review focuses on the molecular stratagems utilized by bacteria, yeast, and mammals in their adaptation to hypoxia . Among this broad range of organisms, changes in oxygen tension appear to be sensed by heme proteins, with subsequent transfer of electrons along a signal transduction pathway which may depend on reactive oxygen species . These heme-based sensors are generally two-domain proteins . Some are hemokinases, while others are flavohemoproteins {flavohemoglobins and NAD(P)H oxidases} . Hypoxia-dependent kinase activation of transcription factors in nitrogen-fixing bacteria bears a striking analogy to the phosphorylation of hypoxia inducible factor-1 (HIF-1) in mammalian cells . Moreover, redox chemistry appears to play a critical role both in the trans-activation of oxygen-responsive genes in unicellular organisms as well as in the activation of HIF-1 . In yeast and bacteria, regulatory operons coordinate expression of genes responsible for adaptive responses to hypoxia and hyperoxia . Similarly, in mammals, combinatorial interactions of HIF-1 with other identified transcription factors are required for the hypoxic induction of physiologically important genes.

J Forensic Sci, 1996 Jul, 41(4), 612 - 6
Postmortem stability of cocaine and cocaethylene in blood and tissues of humans and rabbits; Moriya F et al.; A study was conducted to examine the postmortem stability of cocaine and cocaethylene in rabbit blood and tissues, and to determine whether cocaethylene is produced in decomposed human specimens containing cocaine and endogenous ethanol . Heart blood, liver, brain and femoral muscle taken from rabbits 20 min after oral administration of 20 mg/kg cocaine together with 2 g/kg ethanol were kept at 20-25 degrees C for 5 days . Cocaine and cocaethylene concentrations were in the order brain > liver > muscle > blood, and showed very large intersubject variations at the time of death . Cocaine was degraded rapidly in the blood and liver . However, 12.0 +/- 8.5% and 26.2% +/- 19.4% of the original cocaine was still detectable in the brain and muscle, respectively . Cocaethylene was degraded more slowly than cocaine in all of the specimens . The pH of the blood remained around 7.4 during a 5-day period; all the other specimens showed pH values of 6.2-6.7 on and after the first day postmortem . When 10,000 ng/g cocaine was incubated with decomposed human blood, liver, brain and muscle homogenates containing 0.29-0.60 mg/g endogenous ethanol at 20-25 degrees C and 37 degrees C, no change in cocaine concentration was observed during the study period of 24 h, and no cocaethylene was detected . The pH values of the homogenates were within the range 4.2 to 5.2 at the beginning of the experiment . It was found that: 1) cocaethylene was more stable in postmortem specimens than cocaine; 2) muscle as well as brain was specimen of choice for detecting cocaine and cocaethylene postmortem; 3) cocaine was resistant to decomposition under acidic conditions; and 4) putrefactive bacteria had no ability to produce cocaethylene even in the presence of cocaine and endogenous ethanol.

Mol Biol Evol, 1996 Jul, 13(6), 719 - 34
Molecular biology and evolution of resistance of toxicants; Taylor M et al.; To the prevailing biochemical/physiological classification of mechanisms of organismal resistance to toxicants, an additional molecular dimension is proposed . Predictions are developed regarding the relative prevalence of different classes of mutations and are found to compare favorably with reports from the literature . In particular, point mutations in target loci were the dominant form of resistance for both lab and field selection . Amplifications of target loci were less common than structural mutations, and more common for lab-selected than for field-selected strains . Amplification was the most common mechanism of up-regulation of metabolizing enzymes . In comparison, only one mutation involving cis-regulation and several involving trans-acting regulation were found . Mutations involving gene disruption and down-regulation were uncommon, but were found in appropriate cases, i.e., when toxicants stimulated rather than inhibited target function and when metabolizing enzymes converted toxicants into more toxic metabolites . Additional phenomena of likely but uncertain importance are genetic "succession," recombinational limitation, and negative cross-resistance . More work on these phenomena and on quantification of fitness costs of resistance is recommended.

J Invest Dermatol, 1996 Jul, 107(1), 108 - 12
Dermatophytes contain a novel lipid-like leukocyte activator; Kahlke B et al.; In the early phase of dermatophytosis, neutrophils are regularly detected microscopically in the infected skin . Although neutrophil recruitment may at least in part occur indirectly by complement activation, we asked whether dermatophytes might release chemoattractants for neutrophils . We cultivated various strains of different dermatophytes and tested fungal extracts for the presence of neutrophil chemotactic activity . As a result, we detected neutrophil chemotactic activity only in diethylether extracts, but not in aqueous extracts . We purified this lipid-like leukocyte activator (LILA) to apparent homogeneity by reversed-phase high performance liquid chromatography and found that purified LILA does not show ultraviolet absorption at wavelengths > 210 nm . Biologic studies revealed that LILA is as effective as formyl-methionyl-leucyl-phenylalanine in eliciting neutrophil chemotaxis, degranulation, and activation of the respiratory burst . Desensitization experiments in chemotaxis and degranulation with leukotriene B4, platelet-activating factor, or 5-oxo-eicosanoids revealed that LILA does not cross-desensitize with any of these other lipid-like attractants and thus possibly acts via a distinct as yet postulated neutrophil receptor . It is hypothesized that LILA, similarly to formylated methionyl peptides in bacteria, represents a dermatophyte- and possibly fungus-specific lipid compound that allows the host phagocytes to specifically recognize fungal infection . This system would be similar to the recognition of bacteria by phagocytes via N-formylated methionyl peptides, which represent a characteristic and unique system to identify bacteria.

Kyobu Geka, 1996 Jul, 49(8 Suppl), 646 - 51
{Surgical treatment of infective endocarditis}; Matsubayashi K et al.; From 1981 to 1996, 48 consecutive patients, aged range 1 to 72 years, underwent surgical treatment for infective endocarditis . The infection was in the aortic valve in 10 patients, the mitral valve in 17, the aortic and mitral valves in 7, mixed aortic, mitral and tricuspid valves in one, the tricuspid valve in 9, the pulmonary valve in 3, and the other in 2, thirty-seven patients had native valve endocarditis (NVE) of which 22 cases were in the active stage . Seven cases had active prosthetic valve endocarditis (PVE) and 4 had VSD patch infection . The overall hospital mortality rate was 14.6% (7/48) . The hospital mortality rate of NVE was 2.7% (1/37) and that of active NVE was 4.5% (1/22) . That of PVE was 71.4% (5/7) and one of 4 cases with VSD patch infection was lost, so the mortality rate of the prosthetic material infection was 54.5% (6/11) . Only 1 patient required reoperation for persistent infection . There were 2 late deaths caused by noncardiac disease . Thirty-nine of the total IE patients are now survived . These data demonstrate excellent results in patient with NVE undergoing the surgical treatment at the early phase, and support the premise that patients with active PVE should have also early surgical intervention.

Infect Immun, 1996 Jul, 64(7), 2657 - 65
The major fimbrial subunit of Bordetella pertussis binds to sulfated sugars; Geuijen CA et al.; Bordetella pertussis fimbriae are composed of major and minor subunits, and recently it was shown that the minor fimbrial subunit binds to Vla-5, a receptor located on monocytes (W . Hazenbos, C . Geuijen, B . van den Berg, F . Mooi, and R . van Furth, J . Infect . Dis . 171:924-929, 1995) . Here we present evidence that the major subunits bind to sulfated sugars, which are ubiquitous in the respiratory tract . Binding was observed to chondroitin sulfate, heparan sulfate, and dextran sulfate but not to dextran . Removal of the minor subunit from fimbriae did not significantly affect binding to sulfated sugars, indicating that the major subunit alone is sufficient for this binding . Fimbriae were also able to bind HEp-2 cells, which are known to display glycoconjugates on their surface . This binding was not dependent on the presence of the minor subunit . However, binding was dependent on the sulfation state of the glycoconjugates, since inhibition of the sulfation resulted in a significant reduction of fimbria binding . The specificity of fimbria binding was further characterized by using heparan sulfate-derived disaccharides in inhibition assays . Two disaccharides were highly effective inhibitors, and it was observed that both the degree of sulfation and the arrangement of the sulfate groups on the disaccharides were important for binding to fimbriae . B . pertussis bacteria also bound to sulfated sugars and HEp-2 cells, and analysis of B . pertussis mutants indicated that both filamentous hemagglutinin and fimbriae were required for this binding . A host protein present in the extracellular matrix, fibronectin, has binding activities similar to those of B . pertussis fimbriae, binding to both Vla-5 and sulfated sugars . Two regions in the major fimbrial subunit were identified which showed similarity with fibronectin peptides which bind to sulfated sugars . Thus, B . pertussis fimbriae exemplify molecular mimicry and may co-opt host processes by mimicking natural ligand-receptor interactions.

Infect Immun, 1996 Jul, 64(7), 2643 - 8
Acidic pH changes receptor binding specificity of Helicobacter pylori: a binary adhesion model in which surface heat shock (stress) proteins mediate sulfatide recognition in gastric colonization; Huesca M et al.; The gastric pathogen helicobacter pylori is one of a number of bacteria which bind specifically to gangliotetraosylceramide, gangliotriaosylceramide, and phosphatidylethanolamine in vitro at neutral pH . Since this organism encounters an acid pH during initial infection of the stomach, we have monitored the effect of pH on receptor binding specificity and found induction of specific binding to sulfoglycolipids (sulfatide) following brief treatment at low pH . We have previously shown that heat shock proteins (hsps) bind to sulfatide, and the suspicion that this was a stress-induced response is supported by the fact that a similar change in H . pylori binding specificity was observed if the organisms were briefly exposed to heat shock treatment . Following the stress stimulus, the change in glycolipid binding specificity was prevented by the inclusion of inhibitors of protein synthesis or by incubation with anti-hsp antibodies . Expression of hsps in the surface extract and surface reactivity with anti-hsp antibodies correlated with the change in glycolipid binding specificity . Despite the presence of high levels of H . pylori cell surface urease activity which may neutralize the microenvironmental pH, the acid-induced change in binding specificity was enhanced in the presence of urea . These studies suggest that cell surface hsps mediate sulfatide recognition by this organism under stress conditions . A binary receptor model is proposed for gastric colonization by H . pylori.

Infect Immun, 1996 Jul, 64(7), 2585 - 94
Identification of Legionella pneumophila mutants that have aberrant intracellular fates; Swanson MS et al.; After uptake by macrophages, Legionella pneumiophila evades phagosome-lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum . A collection of bacterial mutants defective for growth in macrophages were isolated, and the intracellular fate of each mutant strain was analyzed by fluorescence microscopy . To measure intracellular replication, bacteria inside macrophages were stained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) . Evasion of the endocytic pathway was quantified by immunofluorescence localization of lp120 {correction of IgpI20} (LAMP-1), a membrane protein of late endosomes and lysosomes, or by measuring colocalization of bacteria with a fluorescent tracer, Texas red-ovalbumin, preloaded into lysosomes . Replication vacuoles were quantified by immunofluorescence localization of BiP, an endoplasmic reticulum protein . By these approaches, four phenotypic groups of mutants were classified . One class formed replication vacuoles less efficiently than the wild type did; another formed replication vacuoles, but replication was abortive; in another class, most phagosomes containing bacteria acquired markers of the endocytic pathway but a minority formed replication vacuoles and the bacteria replicated; finally, a fourth class, the one most defective for intracellular growth, occupied vacuoles that acquired markers of the endocytic pathway.

Infect Immun, 1996 Jul, 64(7), 2449 - 56
Coinoculation with Hartmannella vermiformis enhances replicative Legionella pneumophila lung infection in a murine model of Legionnaires' disease; Brieland J et al.; The effect of inhaled amoebae on the pathogenesis of Legionnaires' disease was investigated in vivo . A/J mice, which are susceptible to replicative Legionella pneumophila infections, were inoculated intratracheally with L . pneumophila (10(6) bacteria per mouse) or were coinoculated with L . pneumophila (10(6) bacteria per mouse) and Hartmannella vermiformis (10(6) amoebae per mouse) . The effect of coinoculation with H . vermiformis on bacterial clearance, histopathology, cellular recruitment into the lung, and intrapulmonary levels of cytokines including gamma interferon and tumor necrosis factor alpha was subsequently assessed . Coinoculation with H . vermiformis significantly enhanced intrapulmonary growth of L . pneumophila in A/J mice . Histopathologic and flow cytometric analysis of lung tissue demonstrated that while A/J mice inoculated with L . pneumophila alone develop multifocal pneumonitis which resolves with minimal mortality, mice coinoculated with H . vermiformis develop diffuse pneumonitis which is associated with diminished intrapulmonary recruitment of lymphocytes and mononuclear phagocytic cells and significant mortality . Furthermore, coinoculation of mice with H . vermiformis resulted in a fourfold enhancement in intrapulmonary levels of gamma interferon and tumor necrosis factor alpha compared with mice infected with L . pneumophila alone . The effect of H . vermiformis on intrapulmonary growth of L . pneumophila in a resistant host (i.e., BALB/c mice) was subsequently evaluated . While BALB/c mice do not develop replicative L . pneumophila infections following inoculation with L . pneumophila alone, there was an eightfold increase in intrapulmonary L . pneumophila in BALB/c mice coinoculated with H . vermiformis . These studies, demonstrating that intrapulmonary amoebae potentiate replicative L . pneumophila lung infection in both a susceptible and a resistant host, have significant implications with regard to the potential role of protozoa in the pathogenesis of pulmonary diseases due to inhaled pathogens and in the design of strategies to prevent and/or control legionellosis.

Biochem J, 1996 Jul 1, 317 ( Pt 1), 147 - 55
Purification and characterization of assimilatory nitrite reductase from Candida utilis; Sengupta S et al.; Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1) . They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively . The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity . Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa . The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration . The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase . The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C . utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis . The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status . The enzyme is stable over a range of temperatures . Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme . Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides . Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.

Dig Dis Sci, 1996 Jul, 41(7), 1337 - 45
Endoscopic-pathologic correlates of Candida esophagitis in acquired immunodeficiency syndrome; Wilcox CM et al.; Although Candida esophagitis is one of the most common opportunistic infections in patients with acquired immunodeficiency syndrome (AIDS), there has been no systematic study of the endoscopic and pathologic manifestations of this disease . During a 53-month period, 141 patients with AIDS and Candida esophagitis were studied . All patients had the severity of esophagitis graded prospectively and esophageal mucosal biopsies performed . Tissue biopsies were evaluated for histologic evidence of ulceration, extent of candidiasis, and presence of viral cytopathic effect . Follow-up was obtained . There appeared to be a uniform endoscopic appearance; with increasing severity, the scattered mucosal plaques coalesced, resulting in circumferential disease and luminal impingement . The pathologic pattern of Candida esophagitis was homogenous . Plaque material was composed primarily of desquamated superficial hyperplastic hyperkeratotic squamous epithelium and inflammatory cells, with infiltration by fungal elements and bacteria consistent with superinfection . Although endoscopic and histopathologic ulcer was commonly seen in these patients (32%), only four patients had ulcer believed secondary to Candida esophagitis alone . In conclusion, in patients with AIDS, Candida esophagitis is a superficial mucosal infection resulting in characteristic endoscopic and histopathologic patterns.

J Bacteriol, 1996 Jul, 178(13), 3939 - 48
Caulobacter and Asticcacaulis stalk bands as indicators of stalk age; Poindexter JS et al.; The prosthecae (stalks) of dimorphic caulobacters of the genera Caulobacter and Asticcacaulis are distinguished among such appendages by the presence of disk-like components known as stalk bands . Whether bands are added to a cell's stalk(s) as a regular event coordinated with the cell's reproductive cycle has not been settled by previous studies . Analysis of the frequency of stalks with i, i + 1, i + 2, etc . bands 'among more than 7,000 stalks of Caulobacter crescentus revealed that in finite (batch) cultures (in which all offspring accumulate), the proportion of stalks with i + 1 hands was regularly 50% of the proportion of stalks with i bands . This implied that the number of bands correlated with the number of reproductive cycles completed by a stalked cell . In chemostat-maintained perpetual cultures, the proportion was greater than 50% because stalked cells, with their shorter reproductive cycle times, contributed a larger proportion of offspring to the steady-state population than did their swarmer siblings . In Asticcacaulis biprosthecum cells, which bear twin prosthecae, the twins on a typical cell possessed the same number of bands . For both genera, stalk bands provide a unique morphological feature that could be employed in an assessment of age distribution and reproductive dynamics within natural populations of these caulobacters.

Am J Respir Crit Care Med, 1996 Jul, 154(1), 116 - 23
Mortality of nosocomial pneumonia in ventilated patients: influence of diagnostic tools; Timsit JF et al.; The overmortality induced by nosocomial infections, especially pneumonia in ventilated patients (VNP), is still a matter of controversy because it is difficult to know precisely the respective effects of VNP per se and both the underlying illness and the severity of the disease that indicates ICU stay . During a 3-yr period, for each patient mechanically ventilated for more than 48 h we recorded underlying illness, reason for mechanical ventilation, clinical and therapeutic data collected during the first 48 h of ventilation, and death in the ICU . Patients with suspicion of VNP (S-VNP) according to clinical, radiologic, and biologic criteria underwent bronchoscopy with protected specimen brush (PSB) and bronchoalveolar lavage culture (BAL-C) . VNP was confirmed (C-VNP) if PSB > or = 10(3) cfu/ml and/or BAL-C > or = 10(4) cfu/ml . Prognostic multivariate analysis was performed introducing S-VNP and C-VNP as time-dependent covariates . Of the 387 studied patients, 112 S-VNP and 56 C-VNP were observed with overall mortality of 43% (168 patients) . MacCabe, APACHE II score, shock, use of sedatives and absence of enteral nutrition were additively associated with an increased mortality as well as C-VNP (relative risk {RR}: 1.8, p = 0.007) . Nevertheless, when S-VNP and C-VNP were simultaneously introduced in the Cox model, only S-VNP remained associated with increased mortality . In patients suspected of VNP, confirmation of VNP using PSB and/or BAL-C adds no prognostic information . Whether this could be explained by the lack of sensitivity of protected distal samples or the severity of underlying conditions of S-VNP patients is still an open issue . A multivariate analysis based on follow-up data during the ICU course of ventilated patients will be initiated in the near future.

Lett Appl Microbiol, 1996 Jul, 23(1), 9 - 12
Reduction of Brochothrix thermosphacta on beef surfaces following immobilization of nisin in calcium alginate gels; Cutter CN et al.; Lean and adipose beef carcass tissues inoculated with Brochothrix thermosphacta (BT) (approx . 4.50 log10 cfu cm-2) were left untreated (U) or treated with 100 micrograms ml-1 nisin (N), calcium alginate (A) or 100 micrograms ml-1 nisin immobilized in a calcium alginate gel (AN) . Tissue samples were refrigerated after treatments and bacterial populations and nisin activity were determined at 0, 1, 2 and 7 d . U, A and N treatments of lean and adipose tissues did not suppress bacterial growth ( > 6 log10 cfu cm-2 by day 7) while treatments of lean and adipose tissues with AN suppressed bacteria ( > 2.42 log10 cfu cm-2 by day 7) . Bacteriocin titres from both tissues were higher in AN vs N samples after the 7 d incubation . This study demonstrates that immobilization of nisin in a gel may be a more effective delivery system of a bacteriocin to the carcass surface than direct application.

Lett Appl Microbiol, 1996 Jul, 23(1), 64 - 6
A selective medium for the isolation of Arcobacter from meats; de Boer E et al.; A method, including enrichment in Arcobacter Selective Broth (ASB) and isolation on semisolid Arcobacter Selective Medium (ASM) under aerobic conditions at 24 degrees C, is described for the isolation of Arcobacter from retail meat products . Selective agents used in ASB and ASM were cefoperazone, trimethoprim, piperacillin and cycloheximide . Arcobacters were isolated from 53 (24.1%) of 220 poultry meat products and also, at lower incidence from samples of beef and pork . The isolates were identified as A . butzleri or A . butzleri-like and belonged to a wide variety of serotypes and biotypes.

J Virol, 1996 Jul, 70(7), 4724 - 8
Prion protein PrPc interacts with molecular chaperones of the Hsp60 family; Edenhofer F et al.; Prions mediate the pathogenesis of certain neurodegenerative diseases, including bovine spongiform encephalopathy in cattle and Creutzfeldt-Jakob disease in humans . The prion particle consists mainly, if not entirely, of PrPSc, a posttranslationally modified isoform of the cellular host-encoded prion protein (PrPc) . It has been suggested that additional cellular factors might be involved in the physiological function of PrPc and in the propagation of PrPSc . Here we employ a Saccharomyces cerevisiae two-hybrid screen to search for proteins which interact specifically with the Syrian golden hamster prion protein . Screening of a HeLa cDNA library identified heat shock protein 60 (Hsp60), a cellular chaperone as a major interactor for PrPc . The specificity of the interaction was confirmed in vitro for the recombinant proteins PrPc23-231 and rPrP27-30 fused to glutathione S-transferase with recombinant human Hsp60 as well as the bacterial GroEL . The interaction site for recombinant Hsp60 and GroEL proteins was mapped between amino acids 180 and 210 of the prion protein by screening with a set of recombinant PrPc fragments . The binding of Hsp60 and GroEL occurs within a region which contains parts of the putative alpha-helical domains H3 and H4 of the prion protein.

J Acquir Immune Defic Syndr Hum Retrovirol, 1996 Jul, 12(3), 233 - 48
T-cell homeostasis, competition, and drift: AIDS as HIV-accelerated senescence of the immune repertoire; Mittler JE et al.; The observation that the density of CD8+ T-lymphocytes increases as the density of CD4+ T-cells declines in adult HIV-1/AIDS patients, together with evidence that the total density of T-cells is regulated (homeostasis) has led to the suggestion that competition between lineages, and classes of T-cells contributes to the pathology of HIV . We use a mathematical model of the interactions between populations of T-cells, HIV, and other parasites to explore the effects of T-cell homeostasis and competition on the progression to AIDS . We demonstrate that as a consequence of parasite-mediated T-cell replication, of competition within and between different T-cell clones, and random processes (T-cell drift), some CD4+ lineages will be represented by relatively few cells, dearths, and some lineages may be lost, leaving holes in the immune repertoire . By killing CD4+ T-lymphocytes, HIV accelerates the rate at which these dearths and holes accumulate and leads to an early breakdown of the immune control of HIV and other parasites, AIDS . When this model allows for intense, but not complete, competition between the CD4+ and CD8+ T-cell populations, it can account for most of the features of an HIV-1 infection in adults, including the gradual decline in CD4+ T-cell densities and concomitant increase in HIV density, as well as the variability in time from infection to AIDS and the decline in the time from infection to AIDS in older patients.

J Med Microbiol, 1996 Jul, 45(1), 6 - 9
Heterogeneity of human intestinal spirochaetes demonstrated by one-dimensional polyacrylamide gel electrophoresis of proteins visualised by (35)S-methionine labelling and Coomassie blue staining; Barrett SP et al.; The relatedness of strains of a human intestinal spirochaete was investigated by comparison of electrophoretic protein profiles produced by Coomassie Blue staining of proteins separated by polyacrylamide gel electrophoresis (PAGE) of lysed organisms and by examination of autoradiographs following PAGE of lysed (35)S-methionine-labelled organisms . A wide diversity of strains was revealed by both techniques but clustering of strains was different by the two methods . These findings support the view that the human intestinal spirochaetes comprise a group of bacteria of considerable heterogeneity.

J Biol Chem, 1996 Jun 28, 271(26), 15695 - 702
Identification of the spectrin subunit and domains required for formation of spectrin/adducin/actin complexes; Li X et al.; Adducin is an actin-binding protein that has been proposed to function as a regulated assembly factor for the spectrin/actin network . This study has addressed the question of the subunit and domains of spectrin required for formation of spectrin/adducin/actin complexes in in vitro assays . Quantitative evidence is presented that the beta-spectrin N-terminal domain plus the first two alpha-helical domains are required for optimal participation of spectrin in spectrin/adducin/actin complexes . The alpha subunit exhibited no detectable activity either alone or following association with beta-spectrin . The critical domains of beta-spectrin involved in complex formation were determined using recombinant proteins expressed in bacteria . The N-terminal domain (residues 1-313) of beta-spectrin associated with F-actin with a Kd of 26 microM, and promoted adducin binding to F-actin with half-maximal activation at 110 nM . Addition of the first alpha-helical domain (residues 1-422) lowered the Kdfor F-actin by 4-fold to 6 microM, but also reduced the capacity by 3-fold and had no effect on interaction with adducin . Further addition of the second alpha-helical domain (residues 1-528) did not alter binding to F-actin but resulted in a 2-fold increased activity in promoting adducin binding with half-maximal activation at 50 nM . Addition of up to eight additional alpha-helical domains (residues 1-1388) resulted in no further change in F-actin binding or association with adducin . These results demonstrate an unanticipated role of the first repeat of beta-spectrin in actin binding activity and of the second repeat in association with adducin/actin, and imply the possibility of an extended contact between adducin, spectrin, and actin involving several actin subunits.

Biochem Biophys Res Commun, 1996 Jun 25, 223(3), 770 - 7
Anaerobic O-demethylations of methoxynaphthols, methoxyfuran, and fluoroanisols by Sporomusa ovata; Stupperich E et al.; In vitro experiments with 3,4-dimethoxybenzoate-induced Sporomusa enzymes a broad O-methyl ether cleavage capacity . The O-demethylase activity hydrolized the methyl-oxygen linkages of methoxynaphtholes of the heterocycles 2-methoxyfuran or 2-methoxythiophene as well as of several dimethoxy and monomethoxy aryls under anaerobic conditions . Also, fluoro and chloro substituents of anisoles enhanced the O-demethylation rate, indicating that an electron delocalized aromatic structure supported the methyl ether activation mechanism . Monomethoxy aromatics with additional chargeable groups, however, were less effectively transformed by the O-demethylase activity . No transformations into hydroxylated products occurred with 4-(trifluoromethoxy)benzyl alcohol, 4-(trifluoromethoxy)fluorobenzene, 2,5-dimethoxytetrahydrofuran, or alkyl-O-methyl ethers . The inert ethers did not affect the 3,4-dimethoxybenzoate metabolism . Ether activation or the following methyl transfer to the methyl acceptor tetrahydrofolate involved a prominent 31 kDa peptide from the cytoplasmic cell fraction, because this particular peptide was lacking in cells grown with methanol, betaine or fructose.

FEBS Lett, 1996 Jun 24, 389(1), 96 - 101
Genomes with distinct function composition; Tamames J et al.; The functional composition of organisms can be analysed for the first time with the appearance of complete or sizeable parts of various genomes . We have reduced the problem of protein function classification to a simple scheme with three classes of protein function: energy-, information- and communication-associated proteins . Finer classification schemes can be easily mapped to the above three classes . To deal with the vast amount of information, a system for automatic function classification using database annotations has been developed . The system is able to classify correctly about 80% of the query sequences with annotations . Using this system, we can analyse samples from the genomes of the most represented species in sequence databases and compare their genomic composition . The similarities and differences for different taxonomic groups are strikingly intuitive . Viruses have the highest proportion of proteins involved in the control and expression of genetic information . Bacteria have the highest proportion of their genes dedicated to the production of proteins associated with small molecule transformations and transport . Animals have a very large proportion of proteins associated with intra- and intercellular communication and other regulatory processes . In general, the proportion of communication-related proteins increases during evolution, indicating trends that led to the emergence of the eukaryotic cell and later the transition from unicellular to multicellular organisms.

FEBS Lett, 1996 Jun 24, 389(1), 25 - 31
A structural model for the membrane-integral domain of succinate: quinone oxidoreductases; Hagerhall C et al.; Many succinate:quinone oxidoreductases in bacteria and mitochondria, i.e . succinate:quinone reductases and fumarate reductases, contain in the membrane anchor a cytochrome b whose structure and function is poorly understood . Based on biochemical data and polypeptide sequence information, we show that the anchors in different organisms are related despite an apparent diversity in polypeptide and heme composition . A general structural model for the membrane-integral domain of the anchors is proposed . It is an antiparallel four-helix bundle with a novel arrangement of hexa-coordinated protoheme IX . The structure can be applied to a larger group of membrane-integral cytochromes of b-type and has evolutionary and functional implications.

Sci Total Environ, 1996 Jun 21, 185(1-3), 125 - 49
The form and bioavailability of non-ionic organic chemicals in sewage sludge-amended agricultural soils; Beck AJ et al.; The application of sewage sludges to agricultural land may increase the concentrations of many toxic organic chemicals in soils which could have adverse effects on wildlife and human health if these compounds enter foodchains . Chlorobenzenes (CBs), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) are amongst those compounds currently receiving most attention . The "form' in which these, and other organic chemicals, are present in soils and their potential to be lost by various processes including leaching, volatilisation and (bio)degradation is shown to be dependent on the physicochemical characteristics of the soil and sewage sludge, environmental conditions and the properties of the chemicals themselves . The distinction is made between those compounds that are labile, reversibly sorbed and irreversibly sorbed by sewage sludge-amended soils . The implications of the form in which the chemicals are present in soil for their "availability' to transfer from the soil to bacteria, fungi, earthworms, grazing livestock and food crops followed by the potential for further transfers, metabolism or bioaccumulation are discussed . The importance of the timing and method of sewage sludge application to soil on "form' and "availability' are also considered.

J Clin Invest, 1996 Jun 15, 97(12), 2842 - 8
Altered cardiac troponin T in vitro function in the presence of a mutation implicated in familial hypertrophic cardiomyopathy; Lin D et al.; Familial hypertrophic cardiomyopathy (HCM) can be caused by dominant missense mutations in cardiac troponin T (TnT), alpha-tropomyosin, C-protein, or cardiac myosin heavy chain genes . The myosin mutations are known to impair function, but any functional consequences of the TnT mutations are unknown . This report describes the in vitro function of troponin containing an IIe91Asn mutation in rat cardiac TnT, corresponding to the HCM-causing Ile79Asn mutation in man . Mutant and wild-type TnT cDNAs were expressed in bacteria and the proteins purified and reconstituted with the other troponin subunits, the mutation had no effect on troponin's affinity for tropomyosin, troponin-induced binding of tropomyosin to actin, cooperative binding of myosin subfragment 1 to the thin filament, CA(2+)-sensitive regulation of thin filament-myosin subfragment 1 ATPase activity, or the CA2+ concentration dependence of this regulation . However, the mutation resulted in 50% faster thin filament movement over a surface coated with heavy meromyosin in in vitro motility assays . The increased sliding speed suggests an unexpected role for the amino terminal region of TnT in which this mutation occurs . The relationship between this faster motility and altered cardiac contraction in patients with HCM is discussed.

J Immunol, 1996 Jun 15, 156(12), 4815 - 20
Expression of C-reactive protein by alveolar macrophages; Dong Q et al.; C-reactive protein (CRP) is well characterized as one of the serum acute phase proteins, the levels of which increase dramatically after infection . CRP has been shown to be involved in multiple immunoregulatory functions . For example, it activates the classical complement cascade, opsonizes bacteria for phagocytosis, and stimulates phagocytic cells . Although CRP is predominantly produced and secreted by hepatocytes, other cells including subsets of lymphocytes, Kupffer cells, and blood monocytes have been shown to synthesize this protein as well . We hypothesized that CRP may be produced in the lung, and therefore it could function directly in pulmonary host defense . Western blot analysis showed that CRP was present in the lung tissue, lung lavage, and alveolar macrophages . This result was further confirmed by immunohistochemical staining of lung sections that showed the localization of CRP in alveolar macrophages . The CRP mRNA was detected subsequently by reverse-transcriptase PCR (RT-PCR), and a single amplified product was obtained from alveolar macrophages as well as from whole lung tissue . Both were the same size as the amplified product obtained from liver mRNA . Furthermore, in situ hybridization with CRP riboprobe demonstrated specific staining of alveolar macrophages both in lung sections and isolated cells . In addition, in situ hybridization showed that CRP mRNA levels in isolated alveolar macrophages were up-regulated by in vitro LPS stimulation . In summary, these results indicate that CRP is produced by alveolar macrophages, and suggest that CRP may be involved in the pulmonary immune response.

J Mol Biol, 1996 Jun 14, 259(3), 480 - 501
Determination of the gene sequence and the three-dimensional structure at 2.4 angstroms resolution of methanol dehydrogenase from Methylophilus W3A1; Xia Z et al.; The DNA sequences for the genes encoding the heavy and light subunits of methanol dehydrogenase from Methylophilus methylotrophus W3A1 have been determined . The deduced amino acid sequence has enabled the structure of the enzyme to be refined at 2.4 angstrom resolution against X-ray data collected on a Hamlin area detector . The structure was refined using the programs PROFFT and X-PLOR with several model building step interspersed . The final model contains two heavy chains (571 amino acids), two light chains (69 amino acids), two molecules of pyrroloquinoline quinone, two Ca2+ and 521 solvent molecules . Each half molecule contains four disulfide linkages and four cis peptides . One of the disulfides is formed from two adjacent cysteine residues linked by a trans peptide which creates a novel eight-membered ring . The heavy subunit is an 8-fold beta-propeller, each "blade" of which is a four-stranded antiparallel twisted beta-sheet . The light chain is an elongated subunit stretching across the surface of the heavy subunit, with residues 1 to 32 containing four beta-turns and residues 33 to 62 forming a helix; however, it neither interacts with the active site, nor the other HL dimer and its functional role is obscure . Around the 8-fold beta-propeller there is a repeating pattern of tryptophan residues located in the outer strand of seven of the eight beta-leaflets, each packed between adjacent leaflets . Each of these tryptophan residues is centered in the beta-strand and participates in the main chain hydrogen bonding of the sheet . Five of the seven tryptophan residues have closely similar interactions with the adjacent beta-leaflet including stacking of the tryptophan indole rings against a peptide plane and formation of a hydrogen bond from NE1 of the indole ring to a main-chain carbonyl . This repeating pattern is conserved over a number of MEDH sequences . The PQQ is located on the pseudo 8-fold rotation axis of the heavy subunit, in a funnel-shaped internal cavity, sandwiched between the indole ring of Trp237 and the two sulfur atoms of the Cys103-Cys104 vicinal disulfide . A hexacoordinate Ca2+ is bound in the active site by one nitrogen and five oxygen ligands, three from the PQQ and the others from two protein side-chains . In the active site an isolated solvent molecule is bound to the O5 of PQQ and to a nearby aspartate side-chain; its position may be the binding site for methanol . The aspartate might than serve as a general base for proton abstraction from the substrate hydroxyl . The C5 atom of PQQ could be activated by electrophilic catalysis by a nearby argenine side-chain or by the calcium ion bound to PQQ.

J Biol Chem, 1996 Jun 14, 271(24), 13953 - 7
Studies on identifying the catalytic role of Glu-204 in the active site of yeast invertase; Reddy A et al.; In a previous study on yeast invertase (Reddy, A., and Maley, F . (1990) J . Biol . Chem . 265, 10817-10820), we identified Asp-23 through the procedures of affinity labeling and site-directed mutagenesis as a catalytic nucleophile . In the present study we undertook to determine other residues involved in the catalytic process . Earlier studies suggested histidine as a potential proton donor in the hydrolysis of sucrose, but by mutagenizing each of the enzyme's four histidines this amino acid was eliminated from consideration . Another candidate appeared to be cysteine, since iodine at about a 2-fold molar excess inactivated invertase by modifying both of the enzyme's cysteine residues . Dithiothreitol treatment restored the sulfhydryl groups and enzyme activity . Replacement of each of the cysteines with alanines revealed that C108A invertase retained full activity whereas C205A was reduced about 4-fold in its kcat . A comparison of the amino acid sequences of fructosylhydrolases revealed a conserved region coincident with Glu-204/Cys-205 . Mutagenizing Glu-204 to Ala resulted in a 3, 000-fold reduction in the kcat of invertase indicating that Glu-204 plays a major role in catalysis . Based on these findings, a mechanism is proposed for the hydrolysis of sucrose which involves Asp-23 as a nucleophile and Glu-204 as an acid/base catalyst.

Nature, 1996 Jun 13, 381(6583), 623 - 5
Intersubunit rotation in active F-ATPase; Sabbert D et al.; The enzyme ATP synthase, or F-ATPase, is present in the membranes of bacteria, chloroplasts and mitochondria . Its structure is bipartite, with a proton-conducting, integral membrane portion, F0, and a peripheral portion, F1 . Solubilized F1 is composed of five different subunits, (alpha beta)3 gamma delta epsilon, and is active as an ATPase . The function of F-ATPase is to couple proton translocation through F0 with ATP synthesis in F1 (ref.3) . Several lines of evidence support the spontaneous formation of ATP on F1 (refs 4,5) and its endergonic release at cooperative and rotating (or at least alternating) sites . The release of ATP at the expense of protonmotive force might involve mechanical energy transduction from F0 into F1 by rotation of the smaller subunits (mainly gamma) within (alpha beta)3, the catalytic hexagon of F1 as suggested by electron microscopy, by X-ray crystal structure analysis and by the use of cleavable crosslinkers . Here we record an intersubunit rotation in real time in the functional enzyme by applying polarized absorption relaxation after photobleaching to immobilized F1 with eosin-labelled gamma . We observe the rotation of gamma relative to immobilized (alpha beta)3 in a timespan of 100 ms, compatible with the rate of ATP hydrolysis by immobilized F1 . Its angular range, which is of at least 200 degrees, favours a triple-site mechanism of catalysis, with gamma acting as a crankshaft in (alpha beta)3 . The rotation of gamma is blocked when ATP is substituted with its non-hydrolysable analogue AMP-PNP.

Mutat Res, 1996 Jun 12, 353(1-2), 151 - 76
Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays; Rueff J et al.; We present here the results obtained within the framework of an EU funded project aimed to develop and validate alternative metabolic activating systems to be used in short-term mutagenicity assays, in order to reduce the use of laboratory animals for toxicology testing . The activating systems studied were established cell lines (Hep G2, CHEL), genetically engineered V79 cell lines expressing specific rat cytochromes P450, erythrocyte-derived systems, CYP-mimetic chemical systems and plant homogenates . The metabolically competent cell lines were used as indicator cells for genotoxic effects as well as for the preparation of external activating systems using other indicator cells . The following endpoints were used: micronuclei, chromosomal aberrations and sister chromatid exchanges, mutations at the hprt locus, gene mutations in bacteria (Ames test), unscheduled DNA synthesis and DNA breaks detected in the comet assay . All metabolic systems employed activated some promutagens . With some of them, promutagens belonging to many different classes of chemicals were activated to genotoxicants, including carcinogens negative in liver S9-mediated assays . In other cases, the use of the new activating systems allowed the detection of mutagens at much lower substrate concentrations than in liver S9-mediated assays . Therefore, the alternative metabolizing systems, which do not require the use of laboratory animals, have a substantial potential in in vitro toxicology, in the basic genotoxicity testing as well as in the elucidation of activation mechanisms . However, since the data basis is much smaller for the new systems than for the activating systems produced from subcellular liver preparations, the overlapping use of both systems is recommended for the present and near future . For example, liver S9 preparations may be used with some indicator systems (e.g., bacterial mutagenicity), and metabolically competent mammalian cell lines may be used with other indicator systems (e.g., a cytogenetic endpoint) in a battery of basic tests.

Mol Gen Genet, 1996 Jun 12, 251(3), 298 - 306
Genetic and physical mapping in Brassica diploid species of a gene cluster defined in Arabidopsis thaliana; Sadowski J et al.; We report the genetic and physical analysis by pulse field gel electrophoresis (PFGE) in three Brassica diploid genomes for a cluster of five genes characterized in a selected segment of 15 kb on chromosome 3 of Arabidopsis thaliana, encoding a Bradyrhizobium CycJ homologue (At1), a rat p67 translation factor homologue (At2), an Em-like (early methionine) protein (At3), chlorophyll synthase (At4) and a yeast Sac1 homologue (A5) . The Arabidopsis gene array was found to be conserved on a single linkage group in each of the Brassica genomes . However, partial complexes were found to be duplicated in other chromosome segments on the same or other linkage groups . Some of the At genes, which could not be genetically mapped because of lack of polymorphism, were assigned to their respective linkage groups by physical mapping . The presence of multiple copies of the A . thaliana gene cluster in the three Brassica genomes further establishes their complex nature, which results from extensive duplication and chromosomal rearrangement . In general, genetic distances between the At genes agreed with values expected for the physical distances determined in Brassica.

Gene, 1996 Jun 12, 172(1), 99 - 104
Expression and functional characterisation of the clpC gene of Mycobacterium leprae: ClpC protein elicits human antibody response; Misra N et al.; This paper reports the expression of a previously described gene {Nath and Laal, Nucleic Acids Res . 18 (1990) 4935}, currently identified as the clpC gene of Mycobacterium leprae, using an in vitro rabbit reticulocyte lysate-coupled transcription/translation system . The produced protein moved as a 95-kDa band on SDS-PAGE . An additional band of 79 kDa was seen which may have resulted from a GTG codon downstream to the initiating ATG in the clpC sequence . A threefold increase in synthesis of the 95-kDa protein was achieved by altering the translation codon context sequence of the ATG start codon . The ClpC (caseinolytic protease C) amino acid sequence, which contained two nucleotide-binding sites, exhibited in vitro ATP binding . Of functional significance was its immunoreactivity in human subjects with mycobacterial infection . Leprosy and tuberculosis patients with active disease had antibodies which recognised ClpC in dot ELISA.

Gene, 1996 Jun 12, 172(1), 149 - 53
Isolation and sequence analysis of the cDNA encoding delta 1-pyrroline-5-carboxylate reductase from Zalerion arboricola; Kelly R et al.; A cDNA encoding delta 1-pyrroline-5-carboxylate reductase (P5CR) was isolated from the pneumocandin (Pmo)-producing fungus, Zalerion arboricola (Za), by complementation of a P5CR-deficient mutant (pro3) of Saccharomyces cerevisiae (Sc) . The cloned cDNA was placed under control of the Sc galactokinase (GAL1) promoter and restored P5CR activity to the pro3 mutant . Sequence analysis revealed that the Za P5CR-encoding cDNA encodes an approx . 35 kDa protein with substantial amino acid (aa) identity to P5CR from another filamentous fungus, Neurospora crassa (Nc) . Za P5CR exhibits a moderate degree of aa identity to P5CR from plants, bacteria, human and Sc . This is the first gene to be isolated from Za.

Gene, 1996 Jun 12, 172(1), 111 - 6
Identification of a native Dichelobacter nodosus plasmid and implications for the evolution of the vap regions; Billington SJ et al.; Studies on the role of various virulence factors of the ovine pathogen, Dichelobacter nodosus, have suffered from the absence of a mechanism for the introduction of DNA into this organism . As an initial step in the development of genetic methods, we have identified and cloned a native 10-kb plasmid, pJIR896, from a clinical isolate . This plasmid was found to be a circular form of vap region 1/3 that is found in the reference strain, A198 . However, pJIR896 lacked the duplicated region present in the A198 sequence and instead contained a 1.7-kb putative insertion sequence, IS1253, which shared similarity to a number of unusual IS elements . A model is proposed for the evolution of vap region 1/3 which involves the integration of a plasmid, such as pJIR896, and subsequent rearrangements resulting from the deletion or transposition of IS1253.

Biochemistry, 1996 Jun 11, 35(23), 7599 - 607
Identification of an active acidic residue in the catalytic site of beta-hexosaminidase; Tse R et al.; Human beta-hexosaminidases A and B (EC 3.2.1.52) are dimeric lysosomal glycosidases composed of evolutionarily related alpha and/or beta subunits . Both isozymes hydrolyze terminal beta-linked GalNAc or GlcNAc residues from numerous artificial and natural substrates; however, in vivo GM2 ganglioside is a substrate for only the heterodimeric A isozyme . Thus, mutations in either gene encoding its alpha or beta subunits can result in GM2 ganglioside storage and Tay-Sachs or Sandhoff disease, respectively . All glycosyl hydrolases ae believed to have one or more acidic residues in their catalytic site . We demonstrate that incubation of hexosaminidase with a chemical modifier specific for carboxyl side chains produces a time-dependent loss of activity, and that this effect can be blocked by the inclusion of a strong competitive inhibitor in the reaction mix . We hypothesized that the catalytic acid residue(s) should be located in a region of overall homology and be invariant within the aligned deduced primary sequences of the human alpha and beta subunits, as well as hexosaminidases from other species, including bacteria . Such a region is encoded by exons 5-6 of the HEXA and HEXB genes . This region includes beta Arg211 (invariant in 15 sequences), which we have previously shown to be an active residue . This region also contains two invariant and one conserved acidic residues . A fourth acidic residue, Asp alpha 258, beta 290, in exon 7 was also investigated because of its association with the B1 variant of Tay-Sachs disease . Conservative substitutions were made at each candidate residue by in vitro mutagenesis of a beta cDNA, followed by cellular expression . Of these, only the beta Asp196Asn substitution decreased the kcat (350-910-fold) without any noticeable effect on the K(m) . Mutagenesis of either beta Asp240 or beta Asp290 to Asn decreased kcat by 10- or 1.4-fold but also raised the K(m) of the enzyme 11- of 3-fold, respectively . The above results strongly suggest that beta Asp196 is a catalytic acid residue in beta-hexosaminidase.

Biochemistry, 1996 Jun 11, 35(23), 7439 - 46
Tetracycline analogs affecting binding to Tn10-Encoded Tet repressor trigger the same mechanism of induction; Lederer T et al.; We examined the influence of substituents in tetracycline (tc) analogs modified at positions 2 and 4-9 and anhydrotetracycline (atc) on induction of the Tn10-encoded Tet repressor (TetR) by a quantitative in vitro induction assay . The equilibrium association constants of the modified tc to TetR were independently determined to distinguish effects on binding from those on induction . We found a correlation between the binding affinity and induction of TetR for most tc analogs . While a substitution at position 5 revealed only minor effects, changes at position 6 increased binding and induction efficiencies up to 20-fold . A chlorine at position 7 or 8 enhanced binding and induction about 4- and 9-fold, respectively . Substituents at position 9 decreased binding up to 5-fold . Epimerization of the dimethylamino function at position 4 in 4-epi-tc resulted in about 300-fold-reduced binding and 80-fold-reduced induction . Substitution of this grouping by hydrogen in 4-de(dimethylamino)-tc resulted in no binding and no induction . The respective atc analog failed to induce as well, although binding was still observed . The dimethylamino function may, thus, play a role in triggering the conformational change of TetR necessary for induction . Substitution of the 2-carboxamido by a nitrilo function did not influence binding and induction efficiencies . Atc showed about 30-fold increased binding and induction, being the most effective inducer tested in this study . The equilibrium association constants of most TetR-{Mg-tc}+ and TetR-({Mg-tc}+)2 analog complexes with tet operator are decreased about 10(2)- and 10(8)-fold, respectively, as compared to those of free TetR . This suggests that these tc analogs share the same molecular mechanism of TetR induction.

Biochemistry, 1996 Jun 11, 35(23), 7308 - 15
Murine DNA cytosine-C5 methyltransferase: pre-steady- and steady-state kinetic analysis with regulatory DNA sequences; Flynn J et al.; We present the first description of KmDNA, KdDNA, Kcat, and Kmethylation for a mammalian DNA methyltransferase . Homogeneous, 190 000 MTDNA (cytosine-5-)-methyltransferase isolated from mouse erythroleukemia cells has turnover constants of 0.15-0.59 h-1 with single-stranded and unmethylated double-stranded oligonucleotides containing a single CpG dinucleotide . These substrates were designed to mimic DNA transcriptional cis elements previously reported to have cytosine C-5-methylated regulation . The rate-limiting step for these substrates is the methylation step itself . In contrast, hemimethylated double-stranded substrates show burst kinetics, consistent with a rapid methylation event (3 h-1) followed by a slower step which determines steady-state Kcat . Hemimethylated and unmethylated double-stranded DNA shows similar binding affinities; these results reveal the molecular basis for the enzyme's preference for hemimethylated DNA to be the methyl transfer step . Substrates with multiple recognition sites do not show burst kinetics and have turnover rate constants of 6 h-1 . Catalytic turnover for the mammalian enzyme is thus approximately 10-fold slower than that for the related bacterial enzymes . Our combined results show quantitatively that one enzyme is certainly capable of both maintenance and de novo methylation and that maintenance of the genomic methylation pattern is preferred over the de novo establishment of new patterns . Direct comparison of the mammalian enzyme with the bacterial DNA cytosine-C5 methyltransferase, M.SssI, indicates dramatic differences in preferences for single-stranded, double-stranded, and hemimethylated double-stranded substrates . Moreover, the specificity hierarchy shown for the M.SssI is derived from very different changes in K(m) and catalysis than those observed for the mammalian DCMTase . These results demonstrate that the M.SssI, and perhaps other DNA cytosine methyltransferases from bacteria, is functionally dissimilar to the mammalian enzyme.

Proc Natl Acad Sci U S A, 1996 Jun 11, 93(12), 6031 - 6
The glycine binding site of the N-methyl-D-aspartate receptor subunit NR1: identification of novel determinants of co-agonist potentiation in the extracellular M3-M4 loop region; Hirai H et al.; The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is a heterooligomeric membrane protein composed of homologous subunits . Here, the contribution of the M3-M4 loop of the NR1 subunit to the binding of glutamate and the co-agonist glycine was investigated by site-directed mutagenesis . Substitution of the phenylalanine residues at positions 735 or 736 of the M3-M4 loop produced a 15- to 30-fold reduction in apparent glycine affinity without affecting the binding of glutamate and the competitive glycine antagonist 7-chlorokynurenic acid; mutation of both residues caused a >100-fold decrease in glycine affinity . These residues are found in a C-terminal region of the M3-M4 loop that shows significant sequence similarity to bacterial amino acid-binding proteins . Epitope tagging revealed both the N-terminus and the M3-M4 loop to be exposed extracellularly, whereas a C-terminal epitope was localized intracellularly . These results indicate that the M3-M4 loop is part of the ligand-binding pocket of the NR1 subunit and provide the basis for a refined model of the glycine-binding site of the NMDA receptor.

J Biol Chem, 1996 Jun 7, 271(23), 13556 - 60
Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain; Reid T et al.; Using a mouse embryo cDNA library, we conducted a two-hybrid screening to identify new partners for the small GTPase Rho . One clone obtained by this procedure contained a novel cDNA of 291 base pairs and interacted strongly with RhoA and RhoC, weakly with RhoB, and not at all with Rac1 and Cdc42Hs . Full-length cDNAs were then isolated from a mouse brain library . While multiple splicing variants were common, we identified three cDNAs with an identical open reading frame encoding a 61-kDa protein that we named rhotekin (from the Japanese "teki," meaning target) . The N-terminal part of rhotekin, encoded by the initial cDNA and produced in bacteria as a glutathione S-transferase fusion protein, exhibited in vitro binding to 35S-labeled guanosine 5'-3-O-(thio)triphosphate-bound Rho, but not to Rac1 or Cdc42Hs in ligand overlay assays . In addition, this peptide inhibited both endogenous and GTPase-activating protein-stimulated Rho GTPase activity . The amino acid sequence of this region shares approximately 30% identity with the Rho-binding domains of rhophilin and a serine/threonine kinase, PKN, two other Rho target proteins that we recently identified (Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuka, A., and Narumiya, S . (1996) Science 271, 645-648) . Thus, not only is rhotekin a novel partner for Rho, but it also belongs to a wide family of proteins that bear a consensus Rho-binding sequence at the N terminus . To our knowledge, this is the first conserved sequence for Rho effectors, and we have termed this region Rho effector motif class 1.

J Biol Chem, 1996 Jun 7, 271(23), 13356 - 61
The synthesis and assembly of functional high and low light LH2 antenna complexes from Rhodopseudomonas palustris in Rhodobacter sphaeroides; Fowler GJ et al.; Photosynthetic bacteria respond to lowered light intensity by increasing the level of the peripheral light-harvesting (LH2) complex . Several species possess an additional mechanism, responding to variations in light conditions by making different types of LH2 complex . However, the study of these complexes in isolation and in the native membrane environment has not been possible . Therefore two LH2 gene pairs from Rhodopseudomonas palustris, associated, respectively, with high light (pucBAa) and low light (pucBAd) growth conditions, were expressed in Rhodobacter sphaeroides . The high light LH2 complex PucBAa was synthesized at appreciable levels in R . sphaeroides, had near-infrared absorption bands at approximately 800-855 nm, and was able to transfer energy efficiently to the native LH1 complex . In contrast, the low light complex PucBAd was found at comparatively low levels, had absorption bands at approximately 797-830 nm, and did not transfer energy to the native LH1 complex efficiently . These observations are discussed in the light of site-directed studies on the R . sphaeroides LH2 complex, and the recently elucidated Rhodopseudomonas acidophila 10050 LH2 structure . Potentially important residues for energy transfer between LH2 and LH1 complexes are identified, as well as some of the factors that influence stability and assembly of LH2 complexes, such as the N-terminal sequences of their protein subunits and their carotenoid binding sites.

Biochim Biophys Acta, 1996 Jun 5, 1312(1), 39 - 47
Topological organization of the cytosolic activating complex of the superoxide-generating NADPH-oxidase . Pinpointing the sites of interaction between p47phoz, p67phox and p40phox using the two-hybrid system; Fuchs A et al.; Activation of the superoxide-generating NADPH-oxidase in phagocytic cells requires the assembly of a membrane-bound flavocytochrome b and cytosolic factors p47phox and p67phox under the control of the GTP-binding protein, Rac . A novel cytosolic component p40phox was recently identified . Most of the components of the complex contain SH3 domains and/or polyproline motifs which are likely to mediate protein-protein interactions occurring in the formation of the active NADPH-complex . The two-hybrid system was used to explore associations between the cytosolic factors . Various constructs of p47phox, p67phox and p40phox cDNAs coding for functional domains were inserted into two-hybrid system vectors, expressing fusion proteins either with the DNA binding protein Lex A or with the activation domain of Gal 4 . The site of interaction of p67phox with p47phox was restricted to the C-terminal SH3 domain of p67phox and to the polyproline motif of p47phox . The polyproline motif of p47phox was also found to mediate interaction with the SH3 domain of p40phox, as well as intramolecular interaction within p47phox . The site of interation of p67phox with p40phox was found to be in the 150 amino acid stretch between the two SH3 domains of p67phox . As the C-terminal tail of p40phox which interacts with p67phox contains neither a SH3 domain nor a polyproline consensus site, it is concluded that a novel type of interaction occurs between p40phox and p67phox . Taken together, the results of the two-hybrid experiments led us to formulate a model for oxidase activation, induced by phosphorylation, in which p40phox tends to prevent spontaneous activation.

Gut, 1996 Jun, 38(6), 841 - 5
Association of cytotoxin production and neutrophil activation by strains of Helicobacter pylori isolated from patients with peptic ulceration and chronic gastritis; Zhang QB et al.; BACKGROUND: Helicobacter pylori is associated with neutrophil infiltration within the gastroduodenal mucosa . Neutrophil activation provides a major source of oxygen free radicals, which have been implicated in the pathogenesis of peptic ulceration . AIM: To investigate if cytotoxin producing strains of H pylori are associated with the generation of oxidative burst in polymorphonuclear neutrophils (PMNs) . PATIENTS: 76 patients undergoing endoscopy of whom 45 had peptic ulcer and 31 chronic gastritis only were studied . METHODS: Strains of H pylori were cultured in Brucella broth . After 48 hours, bacteria were harvested by centrifugation and a bacterial suspension prepared as a stimulus for PMN oxidative burst using chemiluminescence . PMNs were prepared from health blood donors . To test the ability of strains to produce cytotoxin, culture supernatants of each were concentrated by polyethylene glycol and tested on cultured Vero cells for intracellular vacuolation . RESULTS: 30 of 45 (66.7%) peptic ulcer patients induced cell vacuolation versus nine of 31 (29%) strains from patients with chronic gastritis only (p < 0.01) . Cytotoxin positive strains of H pylori regardless of the presence or absence of peptic ulcer displayed an increased induction of respiratory burst in PMNs compared with toxin negative strains from patients with chronic gastritis only (p < 0.05) . Among the toxin negative strains, those from patients with peptic ulcer did not show a significant increase of the oxidative burst than those from patients without peptic ulcer (NS) . CONCLUSION: Toxinogenicity of strains of H pylori seems to be correlated with neutrophil respiratory burst and peptic ulceration . The ability of some strains of H pylori to produce cytotoxin and to induce the oxidative burst in neutrophils may be important in the pathogenesis of peptic ulcer disease.

Rev Sci Tech, 1996 Jun, 15(2), 551 - 61
Appearance and spread of diseases among bivalve molluscs in the northern hemisphere in relation to international trade; Renault T; Bivalve mollusc culture is a well-developed marine aquaculture activity in many countries around the world, notably in the northern hemisphere . During the development of this activity, numerous countries have been confronted with infectious diseases of varying severity and duration . Research has been conducted to determine the aetiology, epidemiology and control measures for these epizootics . Major epizootics in bivalve molluscs have been caused by viruses, bacteria, fungi and protozoan parasites . Moreover important commercial relations exist in marine mollusc culture between different geographical areas . This must be taken into account in explaining the appearance and the spread of some infectious diseases in several countries around the world . The author concentrates on some viral and protozoan diseases of bivalve molluscs reported in the northern hemisphere, in view of their economic impact and their spread related to movement of molluscs through trade.

Int Dent J, 1996 Jun, 46(3), 131 - 8
Endodontic failures--changing the approach; Cheung GS; The underlying reason for endodontic failures is almost invariably due to bacterial infection . The bacteria may be situated within a previously missed or uninstrumented portion of a root canal, infiltrate via a leaky coronal restoration and root filling, or cause contamination from an extra-radicular infection . Management of the failing root canal filling begins with the identification of the source of persistent infection . Should the infection be present within the root canal system, such as a missed canal, orthograde retreatment is the choice of treatment . This is also true for asymptomatic cases which had been inadequately obturated and which require the placement of a dowel into the canal for restorative reasons . Periapical surgery is best reserved for cases with no sign of healing after orthograde retreatment and those with extra-radicular infection . This paper discusses the relationship between endodontics and restorative dentistry, treatment planning for endodontic failures, and the reported rates of success with orthograde and surgical retreatments.

Jpn Circ J, 1996 Jun, 60(6), 382 - 8
Tsutsugamushi myocarditis with congestive heart failure and persistent atrial standstill; Jeong MH et al.; Acute infectious myocarditis is primarily by viruses and bacteria, but sometimes by rickettsia . Tsutsugamushi disease is a febrile illness caused by Rickettsia tsutsugamushi, and has been prevalent in Korea since 1985 . Characteristics of tsutsugamushi disease are fever, rash and eschar . Tsutsugamushi myocarditis is rare . Cardiac involvement may include ST-T changes, PR prolongation, mild mitral regurgitation, and perivascular inflammation with myocardial necrosis . We describe here a 50-year-old woman who complained of fever, orthopnea and chest pain . Work-up of the patient revealed abdominal scar, positive tsutsugamushi antibody, congestive heart failure with severe mitral and tricuspid regurgitation, persistent atrial standstill on electrophysiologic study, junctional rhythm and ST-T changes mimicking anterior myocardial infarction and myocardial inflammation with perivasculitis on endomyocardial biopsy . The patient's condition improved with doxycycline and inotropics . Persistent atrial standstill during was found at the one-year follow-up.

J Steroid Biochem Mol Biol, 1996 Jun, 58(3), 251 - 8
Ability of various members of the hsp70 family of chaperones to promote assembly of the glucocorticoid receptor into a functional heterocomplex with hsp90; Hutchison KA et al.; To be in a conformation that binds steroid, the hormone-binding domain of the glucocorticoid receptor (GR) must be bound to the 90 kDa heat shock protein (hsp90) . Rabbit reticulocyte lysate contains a protein chaperone system that assembles the receptor into a heterocomplex with hsp90 and converts it from a non-steroid-binding to a steroid-binding form . Assembly of the GR-hsp90 heterocomplex requires hsp70, and in this work we examine the activities of four members of the hsp70 protein family in GR-hsp90 heterocomplex assembly . Rabbit reticulocyte lysate was depleted of hsp70 by passing it through a column of ATP agarose, resulting in the inactivation of its GR-hsp90 heterocomplex assembly activity . Addition of purified animal (mouse) or plant (wheat germ) hsp70 to the hsp70-depleted lysate permits assembly of a GR-hsp90 heterocomplex with a high affinity steroid binding site . However, purified hsp70 homologues from bacteria (DnaK) or the endoplasmic reticulum (BiP) do not promote heterocomplex formation, despite the fact that both DnaK and BiP bind to the GR in the assay system . When added to whole (i.e . hsp70-containing) reticulocyte lysate, DnaK and BiP inhibit GR-hsp90 heterocomplex assembly . Wheat germ lysate forms a heterocomplex between mouse GR and plant hsp90, but the addition of purified rabbit hsp70 to the wheat germ lysate does not increase the amount of receptor-wheat hsp90 complex produced, despite the fact that the rabbit hsp70 binds to the GR when it is added to the wheat chaperone system . The conclusion is that binding of hsp70 to receptors does not necessarily reflect a physiologically meaningful interaction . When native receptor heterocomplexes isolated from cytosols contain hsp70, it is likely that the hsp70-bound receptors represent a minority of receptors that have not yet proceeded fully through the receptor heterocomplex assembly process, which includes the dissociation of hsp70 after the binding of hsp90.

J Biochem (Tokyo), 1996 Jun, 119(6), 1106 - 13
Purification, characterization, and sequencing of two cysteine proteinase inhibitors, Sca and Scb, from sunflower (Helianthus annuus) seeds; Kouzuma Y et al.; Two proteinaceous cysteine proteinase inhibitors (cystatins) referred to as Sca and Scb were purified to homogeneity from the seeds of sunflower (Heliantas annuus) by gel filtration on Sephadex G-75 followed by a series of ion-exchange column chromatographies and reverse-phase HPLC (RP-HPLC) . The isoelectric points (pI) of Sca and Scb were estimated to be 5.6 and 9.6, respectively . The inhibitory potencies of these two cystatins were examined with cysteine proteinases from various sources, such as plants, mammals, and bacteria . Papain was strongly inhibited by both Sca and Scb with Ki values of 5.6 x 10(-9) and 1.7 x 10(-10) M, respectively . Sca and Scb were also found to be potent inhibitors of ficin (Ki values of 1.9 x 10(-6) and 2.8 x 10(-9) M, respectively) . Rat cathepsin H was inhibited strongly by Scb and slightly by Sca . Although rat cathepsins B and L were significantly inhibited by Scb, they were scarcely affected by Sca . Neither Sca nor Scb inhibited Arg-gingipain, an arginine-specific cysteine proteinase of Porphyromonas gingivalis . The complete amino acid sequences of the two inhibitors were determined by protein chemical methods . The proteins Sca and Scb consist of 83 and 101 amino acid residues with M(r) of 9,330 and 11,187, respectively, and there are identical residues at 34 positions in the two sequences, that is at 42% of the residues compared . Comparison of their sequences with those of other cystatins revealed that Sca shares 59-73% identical residues with other phytocystatins, while Scb shows less identity to other phytocystatins, sharing only 28-38% identical residues . Furthermore, only 20-27% of the residues of both cystatins, Sca and Scb, are identical to those of the animal cystatins.

J Neurosci Nurs, 1996 Jun, 28(3), 140 - 52; quiz 152-4
Replacement therapy: arginine vasopressin (AVP), growth hormone (GH), cortisol, thyroxine, testosterone and estrogen; Mitchell DH et al.; Replacement therapy is routinely used to treat hormone deficiencies of patients who have had surgery or radiation therapy that damages the hypothalamus or pituitary gland . Hormones commonly replaced include: arginine vasopressin (AVP), growth hormone (GH), cortisol, thyroxine (T4), testosterone and estrogen . AVP, synthesized in the hypothalamus, is stored in and released by the posterior lobe of the pituitary gland . GH is synthesized and released by the anterior pituitary gland . The other hormones are produced and released by target glands each of which is stimulated by a specific anterior pituitary hormone, which in turn is controlled by release of a specific hypothalamic hormone . Feedback control by a high circulating concentration of the target gland's hormone regulates hypothalamic hormone release . Deficiency of AVP, important for water balance in the body, is restored with the synthetic analogue, 1-desamino-8-D-arginine vasopressin (DDAVP); it is given as a nasal spray or by injection . GH is required for normal growth in the developing child; recombinant GH, produced in bacteria, is injected subcutaneously . Adrenocorticotropic hormone (ACTH) controls release of cortisol which is produced by the adrenal cortex and enables the body to cope with stress; cortisol is replaced with prednisolone given orally . Thyroid stimulating hormone (TSH) controls release of the thyroid hormones, T4 and triiodothyronine (T3), which promote growth and development, and regulate energy metabolism; for replacement of T4, oral synthetic L-thyroxine is given . Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control release of testosterone, which promotes maturation of sperm and development of male sexual characteristics; replacement testosterone is administered intramuscularly . In females, FSH and LH control release of estrogens and progesterone which prepare the reproductive tract for release of the ovum, fertilization, implantation and development of the embryo, replacement by estrogen and progesterone preparations which are orally effective is given in a cyclic manner . A transdermal delivery system is available . Nursing implications include cautions and contraindications, potential problems of over-replacement, drug interactions as well as patient teaching points.

J Clin Periodontol, 1996 Jun, 23(6), 587 - 94
Chemiluminescent assay of alkaline phosphatase in human gingival crevicular fluid: investigations with an experimental gingivitis model and studies on the source of the enzyme within crevicular fluid; Chapple IL et al.; The purpose of this study was to investigate how levels of gingival crevicular fluid (GCF) alkaline phosphatase (ALP) change in relation to levels of plaque and gingival inflammation in 20 adults during a 21 day period of experimental gingivitis . The source of ALP within GCF was also investigated using a repeat sampling protocol; by determining enzyme levels derived from 30 putative periodontal pathogenic and non-pathogenic species; and by examining inhibition profiles from a variety of host and bacterial ALP isoenzymes . Total 30-s GCF ALP levels increased significantly (p < 0.002) during experimental gingivitis and preceded an increase in gingival index (GI) by approximately 7 days . Enzyme levels correlated with GCF volume (R = 0.7; p < 0.0001), but repeat sampling indicated that entry of ALP into the gingival crevice was independent of the rate of fluid flow . Only 5 of the bacterial species investigated produced clearly detectable levels of ALP in culture supernatants, these were P . gingivalis (381), P . intermedia (581), P . nigrescens (8944), Dentin P . gingivalis (TW 471: clinical isolate) and C . ochracea (25) . Levamisole inhibition and studies on suspensions of washed plaque demonstrated that host-derived ALP contributed to > 80% of the enzyme in GCF . We conclude that elevated 30-s GCF ALP levels measured using the chemiluminescent assay reported, are detectable before increases in gingival indices and appear to be a better marker of gingival inflammation than ALP concentrations . The major source of ALP within GCF is host derived and in early inflammatory disease is likely to be of polymophonuclear leukocyte origin.

Mol Microbiol, 1996 Jun, 20(5), 1071 - 81
Circadian expression of genes involved in the purine biosynthetic pathway of the cyanobacterium Synechococcus sp . strain PCC 7942; Liu Y et al.; Extensive circadian (daily) control over gene expression in the cyanobacterium Synechococcus sp . strain PCC 7942 is programmed into at least two differentially phased groups . The transcriptional activity of the smaller group of genes is maximal at about dawn and minimal at about dusk . We identified one of the genes belonging to this latter group as purF, which encodes the key regulatory enzyme in the de novo purine synthetic pathway, glutamine PRPP amidotransferase (also known as amidophosphoribosyltransferase) . Its expression pattern as a function of circadian time was confirmed by both luminescence from a purF::luxAB reporter strain and the abundance of purF mRNA . By fusing sequences upstream of the purF coding region to promoterless luxAB genes, we identified a limited upstream region, which potentially regulates purF circadian expression patterns in vivo . We also identified the purL gene immediately upstream of purF . The purL gene encodes FGAM synthetase, the fourth enzyme in the purine nucleotide biosynthesis pathway . Although these genes are expressed as part of a larger operon in other bacteria, reporter gene fusions revealed that purF and purL are transcribed independently in Synechococcus and that they are expressed at different phases of the circadian cycle . This differential expression pattern may be related to the oxygen sensitivity of amidophosphoribosyltransferase.

FEMS Immunol Med Microbiol, 1996 Jun, 14(2-3), 135 - 43
Binding and degradation of lactoferrin by Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens; de Lillo A et al.; The ability of laboratory and clinical strains of Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens to bind and to degrade lactoferrin (Lf) has been assessed . Lf bound readily to whole cells of each species apparently via high-affinity site and one or more low-affinity sites . P . gingivalis showed a lower affinity for Lf than the other two species (P < 0.001) . Virtually all strains of P . gingivalis completely degraded Lf under the conditions employed, whereas P . intermedia and P . nigrescens showed only partial degradation . These data suggest that Lf binds to a high-affinity receptor on all these bacteria and, particularly in the case of P . gingivalis, is then degraded by cell-associated proteases . This property may provide protection to the cell against the effects of Lf in periodontal sites and so is a possible virulence factor in disease . There was no association between the ability to degrade Lf and whether the strains had originated from healthy or diseased oral sites.

Curr Opin Struct Biol, 1996 Jun, 6(3), 353 - 60
Scores for sequence searches and alignments; Henikoff S; Every sequence comparison method requires a set of scores . For aligning protein sequences, substitution scores are based on models of amino acid conservation and properties, and matrices of these scores have substantially improved in recent years . Position-specific scoring matrices provide representations of sequence families that are capable of detecting subtle similarities . Comprehensive evaluations can effectively guide the choice of scores for sequence alignment and searching applications, including those that aid in the prediction of protein structures.

Microbiol Rev, 1996 Jun, 60(2), 366 - 85
Ribosome regulation by the nascent peptide; Lovett PS et al.; Studies of bacterial and eukaryotic systems have identified two-gene operons in which the translation product of the upstream gene influences translation of the downstream gene . The upstream gene, referred to as a leader (gene) in bacterial systems or an upstream open reading frame (uORF) in eukaryotes, encodes a peptide that interferes with a function(s) of its translating ribosome . The peptides are therefore cis-acting negative regulators of translation . The inhibitory peptides typically consist of fewer than 25 residues and function prior to emergence from the ribosome . A biological role for this class of translation inhibitor is demonstrated in translation attenuation, a form or regulation that controls the inducible translation of the chloramphenicol resistance genes cat and cmlA in bacteria . Induction of cat or cmlA requires ribosome stalling at a particular codon in the leader region of the mRNA . Stalling destabilizes an adjacent, downstream mRNA secondary structure that normally sequesters the ribosome-binding site for the cat or cmlA coding regions . Genetic studies indicate that the nascent, leader-encoded peptide is the selector of the site of ribosome stalling in leader mRNA by cis interference with translation . Synthetic leader peptides inhibit ribosomal peptidyltransferase in vitro, leading to the prediction that this activity is the basis for stall site selection . Recent studies have shown that the leader peptides are rRNA-binding peptides with targets at the peptidyl transferase center of 23S rRNA . uORFs associated with several eukaryotic genes inhibit downstream translation . When inhibition depends on the specific codon sequence of the uORF, it has been proposed that the uORF-encoded nascent peptide prevents ribosome release from the mRNA at the uORF stop codon . This sets up a blockade to ribosome scanning which minimizes downstream translation . Segments within large proteins also appear to regulate ribosome activity in cis, although in most of the known examples the active amino acid sequences function after their emergence from the ribosome, cis control of translation by the nascent peptide is gene specific; nearly all such regulatory peptides exert no obvious trans effects in cells . The in vitro biochemical activities of the cat/cmla leader peptides on ribosomes and rRNA suggest a mechanism through which the nascent peptide can modify ribosome behavior . Other cis-acting regulatory peptides may involve more complex ribosomal interactions.

Curr Opin Biotechnol, 1996 Jun, 7(3), 331 - 6
Nucleic acids in the environment; Trevors JT; The past year has witnessed several excellent advances in basic and applied research on nucleic acids in the environment . Improved methods for extracting nucleic acids from environmental samples have been published, as well as information on the use of reporter genes in bacteria, natural genetic transformation in soil and DNA adsorption to soil . These advances will have a significant impact on both future research and the way in which we view nucleic acids in the environment.

Glycoconj J, 1996 Jun, 13(3), 453 - 60
Recognition of glycoconjugates by Helicobacter pylori: an apparently high-affinity binding of human polyglycosylceramides, a second sialic acid-based specificity; Miller-Podraza H et al.; Helicobacter pylori has been reported to agglutinate erythrocytes and to bind to various other cells in a sialic acid-dependent way . The binding was inhibited by sialyllactose or fetuin and other sialylated glycoproteins . The specificity apparently requires bacterial growth on agar, since we found that it was lost after growth in the nutrient mixture Ham's F12 . Instead, the bacteria bound with high affinity and in a sialic acid-dependent way to polyglycosylceramides of human erythrocytes, a still incompletely characterized group of complex glycolipids . Bacteria grown in F12 medium were metabolically labelled with 35S-methionine and analysed for binding to glycolipids on thin-layer chromatograms and to glycoproteins on blots after electrophoresis, with human erythrocyte glycoconjugates in focus . There was no binding to simpler gangliosides including GM3 or sialylparagloboside, or to a mixture of brain gangliosides . In contrast, polyglycosylceramides of human erythrocyte membranes bound at a pmol level . The activity was eliminated by mild acid treatment, mild periodate oxidation or sialidase hydrolysis . Erythrocyte proteins as well as a range of reference glycoproteins did not bind except band 3, which was weakly active . However, this activity was resistant to periodate oxidation . These results indicate a second and novel sialic acid-recognizing specificity which is expressed independently of the previously described specificity.

J Clin Gastroenterol, 1996 Jun, 22(4), 317 - 21
Brush cytology: a reliable method to detect Helicobacter pylori; Dalla Libera M et al.; This study was conducted to verify the reliability of brush cytology in detecting Helicobacter pylori in an unselected group of patients with duodenal ulcer (DU) and nonulcer dyspepsia (NUD) . Endoscopy was performed on 416 consecutive patients: group A, 94 with active DU; group B, 176 patients with DU after omeprazole (n = 78), ranitidine (n = 43), or triple anti-H . pylori therapy (n = 55); and group C, 146 patients with NUD . During endoscopy, the gastric mucosa was brushed and two biopsy samples from the antrum and body were obtained for histology . In 65 patients, culture of the brush-collected materials also was performed as was that from of biopsy samples . The overall frequency of H . pylori presence detected by brush cytology was significantly higher compared with that of histology (p < 0.001), particularly in group A (p < 0.05), group C (p < 0.05), and in patients with DU after omeprazole treatment (p < 0.01), but not in patients with DU after ranitidine or anti-H . pylori treatment . The overall frequency of H . pylori-positive cultures from the brush-collected material was higher compared with cultures from the biopsy samples (38.5% vs . 24.6%), particularly in the NUD group (32.6% vs . 16.3%) . Brush cytology is more sensitive than histology, besides being faster and cheaper, for the assessment of H . pylori infection, particularly when the density of the bacteria is low.

Br J Plast Surg, 1996 Jun, 49(4), 214 - 9
Secondary sternal repair following median sternotomy using interosseous absorbable sutures and pectoralis major myocutaneous advancement flaps; Perkins DJ et al.; A consecutive series of 19 patients were treated for median sternotomy dehiscence by secondary sternal closure with interosseous absorbable sutures and superimposed pectoralis major myocutaneous advancement flaps . These patients were selected for this treatment only on the basis of the quality and quantity of remaining bone stock after debridement . Using this technique there have been no failures of primary therapy with a zero 30-day mortality rate . All patients have achieved good functional and aesthetic results with mechanically stable sternums, wounds confined to the chest and elimination of sepsis . This technique has the advantages of being simple, safe and relatively quick and avoids many of the inherent complications and disadvantages of other techniques and flaps commonly used in the management of this complication.

Trends Biochem Sci, 1996 Jun, 21(6), 199 - 200
The puzzling origin of the genetic code; Cedergren R et al.; Recent results add to the mystery of the origin of the genetic code . In spite of early doubts, RNA can discriminate between hydrophobic amino acids under certain contexts . Moreover, codon reassignment, which has taken place in several organisms and mitochondria, is not a random process . Finally, phylogenies of some aminoacyl-tRNA synthetases suggest that the entire code was not completely assigned at the time of the divergence of bacteria from nucleated cells.

Genetics, 1996 Jun, 143(2), 961 - 72
Single and double infections with Wolbachia in the parasitic wasp Nasonia vitripennis: effects on compatibility; Perrot-Minnot MJ et al.; Wolbachia are cytoplasmically inherited bacteria responsible for reproductive incompatibility in a wide range of insects . There has been little exploration, however, of within species Wolbachia polymorphisms and their effects on compatibility . Here we show that some strains of the parasitic wasp Nasonia vitripennis are infected with two distinct bacterial strains (A and B) whereas others are singly infected (A or B) . Double and single infections are confirmed by both PCR amplification and Southern analysis of genomic DNA . Furthermore, it is shown that prolonged larval diapause (the overwintering stage of the wasp) of a double-infected strain can lead to stochastic loss of one or both bacterial strains . After diapause of a double-infected line, sublines were produced with AB, A only, B only or no Wolbachia . A and B sublines are bidirectionally incompatible, whereas males from AB lines are unidirectionally incompatible with females of A and B sublines . Results therefore show rapid development of bidirectional incompatibility within a species due to segregation of associated symbiotic bacteria.

Microbiology, 1996 Jun, 142 ( Pt 6), 1537 - 41
Collagen-binding activity of Prevotella intermedia measured by a microtitre plate adherence assay; Grenier D; The ability of Prevotella intermedia to bind type I collagen was investigated . A simple method in which bacterial cells were allowed to attach to collagen-coated microtitre plate wells was used to characterize the activity . All strains of P . intermedia tested, as well as those of the closely related species Prevotella nigrescens, showed a capacity to attach to the collagen film . Exponential-phase cultures of P . intermedia demonstrated a greater binding capacity than older cells . Attachment to the collagen film was inhibited by the presence of EDTA, type I and IV collagen, denatured collagen (gelatin), fibrinogen or fibronectin . Pretreatment of bacterial cells with heat (60 degrees C, 30 min) or proteinase K also inhibited the binding . The collagen-binding activity could be solubilized from the bacterial cell surface by incubation with Zwittergent 3-14, a zwitterionic detergent . The collagen-binding capacity of P . intermedia demonstrated in the present study represents a mechanism of colonization allowing these bacteria to attach to a tissue matrix.

Microbiology, 1996 Jun, 142 ( Pt 6), 1459 - 68
Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate in the isocitrate-lyase-negative methylotroph Methylobacterium extorquens AM1; Chistoserdova LV et al.; A region on the Methylobacterium extorquens AM1 chromosome previously shown to complement a chemically induced mutant (PCT48) unable to convert acetyl-CoA into glyoxylate was characterized in detail in order to identify the gene(s) involved in the unknown pathway for acetyl-CoA oxidation . Six complete and two partial ORFs were identified by sequencing . Sequence comparisons suggested these might code for, respectively, a dehydrogenase of unknown specificity, a polypeptide of at least 15 kDa with unknown function, a coenzyme-B12-linked mutase, a catalase, an alcohol dehydrogenase (ADH) of unknown function, a polypeptide of 28 kDa, a ketol-acid reductoisomerase and a propionyl-CoA carboxylase (PCC) . Insertion mutations were introduced into each ORF in order to determine their involvement in C1 and C2 metabolism . Mutations in three genes, encoding the mutase, ADH and PCC, resulted in a phenotype characteristic of mutants unable to oxidize acetyl-CoA, i.e . they were C1-and C2-negative and their growth on these compounds was restored by the addition of glycolate or glyoxylate . Mutants in the genes thought to encode catalase and PCC were found to be deficient in the corresponding enzyme activity, confirming the identity of these genes, while physiological substrates for the mutase and ADH remain unidentified . This work, in which three new genes necessary for conversion of acetyl-CoA into glyoxylate were identified, is an intermediary step on the way to the solution of the unknown pathway for acetyl-CoA oxidation in isocitrate-lyase-negative methylotrophs.

Microbiology, 1996 Jun, 142 ( Pt 6), 1345 - 55
A role for pabAB, a p-aminobenzoate synthase gene of Streptomyces venezuelae ISP5230, in chloramphenicol biosynthesis; Brown MP et al.; Mutagenesis of Streptomyces venezuelae ISP5230 and selection for P-aminobenzoic acid-dependent growth in the presence of sulfanilamide yielded pab mutants (VS519 and VS620) that continued to produce chloramphenicol (Cm), although with increased medium dependence . Transforming the mutants with pDQ102 or pDQ103, which carried a pab-complementing fragment from S . venezuelae ISP5230 in alternative orientations, restored uniformly high Cm production in VS620, but did not alter the medium dependence of Cm production in VS519 . The cloned S . venezuelae DNA fragment was subcloned and trimmed to the minimum size conferring pab complementation . The resulting 2.8 kb BamHl-Sacl fragment was sequenced . Codon preference analysis showed one complete ORF encoding a polypeptide of 670 amino acids . Comparison of the deduced amino acid sequence with database proteins indicated that the N- and C-terminal regions resembled PabA and PabB, respectively, of numerous bacteria . The gene product showed overall sequence similarity to the product of a fused pabAB gene associated with secondary metabolism in Streptomyces griseus . Insertion of an apramycin resistance gene into pabAB cloned in a segregationally unstable vector and replacement of the S . venezuelae chromosomal pabAB with the disrupted copy lowered sulfanilamide resistance from 25 to 5 micrograms mL-1 and blocked Cm production.

Risk Anal, 1996 Jun, 16(3), 367 - 76
The effects of exposure to "synthetic" chemicals on human health: a review; VanDoren PM; This article examines how scientists use human, animal, and bacterial evidence to develop policy recommendations about the health consequences of human exposure to modern chemicals . Human evidence is limited because many epidemiological studies are contaminated with selection effects or unobserved heterogeneity . Changes in the aggregate incidence of morbidity (such as cancer) in the population over time are not a substitute for the lack of good individual-level data because incidence data are contaminated by the medicalization of cancer . Animal tests are also problematic because the expense of conducting experiments leads researchers to use only enough animals to allow detection of large differences in cancer incidence between controls and experimental animals that can only arise if the exposure doses are large . Predictions about the cancer incidence that would result in humans at much lower exposure levels, thus, require statistical inferences that implicitly make choices between false positive and false negative inference errors . Policy recommendations about carcinogens, therefore, are as much the product of value choices as "scientific" knowledge.

Infect Immun, 1996 Jun, 64(6), 2339 - 42
Binding of hemoglobin to the envelope of Porphyromonas gingivalis and isolation of the hemoglobin-binding protein; Fujimura S et al.; The binding activity of the Porphyromonas gingivalis envelope and hemoglobin was examined over a wide range of pH values from 4.5 to 9.0 . The binding activity in low-pH buffers was much higher than that at high pH; the optimum pHs for the binding were found to be 4.5 and 5.0 . Since the hemoglobin bound to the envelope was found to dissociate in the pH 8.5 and 9.0 buffers, the binding is reversible . We hypothesized that hemoglobin-binding protein (HbBP), responsible for the binding to hemoglobin, exists in the envelope and confirmed its presence by dot blot determination with peroxidase-conjugated hemoglobin . Then we attempted to isolate HbBP from the solubilized (by a detergent) materials of the envelope by affinity chromatography . The molecular mass of HbBP was 19 kDa, and the isoelectric point was 4.3.

Infect Immun, 1996 Jun, 64(6), 2188 - 92
Deletion of purE attenuates Brucella melitensis infection in mice; Crawford RM et al.; We previously showed that a purE mutant (delta purE201) of Brucella melitensis 16M is attenuated for growth in cultured human monocytes (E . S . Drazek, H . H . Houng, R . M . Crawford, T . L . Hadfield, D . L . Hoover, and R . L . Warren, Infect . Immun . 63:3297-3301, 1995) . To determine if this strain is attenuated in animals, we compared the growth of the delta purE201 mutant with that of strain 16M in BALB/c mice . The number of bacteria in the spleen and spleen weight peaked for both strains between 1 and 2 weeks postinfection (p.i.), though the number of delta purE201 cells was significantly less than the number of 16M cells recovered from the spleens of infected mice . During the next 6 weeks, delta purE201 was essentially eliminated from infected mice (three of five mice sterile; < 100 CFU in two of live mice at 8 weeks p.i.), whereas bacteria persisted at a high level in the spleens of 16M-infected mice (about 106 CFU per spleen) . The number of bacteria in the livers and lungs of mice infected with either strain paralleled those in the spleen . Mice infected with 16M had a strong inflammatory response, developing dramatic and prolonged splenomegaly (five to eight times normal spleen weight) and producing serum interleukin-6 . In contrast, mice infected with delta purE201 developed only mild, transient splenomegaly at 1 week p.i . and produced no interleukin-6 in their serum . We further characterized the host response to infection by measuring changes in immune spleen cell populations by flow cytometry . CD4- and CD8-positive lymphocytes declined by I week in both experimental groups, while MAC-1-positive cells increased . T-cell subpopulations remained low or declined further, and MAC-1 cells increased to three times normal levels during 8 weeks of infection with 16M but returned to normal by 4 weeks after infection with delta purE201 . These results document infectivity and attenuation of delta purE201 and suggest that it should be further evaluated as a potential vaccine.

Infect Immun, 1996 Jun, 64(6), 2094 - 100
Mixed infection with Porphyromonas gingivalis and Fusobacterium nucleatum in a murine lesion model: potential synergistic effects on virulence; Feuille F et al.; These studies determined the characteristics of tissue destruction in a murine abscess model elicited by mixed infection with the periodontopathogens Fusobacterium nucleatum and Porphyromonas gingivalis . The interbacterial effects of this synergism, the kinetics of the relationship of the bacterial interaction, and the characteristics of the bacteria required for the tissue destruction were studied . Infection of mice with P . gingivalis and F . nucleatum strains elicited lesions of various sizes as a function of infective dose . Primary infection with F . nucleatum plus P . gingivalis at various ratios (i.e., <1:1) resulted in a significantly greater lesion size (P < 0.001) compared with that resulting from primary infection with P . gingivalis alone . At F . nucleatum/P . gingivalis ratios of > or = 1:1, spreading lesion formation and progression were significantly (P < 0.001) decreased, suggesting that bacterial interaction (i.e., coaggregation) may have inhibited the spread of the P . gingivalis infection to a site distant from the initial injection . Infection with F . nucleatum and P . gingivalis simultaneously (at different sites) or F . nucleatum administered within 4 h prior to or 1 h following P . gingivalis infection significantly enhanced the ability of P . gingivalis to form large phlegmonous lesions . Chemical inhibition of the P . gingivalis trypsin-like protease activity or the use of a trypsin-negative P . gingivalis strain abrogated tissue destruction either alone or in combination with F . nucleatum . Therefore, it was possible to examine aspects of virulence of these pathogens in a murine lesion model by either altering bacterial ratios, manipulating the time of infection, or targeting vital bacterial virulence factors.

Infect Immun, 1996 Jun, 64(6), 1968 - 76
Elevated levels of Legionella pneumophila stress protein Hsp60 early in infection of human monocytes and L929 cells correlate with virulence; Fernandez RC et al.; Legionella pneumophila 2064 was selectively radiolabelled in mouse L929 cells and human monocytes to identify proteins expressed early in the course of infection . Polypeptide profiles (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography) of adherent or intracellular bacteria indicated that a 60-kDa stress protein (Hsp60) was preferentially synthesized . Hsp60 synthesis was not induced by medium alone . The synthesis of many polypeptides, including OmpS (major outer membrane protein), diminished over the 1-h period postinfection . However, by 17 h postinfection OmpS and Hsp60 were the dominant proteins synthesized by 2064 . To establish whether induction of Hsp60 was a correlate of virulence, an isogenic avirulent strain (2064M) of 2064 was isolated following selection on a nonpermissive medium . 2064M did not exhibit a stress response when adherent or intracellular in L929 cells or in human monocytes and failed to abrogate phagosome-lysosome fusion . When grown in vitro, 2064M exhibited no deficiencies in the heat shock response and its polypeptide profile resembled that of 2064 . Immunogold electron microscopy was used to localize Hsp60 in L . pneumophila-infected L929 cells . There was an increase in the number of gold particles associated with phagosomes for phagosomes harboring single 2064 bacteria compared with those harboring 2064M . Moreover, by 1 h postinfection, a sixfold increase in the number of gold spheres associated with the membranes of phagosomes was observed for phagosomes harboring 2064 compared with those harboring 2064M . These studies indicate that virulent, but not NaCl-tolerant avirulent, strains of L . pneumophila respond to host-cell-associated environmental signals early in the course of infection . This response includes increased synthesis and possibly extracellular secretion of Hsp60 concomitant with repression of the expression of other genes, including ompS.

Gene, 1996 Jun 1, 171(2), 303 - 4
Sequences of the lizard cDNAs encoding lactate dehydrogenase (LDH) isozymes A (muscle) and B (heart); Mannen H et al.; The nucleotide and deduced amino acid sequences of cDNAs encoding L-lactate dehydrogenase (LDH) isozymes A (muscle) and B (heart) from the lizard, Sceloporus undulatus, were determined . The evolutionary relationships among LDH isozymes from animals, plants and bacteria are presented.

J Bacteriol, 1996 Jun, 178(12), 3572 - 7
Evidence that the hanA gene coding for HU protein is essential for heterocyst differentiation in, and cyanophage A-4(L) sensitivity of, Anabaena sp . strain PCC 7120; Khudyakov I et al.; The highly pleiotropic, transposon-generated mutant AB22 of Anabaena sp . strain PCC 7120 exhibits slow growth, altered pigmentation, cellular fragility, resistance to phage A-4(L), and the inability to differentiate heterocysts . Reconstruction of the transposon mutation in the wild-type strain reproduced the phenotype of the original mutant . Sequencing of the flanking DNA showed that the transposon had inserted at the beginning of a gene, which we call hanA, that encodes Anabaena HU protein (R . Nagaraja and R . Haselkorn, Biochimie 76:1082-1089, 1994) . Mapping of the transposon insertion by pulsed-field gel electrophoresis showed that hanA is located at ca . 4.76 Mb on the physical map of the chromosome and is transcribed clockwise . Repeated subculturing of AB22 resulted in improved growth and loss of filament fragmentation, presumably because of one or more compensatory mutations; however, the mutant retained its A-4(L)r Het- phenotype . The mutation in strain AB22 could be complemented by a fragment of wild-type DNA bearing hanA as its only open reading frame.

J Bacteriol, 1996 Jun, 178(11), 3369 - 73
Sequence of the bchG gene from Chloroflexus aurantiacus: relationship between chlorophyll synthase and other polyprenyltransferases; Lopez JC et al.; The sequence of the Chloroflexus aurantiacus open reading frame thought to be the C . aurantiacus homolog of the Rhodobacter capsulatus bchG gene is reported . The BchG gene product catalyzes esterification of bacteriochlorophyllide a by geranylgeraniol-PPi during bacteriochlorophyll a biosynthesis . Homologs from Arabidopsis thaliana, Synechocystis sp . strain PCC6803, and C . aurantiacus were identified in database searches . Profile analysis identified three related polyprenyltransferase enzymes which attach an aliphatic alcohol PPi to an aromatic substrate . This suggests a broader relationship between chlorophyll synthases and other polyprenyltransferases.

J Bacteriol, 1996 Jun, 178(11), 3246 - 51
Tet(M)-promoted release of tetracycline from ribosomes is GTP dependent; Burdett V; Tet(M) protein, which displays homology to elongation factor G (EF-G), interacts with the protein biosynthetic machinery to render this process resistant to tetracycline in vivo and in vitro . To clarify the basis of the resistance mechanism, the effects of Tet(M) on several reactions which occur during protein synthesis were examined . The mechanism of action of Tet(M) has been clarified by two observations . The protein relieves tetracycline inhibition of factor-dependent tRNA binding and dramatically reduces the affinity of ribosomes for tetracycline when GTP is present . This reduction in drug affinity appears to be due to a large increase in the rate of tetracycline dissociation . Addition of Tet(M) to ribosome-tetracycline complexes results in displacement of bound drug . And, while Tet(M) and EF-G GTPase activities are tetracycline resistant, the two proteins differ in their sensitivities to fusidic acid, with the latter activity inhibited by the drug . Furthermore, while Tet(M) protects translation from tetracycline inhibition in a defined system, it is unable to substitute for either EF-G or elongation factor Tu.

J Bacteriol, 1996 Jun, 178(11), 3232 - 7
Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile; Erauso G et al.; The plasmid pGT5 (3,444 bp) from the hyperthermophilic archaeon Pyrococcus abyssi GE5 has been completely sequenced . Two major open reading frames with a good coding probability are located on the same strand and cover 85% of the total sequence . The larger open reading frame encodes a putative polypeptide which exhibits sequence similarity with Rep proteins of plasmids using the rolling-circle mechanism for replication . Upstream of this open reading frame, we have detected an 11-bp motif identical to the double-stranded origin of several bacterial plasmids that replicate via the rolling-circle mechanism . A putative single-stranded origin exhibits similarities both to bacterial primosome-dependent single-stranded initiation sites and to bacterial primase (dnaG) start sites . A single-stranded form of pGT5 corresponding to the plus strand was detected in cells of P . abyssi . These data indicate that pGT5 replicates via the rolling-circle mechanism and suggest that members of the domain Archaea contain homologs of several bacterial proteins involved in chromosomal DNA replication . Phylogenetic analysis of Rep proteins from rolling-circle replicons suggest that diverse families diverged before the separation of the domains Archaea, Bacteria, and Eucarya.

Surgery, 1996 Jun, 119(6), 657 - 63
Effects of granulocyte colony-stimulating factor in severe pancreatitis; Rao R et al.; BACKGROUND . The effect of granulocyte colony-stimulating factor (G-CSF) on the rate of secondary infections in acute pancreatitis was evaluated in a canine model . Infectious complications are the major determinant of morbidity and mortality in severe pancreatitis . Bacterial translocation has been shown to be a cause of these secondary infections . The relative immunosuppression found with pancreatitis may promote translocation and the spread of bacteria to the pancreas . METHODS . Thirty-four mongrel dogs were studied . Pancreatitis was induced in 18 dogs; 9 were treated with 100 micrograms G-CSF/day and 9 were given only saline solution . Laparotomy alone was done in 16 dogs of which one half were given 100 micrograms G-CSF/day and one half were given saline solution . Daily blood counts and cultures were obtained . All dogs were killed on day 7, and the mesenteric lymph nodes, pancreas, liver, spleen, and peritoneal fluid were cultured and studied histologically . RESULTS . G-CSF caused a significant and sustained increase in mature granulocytes in dogs given pancreatitis . No difference was found in the rate of translocation to mesenteric lymph nodes in dogs given G-CSF (n = 4) versus dogs given saline solution (n = 6) . However, a significant decrease occurred in the spread of bacteria to distant sites in dogs given G-CSF (1 versus 15, p < 0.05) . CONCLUSIONS . Although G-CSF does not decrease the rate of translocation, it does decrease the rate of distant infection in severe acute pancreatitis.

Proc Soc Exp Biol Med, 1996 Jun, 212(2), 99 - 109
Superantigens: structure and relevance to human disease; Johnson HM et al.; Superantigens are a class of immunostimulatory molecules produced by bacteria and viruses . Their potent immune effects are due to their unique ability to bind to the major histocompatibility complex (MHC) outside the antigen-binding cleft and to stimulate T cells in a T-cell receptor (TCR) Vbeta-specific manner . Structural studies have revealed the binding sites involved in the MHC/superantigen/TCR complex . The bacterial superantigens are responsible for a number of syndromes, including food poisoning and toxic shock syndrome, but their effects may be not only acute but also chronic and complex . Recent evidence suggests that superantigens may be relevant to the pathogenesis of autoimmune and immunodeficiency disorders . To illustrate the detrimental effects of superantigens on disease outcome, evidence demonstrating the modulation of experimental allergic encephalomyelitis, an animal model for multiple sclerosis, by superantigen, as well as the potential role of superantigens in HIV pathogenesis of AIDS, will be presented . The information presented may provide valuable insight into the role of superantigens in autoimmunity and HIV infection.

J Virol, 1996 Jun, 70(6), 3478 - 87
Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA; Ruggli N et al.; The complete nucleotide sequence of the genome of classical swine fever virus (CSFV) strain Alfort/187 was determined from three cDNA libraries constructed by cloning of DNA fragments obtained from independent sets of reverse transcription and PCR . The cDNA fragments were then assembled and inserted downstream of a T7 promoter in a P15A-derived plasmid vector to obtain the full-length cDNA clone pA187-1 . The first nucleotide of the CSFV genome was positioned at the transcription start site of the T7 promoter . Cleavage at an SrfI restriction site introduced at the exact 3' end of the cloned viral cDNA allowed the in vitro synthesis of full-length viral RNA by runoff transcription . This RNA proved to be infectious after transfection into porcine kidney cells . Infectivity was not increased after capping of the synthetic RNA . Virus recovered from transfected cells was titrated in porcine kidney cells by endpoint dilution using indirect immunofluorescence and a CSFV-specific monoclonal antibody . RNA transcripts generated from plasmid DNA isolated from bacteria which had been cultured and cloned 10 times remained infectious, indicating that the full-length clone is stable in bacterial cells . A silent point mutation introduced at position 11842 of the genome was retained in the recombinant virus recovered from transfected cells . An infectious chimeric construct was obtained by replacing a 696-bp fragment in pA187-1 with the corresponding cDNA fragment from the CSFV strain CAP . The stably cloned full-length CSFV cDNA allows site-specific mutagenesis of the viral genome and thus will be useful for detailed molecular characterization of the virus as well as for studies of viral pathogenesis.

Blood, 1996 Jun 1, 87(11), 4607 - 17
The proto-oncogene HLF and the related basic leucine zipper protein TEF display highly similar DNA-binding and transcriptional regulatory properties; Hunger SP et al.; Genes encoding transcription factors are frequently altered by chromosomal translocations in acute lymphoblastic leukemia (ALL), suggesting that aberrant transcriptional regulation plays a prominent role in leukemogenesis . E2A-hepatic leukemia factor (HLF), a chimeric transcription factor created by the t(17;19), consists of the amino terminal portion of E2A proteins, including two experimentally defined transcriptional activation domains (TADs), fused to the HLF DNA binding and protein dimerization basic leucine zipper (bZIP) domain . To understand the mechanisms by which E2A-HLF induces leukemia and the crucial functions contributed by each constituent of the chimera, it is essential to define the normal transcriptional regulatory properties of HLF and related bZIP proteins . To address these questions, we cloned the human homologue of TEF/VBP, a bZIP protein closely related to HLF . Using a binding site selection assay, we found that TEF bound preferentially to the consensus sequence 5'-GTTACGTAAT-3', which is identical to the previously determined HLF recognition site . TEF and HLF activated transcription of consensus site-containing reporter genes in several different cell types with similar potencies . Using GAL4 chimeric proteins, a TAD was mapped to a discrete approximate 40 amino acid region of TEF and HLF within which they share 72% amino acid identity and 85% similarity . The TEF/HLF activation domain (THAD) has a predicted helical secondary structure, but shares no sequence homology with previously reported TADs . The THAD contained most, if not all, of the transcriptional activation properties present in both TEF and HLF and its deletion completely abrogated transcriptional activity of TEF and HLF in both mammalian cells and yeast . Thus, TEF and HLF share indistinguishable DNA-binding and transcriptional regulatory properties, whose alteration in leukemia may be pathogenetically important.

J Am Coll Cardiol, 1996 Jun, 27(7), 1555 - 61
Increased incidence of Chlamydia species within the coronary arteries of patients with symptomatic atherosclerotic versus other forms of cardiovascular disease; Muhlestein JB et al.; OBJECTIVES: The objectives of this study were to test prospectively for an association between Chlamydia and atherosclerosis by comparing the incidence of the pathogen found within atherosclerotic plaques in patients undergoing directional coronary atherectomy with a variety of control specimens and comparing the clinical features between the groups . BACKGROUND: Previous work has suggested an association between Chlamydia pneumoniae infection and coronary atherosclerosis, based on the demonstration of increased serologic titers and the detection of bacteria within atherosclerotic tissue, but this association has not yet been regarded as established . METHODS: Coronary specimens from 90 symptomatic patients undergoing coronary atherectomy were tested for the presence of Chlamydia species using direct immunofluorescence . Control specimens from 24 subjects without atherosclerosis (12 normal coronary specimens and 12 coronary specimens from cardiac transplant recipients with subsequent transplant-induced coronary disease) were also examined . RESULTS: Coronary atherectomy specimens were definitely positive in 66 (73%) and equivocally positive in 5 (6%), resulting in 79% of specimens showing evidence for the presence of Chlamydia species within the atherosclerotic tissue . In contrast, only 1 (4%) of 24 nonatherosclerotic coronary specimens showed any evidence of Chlamydia . The statistical significance of this difference is a p value < 0.001 . Transmission electron microscopy was used to confirm the presence of appropriate organisms in three of five positive specimens . No clinical factors except the presence of a primary nonrestenotic lesion (odds ratio 3.0, p = 0.057) predicted the presence of Chlamydia . CONCLUSIONS: This high incidence of Chlamydia only in coronary arteries diseased by atherosclerosis suggests an etiologic role for Chlamydia infection in the development of coronary atherosclerosis that should be further studied.

J Mol Biol, 1996 May 31, 259(1), 58 - 68
Evidence that a kissing loop structure facilitates genomic RNA dimerisation in HIV-1; Haddrick M et al.; Genomic RNA isolated from retroviral particles is a dimer composed of two identical strands . A region called the dimer linkage signal close to the 5' end of the RNA may be involved in forming the dimer . Several models for the formation of the HIV-1 RNA dimer have been proposed . In the kissing loop model, dimerisation results from base-pairing between homologous sequences in an RNA stem-loop . In the guanine tetrad model interstrand guanine contacts from the dimer . We have made mutations preventing the dimerisation of subgenomic RNAs in vitro by these mechanisms . To prevent the kissing loop dimer forming we changed the complementary loop sequence from 711GCGCGC716 to 711AAACGC716 . To prevent the guanine tetrad dimer forming we changed G819 to U . These mutations were introduced into a clone of HIV-1NL4-3 separately and collectively . All three clones produced infectious virions . Dimeric RNA with similar thermal stabilities was isolated from viruses containing either the single or the double mutations . The results suggest that sequences involved in forming a guanine tetrad are not important for HIV-1 RNA dimerisation . In contrast sequences involved in forming a kissing loop complex are not absolutely required, but are important in forming a stable HIV-1 RNA dimer.

Biochemistry, 1996 May 28, 35(21), 6771 - 6
Role of the prodomain in folding and secretion of rat pancreatic carboxypeptidase A1; Phillips MA et al.; Pancreatic carboxypeptidase A1 (CPA1) is synthesized as an inactive precursor, proCPA1, which is processed to the active enzyme by the proteolytic removal of the 95-amino acid N-terminal prodomain . Purified rat proCPA1 is renatured in vitro after denaturation in guanidine or in guanidine plus reducing agents . In contrast, purified CPA1 is not renatured under any of the conditions tested . While proCPA1 is secreted in yeast when fused to the alpha-factor signal sequence in place of its endogenous signal sequence, mature CPA1 is not secreted and is trapped and degraded intracellularly . Thus, in addition to maintaining CPA1 in the inactive state, the prodomain promotes folding and secretion of the proenzyme . Neither of these functions can be restored by supplying the prodomain to CPA1 in trans . The three-dimensional structure of porcine proCPA reveals a number of extensive contacts made between the prodomain and the enzyme active site which account for its inhibitory properties {Guasch et al . (1992) J . Mol . Biol . 224, 141-157} . Among these contacts are salt bridges formed between Arg-71 and Asp-A36 and between Arg-124 and Asp-A89 . Mutation of any of these four residues inhibits secretion of proCPA1 from yeast and results in its intracellular degradation . The effect of the mutations on secretion suggests that interactions which stabilize the binding of prodomain to the native enzyme active site may also be important for the successful folding of proCPA1.

J Immunol Methods, 1996 May 27, 191(2), 149 - 57
A putative enzyme from various secretions specifically inhibits antibody-antigen interactions; Poethke R et al.; Various human secretions (intestinal secretion, saliva, nasal mucus, lacrimal fluid) have been found to inhibit the binding of antibodies to their antigens . Various characteristics (e.g . time, pH, temperature dependence, affinity and size exclusion chromatography) suggested that the inhibitory activity was attributable to an enzyme . Further investigations revealed that this enzyme reacted with the Fab portion of immunoglobulin G, specifically with the heavy chain . It is assumed that it represents a novel immunoglobulin-specific protease since similar results were not obtained with proteolytic enzymes from human digestive organs e.g . pepsin, trypsin and chymotrypsin . Finally, investigating saliva it was demonstrated that the putative protease was not identical to enzymes from periodontal bacteria which are proteolytic for the Fc portion of immunoglobulins . The findings could be of general importance in the design of immunoassays which are to be applied to human (and possibly animal) secretions.

Structure, 1996 May 15, 4(5), 581 - 97
The crystal structure of the light-harvesting complex II (B800-850) from Rhodospirillum molischianum; Koepke J et al.; BACKGROUND: The light-harvesting complexes II (LH-2s) are integral membrane proteins that form ring-like structures, oligomers of alpha beta-heterodimers, in the photosynthetic membranes of purple bacteria . They contain a large number of chromophores organized optimally for light absorption and rapid light energy migration . Recently, the structure of the nonameric LH-2 of Rhodopseudomonas acidophila has been determined; we report here the crystal structure of the octameric LH-2 from Rhodospirillum molischianum . The unveiling of similarities and differences in the architecture of these proteins may provide valuable insight into the efficient energy transfer mechanisms of bacterial photosynthesis . RESULTS: The crystal structure of LH-2 from Rs . molischianum has been determined by molecular replacement at 2.4 A resolution using X-ray diffraction . The crystal structure displays two concentric cylinders of sixteen membrane-spanning helical subunits, containing two rings of bacteriochlorophyll-a (BChl-a) molecules . One ring comprises sixteen B850 BChl-as perpendicular to the membrane plane and the other eight B800 BChl-as that are nearly parallel to the membrane plane; eight membrane-spanning lycopenes (the major carotenoid in this complex) stretch out between the B800 and B850 BChl-as . The B800 BChl-as exhibit a different ligation from that of Rps . acidophila (aspartate is the Mg ligand as opposed to formyl-methionine in Rps . acidophila) . CONCLUSIONS: The light-harvesting complexes from different bacteria assume various ring sizes . In LH-2 of Rs . molischianum, the Qy transition dipole moments of neighbouring B850 and B800 BChl-as are nearly parallel to each other, that is, they are optimally aligned for Foster exciton transfer . Dexter energy transfer between these chlorophylls is also possible through interactions mediated by lycopenes and B850 BChl-a phytyl tails; the B800 BChl-a and one of the two B850 BChl-as associated with each heterodimeric unit are in van der Waals distance to a lycopene, such that singlet and triplet energy transfer between lycopene and the BChl-as can occur by the Dexter mechanism . The ring structure of the B850 BChl-as is optimal for light energy transfer in that it samples all spatial absorption and emission characteristics and places all oscillator strength into energetically low lying, thermally accessible exciton states.

EMBO J, 1996 May 15, 15(10), 2393 - 406
Cell cycle-controlled proteolysis of a flagellar motor protein that is asymmetrically distributed in the Caulobacter predivisional cell; Jenal U et al.; Flagellar biogenesis and release are developmental events tightly coupled to the cell cycle of Caulobacter crescentus . A single flagellum is assembled at the swarmer pole of the predivisional cell and is released later in the cell cycle . Here we show that the MS-ring monomer FliF, a central motor component that anchors the flagellum in the cell membrane, is synthesized only in the predivisional cell and is integrated into the membrane at the incipient swarmer cell pole, where it initiates flagellar assembly . FliF is proteolytically turned over during swarmer-to-stalked cell differentiation, coinciding with the loss of the flagellum, suggesting that its degradation is coupled to flagellar release . The membrane topology of FliF was determined and a region of the cytoplasmic C-terminal domain was shown to be required for the interaction with a component of the motor switch . The very C-terminal end of FliF contains a turnover determinant, required for the cell cycle-dependent degradation of the MS-ring . The cell cycle-dependent proteolysis of FliF and the targeting of FliF to the swarmer pole together contribute to the asymmetric localization of the MS-ring in the predivisional cell.

Cancer Res, 1996 May 15, 56(10), 2306 - 13
Oltipraz-mediated changes in aflatoxin B(1) biotransformation in rat liver: implications for human chemointervention; Buetler TM et al.; Oltipraz (OPZ) is currently being considered for human use to protect against aflatoxin B1 (AFB)-induced hepatocarcinogenesis based on its proven protective effect in rats . The effectiveness of this treatment presumes that orthologous cytochrome P450 and glutathione S-transferase (GST) isozymes metabolize AFB in humans as they do in rats . In this study, alterations in the expression of multiple forms of cytochrome P450 and GST were evaluated after treatment with OPZ, as well as other known P450 inducers, including 3-methylcholanthrene, pregnenolone-16alpha-carbonitrile, and ciprofibrate . Evidence is presented that the male-specific rat CYP 3A2, an orthologue of human CYP 3A4, may be primarily responsible for AFB activation in rat liver at both high and low AFB substrate concentrations . The CYP 1A2 enzyme does not appear to play a role in AFB activation in rat liver at any substrate concentration, whereas the major human P450 enzyme capable of activating AFB at a low substrate concentration has been identified as CYP 1A2 . Surprisingly, we found that the CYP 1A2 steady-state mRNA level and the CYP 1A2-associated methoxyresorufin-O-demethylase activity were induced approximately 3- and 2-fold, respectively, by OPZ in rat liver . However, because CYP 1A2 does not appear to participate in AFB activation, induction of CYP 1A2 may be insignificant for AFB-induced hepatocarcinogenesis in rat models . In the rat, a heterodimeric alpha class GST enzyme containing the Yc2 subunit is the only polypeptide characterized to date in this species with high catalytic activity for the conjugation of activated AFB with glutathione . The GST Yc2 steady-state mRNA level was induced 5-fold by OPZ treatment . This induction was mirrored by significant increases in both the corresponding protein level and AFB-8,9-epoxide-conjugating enzyme activity, which may contribute significantly to protection against AFB-induced carcinogenesis in the rat . Investigations from this and other laboratories have not revealed any evidence for a Yc2-like GST isozyme with high AFB-8,9-epoxide-conjugating activity in human liver . We have also been unable to demonstrate that the two major human alpha class GST isozymes, A1-1 and A2-2, purified from bacteria expressing the corresponding cDNAs, exhibit any significant AFB-8,9-epoxide-conjugating activity . Our results suggest that humans may not be protected to the same extent as rats against AFB-induced hepatocarcinogenesis by treatment with OPZ and that further investigations are needed to establish the usefulness of OPZ for protection against human exposure to AFB.

Carbohydr Res, 1996 May 14, 285, 69 - 79
Structural studies of the exocellular polysaccharide from Sphingomonas paucimobilis strain I-886; Falk C et al.; The exocellular polysaccharide from Sphingomonas paucimobilis strain I-886 has been studied using methylation analysis, Smith degradation, partial acid hydrolysis, NMR spectroscopy, and mass spectrometry as the principal methods . It is concluded that the repeating unit has the following structure: {formula: see text} The absolute configuration of the uronic acid was deduced from 1H NMR chemical shifts and is most likely D . Some preparations of the polysaccharide also contain phosphate and O-acyl groups which have not been identified or localised.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 4595 - 9
Trafficking of Plasmodium chabaudi adami-infected erythrocytes within the mouse spleen; Yadava A et al.; Plasmodium chabaudi adami causes a nonlethal infection in mice . We found that crisis, the time of rapidly dropping parasitemia, was abrogated by splenectomy, indicating the role of spleen in parasite killing . The factors that mediate spleen-dependent immunity are not known . An earlier study in Plasmodium berghei-infected rats showed an association between increased clearance of heat-treated erythrocytes and the onset of crisis {Wyler, D . J., Quinn, T . C . & Chen, L.-T . (1982) J . Clin . Invest . 67, 1400-1404} . To determine the potential effects of different vascular beds in parasite killing, we studied the distribution of parasitized erythrocytes and bacteria in the spleens of P . chabaudi adami-infected mice during precrisis (a period of rising parasitemia) and during crisis . After intravenous injection, bacteria were localized predominantly in the marginal zone . In contrast, parasitized erythrocytes were found in the red pulp . We also found that during precrisis, a time of no immunity, the uptake of radiolabeled infected erythrocytes by the spleen was increased, not decreased . These data imply that no change occurs in the flow of parasitized erythrocytes through the spleen during the transition to an immune state (crisis) . Our observations suggest that immune effector mechanisms, not circulatory changes, account for spleen-dependent parasite killing during a P . chabaudi adami infection in mice.

Biochemistry, 1996 May 14, 35(19), 6126 - 35
Structure and protein binding interactions of the primary donor of the Chloroflexus aurantiacus reaction center; Ivancich A et al.; Soret resonance, QX resonance, and QY near-infrared Fourier transform (FT) (pre)resonance Raman spectroscopies were used to determine pigment-protein interactions of specific bacteriochlorin molecules in the reaction center from Chloroflexus aurantiacus . FT Raman spectroscopy, using 1064 nm excitation, was used to selectively obtain preresonance and resonance vibrational Raman spectra of the primary donor (P) of reaction centers (RCs) from Chloroflexus aurantiacus in the Po and P.+ states, respectively . The FT Raman spectrum of RCs in their neutral P (Po) state exhibits bands at 1605, 1632, 1648, and 1696 cm-1 which are attributable to P in its resting neutral state . Specifically, the latter three Raman bands can be assigned to the conjugated C2 acetyl and C9 keto carbonyl groups of the bacteriochlorophyll (BChl) molecules constituting P . The observation of at least three such bands is indicative of a non-monomeric nature of P, consistent with the proposal that it is a dimer of BChl molecules . The 1632 cm-1 band is consistent only with a hydrogen bonded BChl acetyl carbonyl, while the 1648 cm-1 band is assigned to a non-hydrogen bonded acetyl carbonyl . The 1696 cm-1 band is consistent only with a non-hydrogen bonded keto carbonyl group; from the unusually high intensity of this latter band compared to the others, we propose that the 1696 cm-1 band contains contributions from two keto carbonyl groups, both free of hydrogen bonds . From published protein sequence alignments of the L and M subunits of Rhodobacter (Rb.) sphaeroides and Chloroflexus aurantiacus we assign the 1632 cm-1 band as arising from the C2 acetyl carbonyl of the analogous PM constituent of P, which is hydrogen bonded to tyrosine M187 in the Chloroflexus RC, and propose a pigment-protein structural model for the primary donor of Chloroflexus aurantiacus . The FT Raman spectrum of RCs in the P degrees+ state indicates that one component of the 1696 cm-1 band has upshifted 21 cm-1 to 1717 cm-1 . Compared to Rb . sphaeroides which showed a 26 cm-1 upshift for the corresponding band, the 21 cm-1 upshift indicates that the + charge is more delocalized over the P.+ species of Chloroflexus; we estimate that ca . 65% of the + charge is localized on one of the two BChl molecules of the Chloroflexus primary donor as compared to ca . 80% for Rb . sphaeroides . The consequences of the proposed structure of the Chloroflexus primary donor in terms of its Po/P.+ redox midpoint potential are discussed.

J Mol Biol, 1996 May 10, 258(3), 501 - 37
Conservation and variability in the structures of serine proteinases of the chymotrypsin family; Lesk AM et al.; The chymotrypsin-like serine proteinases are a widely divergent family of enzymes appearing in animals, bacteria and viruses . All comprise two homologous domains containing six-stranded beta-barrels, with the active site between the domains . What are the structural constraints to which these proteins have been subject during their evolution, and how have the molecules explored the limits these constraints impose? We have analysed the structures of 13 widely divergent serine proteinases determined by X-ray crystallography to high resolution and well refined . We have identified the regions of the molecule that evolution has preserved both within each of the two domains and in the interdomain interface . An alignment of the sequences is presented based on structural superpositions . From this we analyse the conserved structural patterns and the interactions crucial to maintaining the structure and the active side.

Gene, 1996 May 8, 170(2), 227 - 32
Cloning and characterization of several cDNAs for UDP-glucose pyrophosphorylase from barley (Hordeum vulgare) tissues; Eimert K et al.; Eleven cDNA clones encoding UDP-glucose pyrophosphorylase (UGPase) have been isolated from cDNA libraries prepared from seed embryo, seed endosperm and leaves of barley (Hordeum vulgare L.) . The sequences were identical, with the exception of positioning of the poly(A) tail; at least five clones with different polyadenylation sites were found . For a putative full-length cDNA {1775 nucleotides (nt) plus polyadenylation tail}, isolated from an embryo cDNA library, an open reading frame of 1419 nt encodes a protein of 473 amino acids (aa) of 51.6 kDa . An alignment of the derived aa sequence with other UGPases has revealed high identity to UGPases from eukaryotic tissues, but not from bacteria . Within the aa sequence, no homology was found to a UDP-glucose-binding motif that has been postulated for a family of glucosyl transferases . The derived aa sequence of UGPase contains three putative N-glycosylation sites and has a highly conserved positioning of five Lys residues, previously shown to be critical for catalysis and substrate binding of potato tuber UGPase . A possible role for N-glycosylation in the intracellular targeting of UGPase is discussed.

FEBS Lett, 1996 May 6, 385(3), 214 - 20
Ancient divergence of long and short isoforms of adenylate kinase: molecular evolution of the nucleoside monophosphate kinase family; Fukami-Kobayashi K et al.; Adenylate kinases (AK) from vertebrates are separated into three isoforms, AK1, AK2 and AK3, based on structure, subcellular localization and substrate specificity . AK1 is the short type with the amino acid sequence being 27 residues shorter than sequences of the long types, AK2 and AK3 . A phylogenetic tree prepared for the AK isozymes and other members of the nucleoside monophosphate (NMP) kinase family shows that the divergence of long and short types occurred first and then differentiation in subcellular localization or substrate specificity took place . The first step involved a drastic change in the three-dimensional structure of the LID domain . The second step was caused mainly by smaller changes in amino acid sequences.

FEBS Lett, 1996 May 6, 385(3), 193 - 6
Gene duplication and the evolution of photosynthetic reaction center proteins; Lockhart PJ et al.; We investigate the evolutionary relationships between photosynthetic reaction center proteins (D1, D2, L and M) and demonstrate that the pattern of nucleotide substitution in these is more complicated than has been assumed in previous phylogenetic analyses . We show that there are serious violations of methodological assumptions in previous published studies . We conclude that there is equal support for hypotheses indicating (i) a single gene duplication of an ancestral reaction center protein followed by diversification and (ii) two independent gene duplications giving rise to proteins in oxygenic and anoxygenic systems.

Yale J Biol Med, 1996 May-Jun, 69(3), 301 - 16
Acid, protons and Helicobacter pylori; Sachs G et al.; The anti-ulcer drugs that act as covalent inhibitors of the gastric acid pump are targeted to the gastric H+/K+ ATPase by virtue of accumulation in acid and conversion to the active sulfenamide . This results in extremely effective inhibition of acid secretion . Appropriate dosage is able to optimize acid control therapy for reflux and peptic ulcer disease as compared to H2 receptor antagonists . However, clinical data on recurrence show that Helicobacter pylori eradication should accompany treatment of the lesion . These drugs have been found to synergize with many antibiotics for eradication . The survival of aerobes depends on their ability to maintain a driving force for protons across their inner membrane, the sum of a pH and potential difference gradient, the protonmotive force (pmf) . The transmembrane flux of protons across the F1F0 ATPase, driven by the pmf, is coupled to the synthesis of ATP . The internal pH of H . pylori was measured using the fluorescent dye probe, BCECF, and the membrane potential defined by the uptake of the carbocyanine dye, DiSC3 {5} at different pHs to mimic the gastric environment . The protonmotive force at pH 7.0 was composed of a delta pH of 1.4 (-84mV) and a delta potential difference of -131mV, to give a pmf of -215 mV . The effect of variations in external pH on survival of the bacteria in the absence of urea correlated with the effect of external pH on the ability of the bacteria to maintain a pmf . The effect of the addition of 5 mM urea on the pmf was measured at different medium pH values . Urea restored the pmf at pH 3.0 or 3.5, but abolished the pmf at pH 7.0 or higher, due the production of the alkalinizing cation, NH3 . Hence H . pylori is an acid-tolerant neutrophile due to urease activity, but urease activity also limits its survival to an acidic environment . These data help explain the occupation of the stomach by the organism and its distribution between fundus and antrum . This distribution and its alteration by proton pump inhibitors also explains the synergism of proton pump inhibition and antibiotics such as amoxicillin and clarithromycin in H . pylori eradication.

Trends Genet, 1996 May, 12(5), 171 - 5
Control of messenger RNA stability in higher eukaryotes; Ross J; The mRNA decay rate (half-life) is a major determinant of mRNA abundance in organisms from bacteria to mammals . mRNA levels can fluctuate many-fold following a change in mRNA half-life, without any change in transcription, and these fluctuations affect how a cell grows, differentiates and responds to its environment . The half-lives of many mRNAs vary tenfold or more in response to cytokines, hormones, starvation, hypoxia, or viral infection . Three major questions regarding mRNA stability are currently being addressed . What sequences in mRNAs determine half-lives? What enzymes degrade mRNAs? What (trans-acting) factors regulate mRNA stability and how do they function? This review focuses on RNA-binding or regulatory proteins and on candidate messenger ribonucleases (mRNases).

Mol Chem Neuropathol, 1996 May-Aug, 28(1-3), 77 - 81
Immunogenetic studies in autism and related disorders; Warren RP et al.; The major histocompatibility complex comprises a number of genes that control the function and regulation of the immune system . One of these genes, the C4B gene, encodes a product that is involved in eliminating pathogens such as viruses and bacteria from the body . We previously reported that a deficient form of the C4B gene, termed the C4B null allele (no C4B protein produced) had an increased frequently in autism . In this study we attempted to confirm the increased incidence of the C4B null allele in autism and investigated the presence of a C4B null allele in two other childhood disorders, attention-deficit hyperactivity disorder and dyslexia (reading disability) . In addition, we explored the relationship of autism to the DR beta 1 gene, a gene located close to the C4B in autism . We confirmed the finding of an increased frequency of the C4B null allele in autism and found that the related disorders also had an increased frequency of this null allele . In addition, two alleles of the DR beta 1 gene also had significantly increased representation in the autistic subjects.

Ital J Gastroenterol, 1996 May, 28(4), 225 - 8
Acute gastrointestinal bleeding due to Meckel's diverticulum heterotopic gastric mucosa; Maieron R et al.; Meckel's diverticulum is the most common congenital anomaly of the gastrointestinal tract occurring in approximately 2% of the population . In our retrospective study, we analyzed 58 surgical specimens of Meckel's diverticulum operated on in our hospital . Heterotopic gastric mucosa was found in ten . Aim of this study was to establish the aetiopathogenesis of inflammation and consequent haemorrhage in Meckel's diverticulum with heterotopic gastric mucosa . Some studies showed that Helicobacter-like bacteria could play an important role in determining local phlogosis in heterotopic gastric mucosa of Meckel's diverticulum, however, none were found in our biopsy specimens . Analyzing patients with acute intestinal haemorrhage (4 out of 10 with heterotopic gastric mucosa) in Meckel's diverticulum a history of previous oral administration of NSAID's was positive in 3 of them . Although in the recent literature there were few case reports on the use of NSAID's and bleeding from Meckel's diverticulum, our results suggest that even short-term use, in small quantities, of NSAID's can play an important role in determining acute bleeding from Meckel's diverticulum with heterotopic gastric mucosa.

J Periodontal Res, 1996 May, 31(4), 278 - 84
Novel polysaccharide capsular serotypes in Porphyromonas gingivalis; Laine ML et al.; Recently van Winkelhoff et al . (1) described 3 novel serotypes in virulent Porphyromonas gingivalis strains, which were based on different polysaccharide antigens . These antigens probably represent capsular structures and have been designated K1, K2 and K3 . In the present study we report on 3 novel capsular serotypes, which are represented by P . gingivalis strains ATCC 49417, HG 1690 and HG 1691 . The strains, isolated from patients with periodontitis, showed obvious encapsulation in wet India ink preparations . Thermostable antigens could be detected in the supernatant fractions of autoclaved cells . These antigens appeared to be negatively charged, sensitive to periodate degradation, and resistant to proteinase K treatment . On the basis of these characteristics we conclude that the antigens are probably extra-cellular polysaccharides representing a bacterial capsular structure . These K-antigens did not cross-react with K1, K2 or K3 immune-sera of P . gingivalis, with the exception of the K2 antiserum, which partially recognized K5- and K6-antigens . In contrast, K5 and K6 antisera did not react with the K2-antigen . After absorbtion of the K2 antiserum with cells of strains HG 1690 (K5) and HG 1691 (K6) cross-reactivity was no longer present . We propose these novel serotypes to be designated: K4 (ATCC 49417), K5 (HG 1960) and K6 (HG 1691).

J Comp Pathol, 1996 May, 114(4), 419 - 37
The pathology of ovine paratuberculosis: gross and histological changes in the intestine and other tissues; Clarke CJ et al.; In sheep clinically affected with paratuberculosis, two distinct forms of microscopical pathology were recognized, related to a high or a low degree of mycobacterial colonization ("multibacillary" or "paucibacillary" presence) . These forms were characterized by different types of cellular infiltrate in the ileal mucosa and submucosa . Statistical analysis demonstrated strong correlations between the presence of large numbers of acid-fast organisms and macrophage infiltration, and between small numbers and lymphocyte infiltration . Correlations also existed between high numbers of acid-fast bacteria and a positive serum antibody test results; and between the presence of giant cells and lymphocytes in the gut . This study suggests that in ovine paratuberculosis the same clinical and gross pathological changes can result from different pathogenetic mechanisms.

Curr Biol, 1996 May 1, 6(5), 533 - 6
A difficult phase for introns-early . Molecular evolution; Hurst LD et al.; Close analysis of intron phase - the position of introns within codons - is claimed to provide novel evidence supporting the view that introns predate the divergence of bacteria and eukaryotes and, via 'exon shuffling', played a crucial role in protein evolution . But just how compelling is this evidence?

Eur J Gastroenterol Hepatol, 1996 May, 8(5), 421 - 3
Helicobacter pylori eradication with short-term therapy leads to duodenal ulcer healing without the need for continued acid suppression therapy; Goh KL et al.; OBJECTIVE: To determine whether duodenal ulcers continue to heal following successful Helicobacter pylori eradication with short-term eradication therapy without further acid suppression therapy . METHODS: Patients with endoscopically proven duodenal ulcers who were H . pylori positive were randomized to receive omeprazole 40 mg each morning and clarithromycin 500 mg three times daily or famotidine 40 mg twice daily and clarithromycin 500 mg three times daily for 2 weeks . No acid-suppressing agents nor ulcerhealing drugs such as bismuth compounds or sucralfate were prescribed after that . Patients were re-examined endoscopically at week 2 at the end of treatment, and at week 6, 4 weeks after the completion of treatment . RESULTS: Thirty of 44 (68.2%) patients from both treatment arms, in whom the bacteria were subsequently noted to have been eradicated, had healed ulcers at week 2; at Week 6, 42 of 44 (95.5%) were noted to have healed ulcers without further acid-suppressing or ulcer-healing treatment . CONCLUSION: Although a short-term acid-suppressing treatment is insufficient to heal ulcers, where an important putative factor such as H . pylori is eliminated, the ulcer healing process continues without further need for acid-suppressing or ulcer-healing agents.

Med Microbiol Immunol (Berl), 1996 May, 185(1), 31 - 7
High- and low-level cytokine induction in human peripheral blood mononuclear cells by different Borrelia burgdorferi strains; Schulze J et al.; In this report we have compared the ability of 14 Borrelia burgdorferi sensu lato isolates to stimulate monocytes . From these isolates, all three human pathogen genospecies were represented . To determine the stimulatory activity of the different strains, interleukin-1 beta (IL-1 beta) was measured in the supernatant of monocyte cultures . This was achieved with borrelial strains in a ratio of 10 bacteria to 1 monocyte . In the majority of strains the stimulation induced a release of about 8000 pg/ml IL-1 beta, whereas four strains (B31, 297, EB3, 1/B29) induced more than 18,000 pg/ml IL-1 beta . We could, therefore, define two groups: low-level inductors and high-level inductors for IL-1 beta . The strains in the defined groups could not be ascribed to one distinct genospecies or a biological source . Further experiments confirmed the same differential release for tumor necrosis factor-alpha, but not for IL-6 . Studies on IL-1 beta indicated that high- and low-level release of cytokine was due to differences in protein synthesis.

In Vivo, 1996 May-Jun, 10(3), 285 - 92
Liver tumorigenesis by Helicobacter hepaticus: considerations of mechanism; Canella KA et al.; A new animal model for the causation of liver tumors via a bacterial infection presented itself fortuitously in the form of a new species, Helicobacter hepaticus . This species of Helicobacter colonizes the hepatic bile canaliculi in susceptible strains of mice, resulting in hepatitis and hepatocellular and hepatocholangiolar adenomas and carcinomas . The mechanism by which this infection leads to cancer is unknown . Tests with Helicobacter hepaticus have revealed thus far that the bacteria do not secrete a mutagen which is capable of detection by the Ames Assay . Measurement of oxidatively damaged bases in the liver DNA of hepaticus infected mice have shown accumulation of 8-oxodeoxyguanosine with disease progression . Other promutagenic DNA lesions, 7-methylguanine and O6-methylguanine, indicative of nitrosation of endogenous amines by nitric oxide, were not detected . Analysis of carcinomas and adenomas taken from H . hepaticus infected A/JCr mice revealed no mutations in ras oncogenes or in exons 5-8 of the p53 gene . These preliminary results indicate that a non-genotoxic tumor promotion mechanism, possibly implemented by reactive oxygen species from the immune response, is more likely than a genotoxic mechanism.

Curr Opin Rheumatol, 1996 May, 8(3), 203 - 9
Potential infectious agents in the induction of arthritides; Krause A et al.; In the multifactorial etiology of rheumatic diseases, infectious agents are regarded as the major environmental factors that may cause inflammatory arthritides in genetically susceptible hosts . Two not mutually exclusive pathogenetic pathways are hypothesized to explain the initiation and perpetuation of chronic arthritides by infectious agents: persistent infection and induction of immunopathology . In this review we focus on the role of infections in the etiopathogenesis of rheumatoid arthritis . Retroviruses and enteropathogenic bacteria continue to be the most intensively discussed candidates as possible etiologic factors of rheumatoid arthritis . Although there is ample indirect evidence for the involvement of infections in the pathogenesis of autoimmune disease, direct proof is still missing . There may be no single infectious trigger for rheumatoid arthritis, but multiple infectious agents that share antigenic motifs . The "reverse immunology" approach addresses this issue and is discussed in our outlook on future research directions.

J Virol Methods, 1996 May, 59(1-2), 73 - 82
Detection and typing of subgroup F adenoviruses using the polymerase chain reaction; Tiemessen CT et al.; A DNA amplification test was developed for the sensitive detection of the diarrhoea-associated subgroup F adenoviruses in clinical specimens . The test was made highly specific for serotypes 40 and 41 by using a region of the genome (the long-fiber gene) which is not significantly homologous to other human adenoviruses, but which is highly conserved between Ad40 and Ad41 . A positive subgroup F adenovirus diagnosis was characterized by the presence of an amplification product of 152 base pairs, which could be digested into products of predictable length by restriction enzymes XbaI and SpeI . The viruses were typed as either Ad40 or Ad41 by digestion of the amplification product with a restriction enzyme which digested only Ad40 DNA . The specificity of the test was assessed using DNA from other adenoviruses, from human and simian cells, and from bacteria commonly found in the human intestine . There was a strong correlation between results of typing obtained with PCR and restriction enzyme typing of Ad40 and Ad41, and also positivity using subgroup F specific probes in dot blot hybridizations.

J Infect, 1996 May, 32(3), 223 - 5
The organisms reported to cause infective myocarditis and pericarditis in England and Wales; Fairley CK et al.; It is difficult to acquire an overall perspective of the range of organisms responsible for infective myocarditis or pericarditis, and their relative importance, as most studies have involved only case reports or case series of a single organism . This study analyses reports to the Communicable Disease Surveillance Centre, of the Public Health Laboratory Service . Reports where myocarditis or pericarditis was included as the main clinical features between 1990 and 1993 were studied . Between 1990 and 1993, 368 cases of myocarditis and/or pericarditis were reported to CDSC . Viruses were reported to cause 253 (69%) cases, bacteria were responsible for 49 (13%) cases, mycoplasma for 32 (9%) cases, chlamydia for 16 (4%) cases and Mycobacterium tuberculosis for nine (2%) cases . Infection with coxsackie B virus was most frequently associated with a mixed picture of myo/pericarditis, whereas influenzae virus was associated with pericarditis or myocarditis alone . This information will provide clinicians with details of the more likely pathogens responsible for these conditions.

J Clin Periodontol, 1996 May, 23(5), 471 - 6
Inhibitory effects of acid water prepared by an electrolysis apparatus on early plaque formation on specimens of dentine; Ito K et al.; The aim of this study was to compare the effects of acid water prepared by an electrolysis apparatus with placebo treatment on the ultrastructure of early plaque formed on dentine specimens attached to retainers in the oral cavity . Dentine specimens were taken from 12 healthy extracted human 3rd molars . 4 dentine specimens were placed in the both the right and left buccal flanges of retainers fabricated from self-setting acrylic resin . The retainers were placed on both maxillary buccal sites in 6 subjects . The test solution was acid water (AW) prepared by an electrolysis apparatus with a pH of 2.7 and an oxidation-reduction potential of more than 1100 mV . As a positive control, 0.2% chlorhexidine digluconate (CHX) solution was used and normal saline solution as a negative control . 4 specimens placed in the right and left retainers were randomly allocated to 4 treatments as follows: treatment A, washing with AW; treatment B, washing with CHX solution; treatment C, washing with normal saline; treatment D, no washing . Washing was carried out in a plastic beaker containing 30 ml of each solution for 30s 2X daily over a 7-day period . The specimens were then carefully removed from the retainers, the morphology and thickness of the plaque formed examined by SEM, and the developmental condition of the plaque analyzed statistically . The plaque on the specimens in treatments A and B consisted mainly of coccoid forms . Mature plaque formation with complex flora was seen on the specimens in treatments C and D . The mean thickness of the plaque deposits on the dentin specimens as measured on SEM photographs magnified 2000 times was 8.80 mm for treatment . A, while in treatment B it was 3.90 mm . Plaque thickness for treatment C was 24.97 mm, and for treatment D 25.67 mm . The thickness of plaque formed on the sectioned specimens was significantly less for treatments A and B than for treatments C and D . However, there was no statistically significant difference between treatments A and B, and between treatments C and D (p < 0.0001) . The results of this short-term study indicate that AW washing has almost the same potential for inhibition of plaque formation as CHX washing, and is more effective for inhibiting plaque formation than washing with sterile saline . It is therefore concluded that AW may be useful as an anti-plaque agent.

Environ Health Perspect, 1996 May, 104 Suppl 3, 633 - 7
Mechanisms of spontaneous human cancers; Venitt S; The causes of much of human cancer remain obscure . The fraction that is spontaneous is unknown and cannot be calculated until all known external causes have been accounted for . This is not a feasible proposition . However, there is substantial evidence that about 80% of human cancer could be avoided by eliminating tobacco consumption; by dietary changes; by reducing infection with certain viruses, bacteria, and parasitic worms; and, in white populations, by avoiding sunburn . Alcohol, occupational and medical carcinogens, and certain patterns of reproductive behavior also contribute to the cancer burden . Cancers that cannot be attributed to these causes, and for which no other causes can be found, could be considered spontaneous and to arise from endogenous processes . Epidemiological evidence suggests that spontaneous and induced cancers share the same mechanism . Cancer is a genetic disorder of somatic cells . An accumulation of mutant genes that control the cell cycle, maintain genomic stability, and mediate apoptosis is central to carcinogenesis . Spontaneous mutation may cause spontaneous cancer . Endogenous causes of mutation include depurination and depyrimidation of DNA; proofreading and mismatch errors during DNA replication; deamination of 5-methylcytosine to produce C to T base pair substitutions; and damage to DNA and its replication imposed by products of metabolism (notably oxidative damage caused by oxygen free radicals) . Deficiencies in cellular defense mechanisms may also provoke spontaneous mutation . These include defective DNA excision-repair; low levels of antioxidants, antioxidant enzymes, and nucleophiles that trap DNA-reactive electrophiles; and enzymes that conjugate nucleophiles with DNA-damaging electrophiles . Mechanisms underlying many of those cellular defenses are under genetic control . Thus, germ line mutations or polymorphisms of genes that govern them may also contribute to spontaneous cancer.

Int J Urol, 1996 May, 3(3), 207 - 11
In vitro degradation of oxalic acid by human feces; Ito H et al.; BACKGROUND: Oxalic acid-degrading bacteria have been isolated from human faces . Therefore, the possibility exists that oxalic acid in food is degraded in human intestine by such bacteria, and absorption and excretion of oxalic acid is reduced . It may be possible that patients who form idiopathic stones have fewer oxalic acid-degrading bacteria than healthy controls . The purpose of this study is to examine the possibility that oxalic acid in food is degraded in the human intestine . METHODS: Nineteen patients with calcium stones and 13 healthy subjects were included in the study . Samples of feces were diluted with Barber medium containing 1 g/L of oxalic acid dihydrate, and 1%, 0.1% and 0.01% suspensions were prepared . These solutions were incubated under anaerobic conditions at 37 degrees C for seven days . The degradation of oxalic acid was estimated by the decrease of oxalic acid in the medium . RESULTS: The feces of almost all persons with or without calculi degraded oxalic acid to some degree . Incomplete or no oxalic acid degradation was found in 15 of the 19 stone-forming patients and in five of the 13 stone-free controls . CONCLUSION: Large numbers of oxalic acid-degrading bacteria were observed less often in the feces of stone-formers than in the feces of stone-free controls.

New Horiz, 1996 May, 4(2), 224 - 34
Reperfusion injury; Ar'Rajab A et al.; Reperfusion injury, precipitated by lack of oxygen, is likely to play a major role in many clinical conditions, including shock, coronary artery occlusion disease, and solid organ transplantation . Certain tissues, such as the intestinal mucosa, may be especially susceptible because of the specific microvascular anatomy . Structural changes include not only swelling of the organelles but also the entire cell due to the entry of water and electrolytes . Lysosomal ruptures precede cell death . Other key substances which either participate in or are part of oxygen free radical formation in tissue injury are calcium ions, leukocytes, and bacteria . Leukocyte adhesion has been implicated as a critical step in vascular endothelium injury, leading to increased microvascular permeability and thrombosis . Induction of neutropenia or the administration of antileukocyte adhesion monoclonal antibodies, preventing typical injuries, implies a central role of the white blood cells in reperfusion injury . Specifically, oxygen free radical formation in the intestines may trigger or cause injury in other distant organs, e.g., the heart and lungs, and affect overall vascular function . So-called "bacterial translocation" from the intestines to the lymphatic vessels and the bloodstream is a more recently discovered phenomenon whose role is largely unknown . Ischemic preconditioning is still another concept, mainly tested in the canine heart, that has potential clinical applications . Reperfusion of ischemic tissue occurs with solid organ transplantation, often after considerable cold ischemia time . Protective mechanisms include oxygen free radical scavengers, i.e., allopurinol and superoxide dismutase . Other measures proven to be effective during the implantation are blood volume expansion with colloid solutions and/or electrolyte solutions, and the administration of a calcium antagonist . The mechanisms of these measures are likely related to improved renal microcirculation and relief of vasospasm.

Plant Mol Biol, 1996 May, 31(2), 323 - 35
Cloning, molecular and functional characterization of Arabidopsis thaliana allene oxide synthase (CYP 74), the first enzyme of the octadecanoid pathway to jasmonates; Laudert D et al.; Allene oxide synthase, an enzyme of the octadecanoid pathway to jasmonates, was cloned from Arabidopsis thaliana as a full-length cDNA encoding a polypeptide of 517 amino acids with a calculated molecular mass of 58705 Da . From the sequence, an N-terminal transit peptide of 21 amino acids resembling chloroplast transit peptides was deduced . Three out of four invariant amino acid residues of cytochrome P450 heme-binding domains are conserved and properly positioned in the enzyme coding region, including the heme-accepting cysteine (Cys-470) . Southern analysis indicated in A . thaliana only one allene oxide synthase gene to be present . While transcript levels were rapidly and transiently induced after wounding of the leaves, allene oxide synthase activity remained nearly constant at a low level of ca . 0.8 nkat per mg of protein . The cDNA encoding A . thaliana allene oxide synthase was highly expressed in bacteria giving rise to a polypeptide of the calculated molecular mass . The protein was enzymatically active, and verification of the reaction products by GC-MS showed that it was capable of utilizing not only 13-hydroperoxylinolenic acid (13-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid), but also 13-hydroperoxylinoleic acid (13-hydroperoxy-9(Z), 11(E)-octadecadienoic acid) as substrate . The data suggest parallel pathways to jasmonates from linolenic acid or linoleic acid in A . thaliana.

Ann Plast Surg, 1996 May, 36(5), 453 - 7
Transverse glabellar flap for obliteration/isolation of the nasofrontal duct from the anterior cranial base; Disa JJ et al.; Management of fractures involving the nasofrontal duct region of the frontal sinus has focused on preserving function when possible or obliterating the sinus and duct when fracture patterns potentiate ductal obstruction and possible transcranial seeding of bacteria . When frontal sinus preservation is in doubt, controversy surrounds the use of cranialization versus obliteration, and the method of obliteration . Perioperative and late postoperative infections are uncommon, but their occurrence jeopardizes an often complex reconstruction and can be life threatening . This paper describes the design and indications for a pedicled transverse glabellar muscle flap for obliteration of the nasofrontal duct, thereby isolating the anterior cranial base from the aerodigestive system . This vascularized muscle flap utilizes the corrugator supercilii and procerus muscles, which are introduced into the sinus via a small, surgically created window in the superomedial orbital wall without disturbing the central facial aesthetic contours . Six patients with comminuted fractures at the nasofrontal duct level associated with displaced posterior frontal sinus fractures have been treated with the transverse glabellar flap . Follow-up ranges from 8 to 30 months . There have been no early or late postoperative complications . The transverse glabellar flap is a reliable and versatile method of partitioning the upper aerodigestive tract from the anterior cranial base with vascularized tissue, thus minimizing the risk of infectious complications . The resulting donor site deformity is more acceptable than that seen with the traditional pedicled galeal frontalis flap.

Mol Microbiol, 1996 May, 20(3), 657 - 66
Isolation and characterization of insertional mutations in flagellin genes in the archaeon Methanococcus voltae; Jarrell KF et al.; Methanococcus voltae is a flagellated member of the Domain Archaea that has four flagellin genes arranged in two transcriptional units . One transcriptional unit encodes only flaA while the second is a multi-cistronic unit encoding three flagellin genes (flaB1, flaB2, and flaB3) as well as at least seven other open reading frames downstream . The polymerase chain reaction was used to amplify an internal fragment of the flaA gene which was subsequently cloned into an insertion vector developed for M . voltae . Transformation of protoplasts with this vector led to the isolation of mutant strains that had insertions in flaA or flaB2 . Mutant strains carrying insertions in flaA had flagelia that were similar to wild-type cells in both number and appearance when viewed using the electron microscope . In addition, some of these mutant strains had profiles identical to the wild type in immunoblots developed with antisera raised against the 31 kDa flagellin of M . voltae . All flaA mutant strains and the wild-type cells showed immuno-cross-reactive bands at 33 and 31 kDa (corresponding to purified flagellins) as well as at 18 kDa . Some flaA mutant strains also showed an immuno-cross-reactive band at 27 kDa which probably represents a truncated flagellin produced by the insertion vector . However, both types of flaA mutant strains were less motile than the wild type in semi-swarm plate experiments . The mutant strain with an insertion in flaB2 was non-flagellated when examined by electron microscopy and it was non-motile in semi-swarm plate experiments . It represents the first structural mutant strain isolated in a methanogen . This mutant strain lacked the 33, 31, and 18 kDa immuno-cross-reactive bands observed in the wild type and flaA mutant strains, and instead had a novel band at 20 kDa . This band may represent an unmodified flagellin which still has an attached leader peptide . If so, then one of the downstream genes in the multi-cistronic transcriptional unit may encode a leader peptidase for the flagellin system.

Mol Microbiol, 1996 May, 20(3), 581 - 92
The centromere-like parC locus of plasmid R1; Breuner A et al.; The parA partitioning system of plasmid R1 consists of three components: the cis-acting centromere-like parC locus, and two proteins, ParM and ParR . The parC locus contains two sets of five direct repeats (iterons) to which the ParR protein binds . The parA promoter is located in the core region between the two sets of iterons . Mini-R1 replicons carrying parC are stabilized by the simultaneous presence of ParM and ParR . The parC locus present on a co-resident plasmid leads to instability of the mini-R1 replicon (incompatibility) . Here we present a genetic analysis of the stability and incompatibility phenotypes associated with parC . We show that all 10 iterons are required for maximum stabilization and incompatibility . Replacement of the core promoter region between the repeats by a foreign promoter region did not reduce stabilization . Thus, the only structural components in parC seem to be the two sets of iterons . The parA promoter, P parA, is repressed by ParR . We show that all 10 iterons are required for full repression of the promoter . The activity of the promoter was influenced by sequences located outside the core region . An A-rich region located upstream of the -35 element of PparA was found to increase promoter activity . The region encoding the parA mRNA leader region also strongly influenced the expression level of PparA- lacZ fusions . We show that this high expression (hex) element is a transcriptional antiterminator that prevents Rho-dependent termination.

Med Hypotheses, 1996 May, 46(5), 459 - 62
Cancer and malignant resistance of cells as phenomena of adaptation to damaging factors; Monceviciute-Eringiene E; I propose the hypothesis that mechanisms of general biological persistent resistance to damaging factors are closely related to the development of tumour cells . This phenomenon is characteristic of bacterial variants whose resistance to antibiotics and other chemotherapeutic drugs appears through L-transformation . As somatic cells are exposed to carcinogens and develop into tumour cells, they also acquire resistance to the toxic effects of carcinogens through multistage malignant transformation . Many cancerous cells, which have acquired persistent resistance to chemotherapy drugs or irradiation, often reappear locally or in metastases after courses of treatment . Thus, these cells undergo a kind of repeated development of malignancy . After a certain remission period, they begin to multiply more intensively locally, and are more likely to spread by metastasis . All resistant cells have the following characteristics: simplified metabolism, genetic, biochemical and morphological properties; lower requirements from their nutrient medium; rapid growth; parasitic qualities; invasiveness . It is as if they regress into a more primitive mode of existence (atavism) to survive under unfavourable circumstances . Somatic cells, resistant to carcinogens and the cells which undergo progression to more malignant types under the influence of drugs become similar to unicellular organisms or to forms of the latter which are resistant to damaging factors . The more primitive the cells become, the better they survive . Thus, cancer is a special case of the general resistance of cells to damaging factors.

Bone Marrow Transplant, 1996 May, 17(5), 825 - 33
Search for the antigen-specificity of homogeneous IgG components (H-IgG) after allogeneic bone marrow transplantation; Gerritsen EJ et al.; After allogeneic BMT, transient homogeneous Ig components (H-Ig) can be detected in the sera of most graft recipients . So far, data on the antigen-specificity and therefore the function of these H-Ig are not available . Such information may be important for our understanding of the underlying mechanisms that are responsible for these excessive clonal B cell expansions, and it may help to delineate the functional antibody repertoire after BMT . In the present study, sera of 98 paediatric BM graft recipients were investigated for the potential presence of H-Ig of IgG isotype (H-IgG) with specificity towards a panel of antigens, including vaccine and herpes virus antigens, auto-antigens and allo-antigens . The vast majority of H-IgG in sera of BM graft recipients were unreactive when tested for this panel of antigens . However, in four cases, antigen-specificity of H-IgG to tetanus toxoid could be demonstrated after vaccination with that antigen . An explanation for the negative findings may be either that a restricted antibody production had been elicited by other non-tested antigens, eg substances of colonizing and translocating bacteria or of food antigens, or that the H-IgG components may have anti-idiotype or anti-'self' specificity.

J Clin Microbiol, 1996 May, 34(5), 1323 - 6
New drug susceptibility test for Mycobacterium tuberculosis using the hybridization protection assay; Miyamoto J et al.; We developed a novel method for early detection of drug-resistant strains of Mycobacterium tuberculosis by using the hybridization protection assay (HPA) . The number of viable bacteria during the incubation period correlated well with the number of relative light units measured by the HPA . In addition, the relative light unit values of susceptible strains on the first, third and fifth days of incubation were significantly different from those of resistant strains for both isoniazid and rifampin . Our results suggest that after isolation of the organism from clinical specimens, drug-resistant strains of M . tuberculosis are accurately detected by the HPA even after 1 day of incubation with the drug.

J Clin Microbiol, 1996 May, 34(5), 1083 - 5
Clinical evaluation of the Roche AMPLICOR PCR Mycobacterium tuberculosis test for detection of M . tuberculosis in respiratory specimens; Bergmann JS et al.; The reliability of the Roche AMPLICOR Mycobacterium tuberculosis test (AMPLICOR MTB) for the diagnosis of pulmonary tuberculosis was evaluated by testing 956 respiratory specimens from 502 patients and comparing results with results by culture and medical history . Of those 135 specimens that were culture positive for mycobacteria, 61 specimens from 31 patients grew M . tuberculosis . Fifty-two specimens were smear positive for acid-fast bacteria (AFB); M . tuberculosis was isolated from 41 of these specimens . On initial testing, the sensitivity and specificity of the AMPLICOR MTB assay, compared with culture, were 78.7 and 99.3%, respectively . After resolution of discrepancies (by review of medical history), the sensitivity, specificity, and positive and negative predictive values of the AMPLICOR MTB assay were 79.4, 99.6, 92.6, and 98.6%, respectively . Two specimens from two patients with no clinical evidence of tuberculosis were AMPLICOR MTB positive and culture positive for Mycobacterium avium complex . For AFB smear-positive specimens, the sensitivity, specificity, and positive and negative predictive values of AMPLICOR MTB were 97.6, 100, 100, and 90.9%, respectively . For AFB smear-negative specimens, the sensitivity, specificity, and positive and negative predictive values of AMPLICOR MTB were 40.0, 99.5, 69.2, and 98.7%, respectively . Our results support the use of AMPLICOR MTB for rapid diagnosis of tuberculosis in patients whose respiratory specimens are AFB smear positive . Further studies are needed to determine the most clinically relevant and cost-effective use of this assay with AFB smear-negative specimens.

Alcohol Clin Exp Res, 1996 May, 20(3), 551 - 5
Aldehyde dehydrogenases of the rat colon: comparison with other tissues of the alimentary tract and the liver; Koivisto T et al.; Intracolonic bacteria have previously been shown to produce substantial amounts of acetaldehyde during ethanol oxidation, and it has been suggested that this acetaldehyde might be associated with alcohol-related colonic disorders, as well as other alcohol-induced organ injuries . The capacity of colonic mucosa to remove this bacterial acetaldehyde by aldehyde dehydrogenase (ALDH) is, however, poorly known . We therefore measured ALDH activities and determined ALDH isoenzyme profiles from different subcellular fractions of rat colonic mucosa . For comparison, hepatic, gastric, and small intestinal samples were studied similarly . Alcohol dehydrogenase (ADH) activities were also measured from all of these tissues . Rat colonic mucosa was found to possess detectable amounts of ALDH activity with both micromolar and millimolar acetaldehyde concentrations and in all subcellular fractions . The ALDH activities of colonic mucosa were, however, generally low when compared with the liver and stomach, and they also tended to be lower than in small intestine . Mitochondrial low K(m) ALDH2 and cytosolic ALDH with low K(m) for acetaldehyde were expressed in the colonic mucosa, whereas some cytosolic high K(m) isoenzymes found in the small intestine and stomach were not detectable in colonic samples . Cytosolic ADH activity corresponded well to ALDH activity in different tissues: in colonic mucosa, it was approximately 6 times lower than in the liver and about one-half of gastric ADH activity . ALDH activity of the colonic mucosa should, thus, be sufficient for the removal of acetaldehyde produced by colonic mucosal ADH during ethanol oxidation . It may, however, be insufficient for the removal of the acetaldehyde produced by intracolonic bacteria . This may lead to the accumulation of acetaldehyde in the colon and colonic mucosa after ingestion of ethanol that might, at least after chronic heavy alcohol consumption, contribute to the development of alcohol-related colonic morbidity, diarrhea, and cancer.

J Periodontol, 1996 May, 67(5), 515 - 22
Detection of local and systemic cytokines in adult periodontitis; Prabhu A et al.; Periodontitis is a chronic inflammatory disease of the soft and hard supporting tissues of the teeth and is a major cause of tooth loss in adults . The local host response to periodontopathic bacteria results in the release of inflammatory mediators and cytokines . Since cytokines are indicative of effector functions, we compared the pattern of cytokine production in periodontal patients and healthy controls . Specifically, we investigated the simultaneous presence of cytokines produced by T helper (Th)1, Th 2, and inflammatory cells which could be involved in periodontitis . We also compared the expression of these cytokine mRNAs in healthy and diseased tissues . No significant differences were detected at the protein or mRNA levels of the cytokines in the systemic circulation of patients and controls . The surface markers CD16 and CD56 were expressed on significantly fewer peripheral mononuclear cells of patients when compared to controls . gamma delta + T cells were found in half of the diseased tissues, but in none of the healthy tissues of either patients or controls . Finally, significant differences were observed between healthy and inflamed gingival tissues in the cytokine mRNA profile . Expression of IL-6 and IFN-alpha mRNA was significantly higher in diseased tissues compared to healthy tissues in patients.

J Periodontol, 1996 May, 67(5), 472 - 7
pH changes observed in the inflamed gingival crevice modulate human polymorphonuclear leukocyte activation in vitro; Leblebicioglu B et al.; Previous studies have noted a positive correlation between gingival inflammation and crevicular pH, which reportedly varies from 6.5 to 8.5 . In the present study, we characterized the manner in which deviation from the "physiological" pH of blood (7.2) influences activation of chemotaxis, phagocytosis, superoxide generation, and degranulation by human polymorphonuclear leukocytes (PMNs) . Purified PMNs were suspended in HEPES-buffered balanced salts solutions adjusted to pH 6.7, 7.2, 7.7, or 8.2 . In a modified Boyden chamber, the chemotactic response to fMet-Leu-Phe was maximal at pH 7.2 . In comparison, chemotaxis was significantly depressed at pH 7.7 and pH 8.2 (P < 0.05), but was not significantly different at pH 6.7 . Activation of the respiratory burst by fMet-Leu-Phe was optimal at pH 7.2, but was significantly depressed at pH 6.7 and 8.2 (P < 0.05) . pH had little effect on N-acetyl-beta-glucosaminidase release from primary granules . However, lactoferrin release from the secondary granules of fMet-Leu-Phe-activated PMNs was significantly lower at pH 7.2 than at pH 6.7 or 8.2 (P < 0.05) . Moreover, phagocytosis of opsonized bacteria was significantly lower at pH 7.2 than at pH 7.7 . In addition to these effects on functional activation, extracellular pH influenced the magnitude of intracellular Ca2+ mobilization . Peak fMet-Leu-Phe-induced Ca2+ levels were significantly higher at pH 8.2 than at pH 7.2 (P < 0.01) . These findings suggest that the pH of the periodontal environment can selectively influence PMN activation, thereby altering the balance between bacteria and the host response.

Pediatr Infect Dis J, 1996 May, 15(5), 397 - 404
Trends in hospitalizations for diarrhea in United States children from 1979 through 1992: estimates of the morbidity associated with rotavirus; Jin S et al.; OBJECTIVES . To examine trends in the hospitalizations of children for diarrheal disease in the U.S . and to provide estimates for the burden of disease associated with rotavirus diarrhea . METHODS . Data for diarrheal hospitalizations among U.S . children ages 1 month through 4 years were compiled from the National Hospital Discharge Survey for the years 1979 through 1992 . Between 1979 and 1992, 12% of all hospitalizations of U.S . children 1 month through 4 years of age had an International Classification of Diseases code for diarrhea listed in one of the top three positions on the discharge diagnosis . RESULTS . The annual rate of diarrheal hospitalizations, 97 per 10 000 persons (average, 185 742 per year), did not change substantially during the 14-year study period and accounted annually for 724 394 inpatient days (3.9 days per hospitalization) . For most diarrheal hospitalizations (75.9%) no causative agent was specified in the National Hospital Discharge Survey records; of the remaining 24.8%, viruses were most commonly reported (19.3%), followed by bacteria (5.1%) and parasites (0.7%) . The proportion of hospitalizations associated with viral diarrheas rose from 13% to 27% during the 14-year study period, whereas the proportion of hospitalizations for noninfectious diarrhea declined from 79% to 60% . Every year the number of hospitalizations peaked from November through April, the "winter" months, among children ages 4 through 35 months; this peak began in the West during November and December and reached the Northeast by March . CONCLUSIONS . Diarrhea continues to be a common cause of hospitalization among children in the United States and the winter seasonality estimated to be caused in large part by rotavirus would be expected to decrease if rotavirus vaccines currently being developed were introduced . Our analysis of temporal trends in diarrheal hospitalizations provides a unique surrogate with which to estimate the disease burden associated with rotavirus diarrhea.

Antimicrob Agents Chemother, 1996 May, 40(5), 1121 - 5
In vitro effect of tinidazole and furazolidone on metronidazole-resistant Trichomonas vaginalis; Narcisi EM et al.; Trichomonas vaginalis is a common sexually transmitted protozoan parasite . Although often considered simply a nuisance infection, T . vaginalis has been implicated in premature rupture of placental membranes and increases in the risk of acquiring human immunodeficiency virus . Metronidazole, a 5-nitroimidazole, is currently the drug of choice to treat T . vaginalis infection . Because some patients have severe reactions to metronidazole and others are infected with metronidazole-resistant T . vaginalis, we were prompted to investigate alternative therapies . Tinidazole, another 5-nitroimidazole used in other countries to treat T . vaginalis infections, and furazolidone, a nitrofuran presently used to treat giardiasis and infections with some anaerobic enteric bacteria, were investigated for effectiveness against 9 metronidazole-susceptible and 12 metronidazole-resistant T . vaginalis patient isolates . The in vitro aerobic and anaerobic minimum lethal concentrations (MLC) and the time for drug efficacy were determined . Tinidazole killed the metronidazole-susceptible isolates at a low MLC but was effective against only 4 of the 12 metronidazole-resistant isolates . In contrast, furazolidone was effective at a low MLC for all isolates . When tinidazole was effective, it required > 6 h to kill trichomonads . However, furazolidone killed both metronidazole-susceptible and resistant trichomonads within 2 to 3 h of exposure . These data suggest that furazolidone may be a good candidate for treating metronidazole-resistant trichomoniasis and that further investigation of this drug is warranted.

Thorax, 1996 May, 51(5), 530 - 3
Growth of acid fast L forms from the blood of patients with sarcoidosis; Almenoff PL et al.; BACKGROUND: Acid fast cell wall deficient forms (CWDF) of bacteria have been grown from blood, bronchial washings, and ocular anterior chamber fluid from patients with sarcoidosis . A monoclonal antibody raised against Mycobacterium tuberculosis whole cell antigen (H37RV) was used to characterise further CWDF grown from the blood of patients with sarcoidosis . METHODS: Blood from 20 patients with active sarcoidosis and from 20 controls was cultured using methods favourable for the growth of CWDF . Isolates were further characterised by indirect fluorescent antibody analysis using a monoclonal antibody highly reactive with M tuberculosis . RESULTS: CWDF were grown from the blood of 19 of 20 subjects with sarcoidosis . All isolates stained positively with the monoclonal antibody and with a modified Kinyoun stain . No organisms were grown from the blood of controls . CONCLUSIONS: These data demonstrate that CWDF can be grown from the blood of nearly all patients with active sarcoidosis . The results confirm that the organisms are mycobacterial in origin and are similar, if not identical, to M tuberculosis . Their role in the pathogenesis of sarcoidosis is unknown.

J Nurse Midwifery, 1996 May-Jun, 41(3), 218 - 23
Current assessment of the use of intrauterine devices; Westhoff CL; The need for effective, long-term contraception remains a significant issue . Intrauterine devices (IUDs) have evolved to the point where currently available devices are comparable, in safety and efficacy, to oral contraceptives . Due to concerns regarding previous devices, available IUDs remain largely underused by midwives and all other providers of reproductive health care . Recent medical data refute many of the medical-legal misconceptions . Currently available IUDs are safe and effective and represent a suitable contraceptive alternative for the appropriate patientPIP: Modern use of the intrauterine device (IUD) dates back to the early 1900s, when gynecologists in Germany began using contraceptive stem pessaries which extended into the uterus . IUDs then continued to evolve throughout the century . Introduced for use in the 1960s and 1970s, the Dalkon Shield was promoted as suitable for use in nulliparous women . By 1973, however, cases of septic abortion were reported to be associated with its use . The design of the Dalkon Shield was found to facilitate the ascension of bacteria from the vagina into the uterine cavity, leading to the development of pelvic infection . Dalkon Shield sales were discontinued in 1974, but the removal of inserted devices was not called for until the early 1980s . Many women who developed pelvic infections while using a Dalkon Shield joined in a class-action lawsuit against the manufacturer . The resultant negative media attention, as well as increased trepidation over liability by clinicians, contributed significantly to a sharp decrease in the use of all IUDs . The author explains that modern IUDs are now comparable in safety and efficacy to oral contraceptives, and that recent medical data refute many of the medical-legal misconceptions . Available IUDs are still largely underused by midwives and all other providers of reproductive health care due to concerns regarding earlier devices . The mechanism of IUD action; efficacy; pelvic inflammatory disease (PID), infection, and ectopic pregnancy; and product liability and malpractice are discussed .

Bratisl Lek Listy, 1996 May, 97(5), 304 - 7
{Problems and perspectives of wider use of saliva for diagnostic purposes}; Dubayova K et al.; Saliva of individual salivary glands differs in appearance, density and particularly in the chemical composition . Generally, the composition of saliva is affected by the composition of blood plasma, salivary flow rate, hormonal activity, drug administration, smoking and other physiological and pathophysiological states of the organism . In spite of these facts, many of the components are permanently present in saliva (e.g . peptides, enzymes, hormones...) only their concentrations vary . In some special cases unusual constituents can be detected in the saliva as legal and illegal drugs, antibodies (HIV), and abnormal bacteria or viruses . When there is good correlation between the levels of constituents in saliva and blood plasma then the determination of the constituent level in saliva can be used for diagnostic and/or monitoring purposes . But the main advantage of saliva analysis resides in stress-free and harmless collection of saliva in comparison with blood withdrawing . However, the use of saliva for diagnostic purposes is still at its beginning . So far, only few such applications are known, but optimists believe that the saliva analysis has a very prospective chance to substitute or alternate the biochemical analysis of blood plasma due to the mentioned advantage and to attribute more information on the processes in the human body . (Fig . 1, Ref . 16.)

APMIS, 1996 May, 104(5), 389 - 94
Imprint cytology of cat scratch disease . A report of eight cases; Kojima M et al.; The cytologic features of cat scratch disease (CSD) from eight cases in imprint smears are presented . All patients were clinicopathologically diagnosed with CSD as follows: 1) a history of animal exposure was recorded 2 to 4 weeks before lymphadenopathy; 2) the disease occurred in the autumn and winter months; 3) a characteristic histopathology in the biopsied lymph node specimens was observed; and 4) Warthin-Starry silver stain-positive bacteria were detected in four of the seven cases examined . The characteristic cytologic finding was the presence of confluent epithelioid cells with nearby and central scattering of neutrophils against a background of polymorphic inflammatory cells . Furthermore, a varying number of medium-sized to large lymphoid cells with an appearance suggestive of monocytoid B lymphocytes (MBLs) were noted to be associated with the epithelioid cells . These cytologic findings closely paralleled the histologic patterns of epithelioid cell granulomas, with and without MBLs, which we have previously reported are probably associated with the disease.

Acta Cytol, 1996 May-Jun, 40(3), 461 - 4
Fine needle aspiration cytology of mycetoma; EL Hag IA et al.; OBJECTIVE: To describe fine needle aspiration cytology of mycetoma and determine its usefulness in diagnosis . STUDY DESIGN: The study group consisted of 14 patients with different types of mycetoma lesions, which were aspirated . Smears were reviewed without knowing the type of mycetoma, and the findings were compared with those observed in histologic sections . RESULTS: In mycetoma, the causative organisms have a distinct appearance on cytologic smears . They are surrounded and infiltrated by neutrophils in a background of polymorphous, inflammatory cells consisting of neutrophils, histiocytes, lymphocytes, plasma cells, macrophages and foreign body giant cells . This allows differentiation from artifacts and inflammatory lesions caused by other bacteria and fungi . The distinction between eumycetoma and actinomycetoma in fine needle aspiration cytology was found to be as accurate as is histopathology when the grains were present . CONCLUSION: These results demonstrate that mycetoma can be accurately diagnosed by fine needle aspiration cytology . The technique is simple, inexpensive, rapid and sensitive . It can be used in the routine diagnosis of mycetoma, in epidemiologic surveys and in material collection.

J Cell Biol, 1996 May, 133(4), 801 - 7
Oligomeric and subunit structure of the Helicobacter pylori vacuolating cytotoxin; Lupetti P et al.; Disease-associated strains of Helicobacter pylori produce a potent toxin that is believed to play a key role in peptic ulcer disease in man . In vitro the toxin causes severe vacuolar degeneration in target cells and has thus been termed VacA (for vacuolating cytotoxin A) . Cytotoxic activity is associated with a > 600-kD protein consisting of several copies of a 95-kD polypeptide that undergoes specific proteolytic cleavage after release from the bacteria to produce 37- and 58-kD fragments . Quick freeze, deep etch electron microscopy has revealed that the native cytotoxin is formed as regular oligomers with either six- or seven-fold radial symmetry . Within each monomer, two domains can clearly be distinguished, suggesting that the 37- and 58-kD fragments derive from proteolytic cleavage between discrete subunits of the monomer . Analysis of preparations of the toxin that had undergone extensive cleavage into the 37- and 58-kD subunits supports this interpretation and reveals that after cleavage the subunits remain associated in the oligomeric structure . The data suggest a structural similarity with AB-type toxins.

Microb Ecol, 1996 May, 31(3), 249 - 68
Protistan Bacterivory in an Oligomesotrophic Lake: Importance of Attached Ciliates and Flagellates
Carrias J, Amblard C, Bourdier G.
Seasonal and depth variations of the abundance, biomass, and bacterivory of protozoa (heterotrophic and mixotrophic flagellates and ciliates) were determined during thermal stratification in an oligomesotrophic lake (Lake Pavin, France) . Maximal densities of heterotrophic flagellates (1.9x10(3) cells ml-1) and ciliates (6.1 cells ml-1) were found in the metalimnion . Pigmented flagellates dominated the flagellate biomass in the euphotic zone . Community composition of ciliated protists varied greatly with depth, and both the abundance and biomass of ciliates was dominated by oligotrichs . Heterotrophic flagellates dominated grazing, accounting for 84% of total protistan bacterivory . Maximal grazing impact of heterotrophic flagellates was 18.9x10(6) bacteria 1(-1)h-1 . On average, 62% of nonpigmented flagellates were found to ingest particles . Ciliates and mixotrophic flagellates averaged 13% and 3% of protistan bacterivory, respectively . Attached protozoa (ciliates and flagellates) were found to colonize the diatom Asterionella formosa . Attached bacterivores had higher ingestion rates than free bacterivorous protozoa and may account for 66% of total protozoa bacterivory . Our results indicated that even in low numbers, epibiotic protozoa may have a major grazing impact on free bacteria.

Aust Vet J, 1996 May, 73(5), 164 - 9
Characterisation of haemolytic RTX toxins produced by Australian isolates of Actinobacillus pleuropneumoniae; Tarigan S et al.; The haemolytic RTX toxins of 27 isolates of Actinobacillus pleuropneumoniae, representing all serovars that have been isolated in Australia, were characterised . The quantity of protein secreted by these isolates into the media was not significantly different between serovars, but haemolytic activity was detected only in the unconcentrated supernatants from cultures of serovar 1 and 5 isolates . Haemolytic activity in supernatants of serovar 2, 3 and 7 isolates was detected only after the supernatants were concentrated . On Southern hybridisation blots, genomic DNA of serovar 1 and 5 isolates contained regions that were similar to the cloned structural genes for ApxI (apxIA) and for ApxII (apxIIA) . In contrast, genomic DNA of serovar 2, 3 and 7 isolates only contained regions similar to, if not identical with, the cloned apxIIA gene . The haemolytic activity of the culture supernatant depends on the type or composition of media and adaptability of the bacteria to in-vitro cultivation . Low passage cultures of A pleuropneumoniae, which were characterised by waxy colonies, produced significantly weaker haemolytic activity than A pleuropneumoniae after several passages in vitro.

Plant J, 1996 May, 9(5), 649 - 58
CLA1, a novel gene required for chloroplast development, is highly conserved in evolution; Mandel MA et al.; An albino mutant designated cla1-1 (for "cloroplastos alterados', or "altered chloroplasts') has been isolated from a T-DNA-generated library of Arabidopsis thaliana . In cla1-1 plants, chloroplast development is arrested at an early stage . cla1-1 plants behave like wild-type in their capacity to etiolate and produce anthocyanins indicating that the light signal transduction pathway seems to be unaffected . Genetic and molecular analyses show that the disruption of a single gene, CLA1, by the T-DNA insertion is responsible for the mutant phenotype . RNA expression patterns indicate that CLA1 is positively regulated by light and that it has different effects on the steady-state RNA levels of some nuclear- and chloroplast-encoded photosynthetic genes . Although the specific function of the CLA1 gene is still unknown, it encodes a novel protein conserved in evolution between photosynthetic bacteria and plants which is essential for chloroplast development in Arabidopsis.

Biochem J, 1996 May 1, 315 ( Pt 3), 845 - 9
Heat shock proteins and macrophage resistance to the toxic effects of nitric oxide; Hirvonen MR et al.; Nitric oxide (NO) functions as a pathophysiological mediator in mammalian tissues . Activated macrophages produce NO as a non-specific immune response directed against invading bacteria or micro-organisms . The same macrophages that initiate the production of NO also can be toxically affected by NO . Incubation of RAW 264.7 macrophages with lipopolysaccharide (LPS) and/or interferon-gamma (INF-gamma) induced the formation of NO by the activation of a cytokine-inducible NO synthase (NOS) . The viability of these macrophages was inversely correlated with the formation of nitrite, a final NO-oxidation product measurable in the incubation medium . The addition of an NOS inhibitor, NG-monomethyl-L-arginine, diminished NO formation and preserved cell viability in a dose- and time-dependent fashion . Treatment of macrophages with ten cycles of non-lethal doses of LPS and INF-gamma, each followed by subculturing of the surviving cells, resulted in cell resistance to the NO toxic insult induced by LPS and INF-gamma . These resistant macrophages showed a 2-fold increase in the expression of the constitutive heat shock protein (HSC 70) which is known to be involved in protecting cells against the action of various metabolic insults . Our results establish a link between cell resistance to the toxic effects of NO, and the expression of heat shock proteins in RAW 264.7 macrophages.

Carcinogenesis, 1996 May, 17(5), 1179 - 81
Kinds of mutations induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in the hprt gene of Chinese hamster ovary cells; Hyttinen JM et al.; The kinds of mutations induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in the protein coding region of the hprt gene of Chinese hamster ovary (CHO) cells were determined by direct sequencing of polymerase chain reaction (PCR)-amplified cDNA . Primary mutations were found in 15 of 19 of the mutants: 11 were G:C-->T:A transversions, two were A:T-->T:A transversions and two were deletions of single G:C base pairs (-1 frameshifts) . The remaining four mutants had large alterations in the cDNA that were explained by mRNA splicing errors . A group of control mutants had more diverse hprt cDNA alterations than MX-induced mutants . Transversions yielding an A:T base pair were the predominant type of MX-induced mutations, in agreement with previous findings in bacteria . This specificity may be explained by the 'A rule', that DNA polymerases preferentially insert adenine nucleotides opposite non-instructional lesions.

Can J Microbiol, 1996 May, 42(5), 467 - 78
The Azospirillum brasilense rpoN gene is involved in nitrogen fixation, nitrate assimilation, ammonium uptake, and flagellar biosynthesis; Milcamps A et al.; The rpoN (ntrA) gene (encoding sigma 54) of Azospirillum brasilense Sp7 was isolated by using conserved rpoN primers and the polymerase chain reaction, and its nucleotide sequence was determined . The deduced amino acid sequence of the RpoN protein was found to share a high degree of homology with other members of the sigma 54 family . Two additional open reading frames were found in the Azospirillum brasilense rpoN region, with significant similarity to equivalent regions surrounding the rpoN locus in other bacteria . An rpoN mutant of Azospirillum brasilense Sp7 was constructed by gene replacement and found to be defective in nitrogen fixation, nitrate assimilation, and ammonium uptake . Lack of ammonium uptake was also found in previously isolated Azospirillum brasilense ntrB and ntrC mutants, further supporting the role of the ntr system in this process . In addition, the rpoN mutant was found to be nonmotile, suggesting a role of RpoN in Azospirillum brasilense flagellar biosynthesis.

FEMS Microbiol Rev, 1996 May, 18(2-3), 237 - 48
The unique DNA topology and DNA topoisomerases of hyperthermophilic archaea; Forterre P et al.; Hyperthermophilic archaea exhibit a unique pattern of DNA topoisomerase activities . They have a peculiar enzyme, reverse gyrase, which introduces positive superturns into DNA at the expense of ATP . This enzyme has been found in all hyperthermophiles tested so far (including Bacteria) but never in mesophiles . Reverse gyrases are formed by the association of a helicase-like domain and a 5'-type 1 DNA topoisomerase . These two domains might be located on the same polypeptide . However, in the methanogenic archaeon Methanopyrus kandleri, the topoisomerase domain is divided between two subunits . Besides reverse gyrase, Archaea contain other type 1 DNA topoisomerases; in particular, M . kandleri harbors the only known procaryotic 3'-type 1 DNA topoisomerase (Topo V) . Hyperthermophilic archaea also exhibit specific type II DNA topoisomerases (Topo II), i.e . whereas mesophilic Bacteria have a Topo II that produces negative supercoiling (DNA gyrase), the Topo II from Sulfolobus and Pyrococcus lack gyrase activity and are the smallest enzymes of this type known so far . This peculiar pattern of DNA topoisomerases in hyperthermophilic archaea is paralleled by a unique DNA topology, i.e . whereas DNA isolated from Bacteria and Eucarya is negatively supercoiled, plasmidic DNA from hyperthermophilic archaea are from relaxed to positively supercoiled . The possible evolutionary implications of these findings are discussed in this review . We speculate that gyrase activity in mesophiles and reverse gyrase activity in hyperthermophiles might have originated in the course of procaryote evolution to balance the effect of temperature changes on DNA structure.

FEMS Microbiol Rev, 1996 May, 18(2-3), 173 - 88
On the origin of respiration: electron transport proteins from archaea to man; Schafer G et al.; All aerobic organisms use the exergonic reduction of molecular oxygen to water as primary source of metabolic energy . This reaction is catalyzed by membrane residing terminal heme/Cu-oxidases which belong to a superfamily of widely varying structural complexity between mitochondrial and bacterial members of this family . Over the last few years, considerable information from this and other laboratories accumulated also on archaeal respiratory chains and their terminal oxidases . In the following, the molecular and catalytic properties of the latter are discussed and compared to those from bacteria and eucarya under the aspect of their energy conserving capabilities and their phylogenetic relations . The Rieske iron-sulfur proteins being important functional constituents of energy transducing respiratory complexes are included in this study . A number of essential conclusions can be drawn . (1) Like bacteria, archaea can also contain split respiratory chains with parallel expression of separate terminal oxidases . (2) The functional core of all oxidases is the highly conserved topological motif of subunit I consisting of at least 12 membrane spanning helices with the 6 histidine residues of the heme/Cu-binding centers in identical locations . (3) Some archaeal oxidases are organized in unusual supercomplexes with other cytochromes and Rieske {2Fe2S} proteins . These complexes are likely to function as proton pumps, whereas on a structural basis several subunit I equivalents alone are postulated to be unable to pump protons . (4) The genes of two archaeal Rieske proteins have been cloned from Sulfolobus; phylogenetically they are forming a separate archaeal branch and suggest the existence of an evolutionary ancestor preceding the split into the three urkingdoms . (5) Archaeal oxidase complexes may combine features of electron transport systems occurring exclusively as separate respiratory complexes in bacteria and eucarya . (6) As far back as the deepest branches of the phylogentic tree, terminal oxidases reveal a degree of complexity comparable to that found in higher organisms . (7) Sequence analysis suggests a monophyletic origin of terminal oxidases with an early split into two types found in archaea as well as bacteria . This view implies an origin of terminal oxidases prior to oxygenic photosynthesis in contrast to the widely accepted inverse hypothesis.

J Bacteriol, 1996 May, 178(10), 2757 - 66
Bradyrhizobium (Arachis) sp . strain NC92 contains two nodD genes involved in the repression of nodA and a nolA gene required for the efficient nodulation of host plants; Gillette WK et al.; The common nodulation locus and closely linked nodulation genes of Bradyrhizobium (Arachis) sp . strain NC92 have been isolated on an 11.0-kb EcoRI restriction fragment . The nucleotide sequence of a 7.0-kb EcoRV-EcoRI subclone was determined and found to contain open reading frames (ORFs) homologous to the nodA, nodB, nodD1, nodD2, and nolA genes of Bradyrhizobium japonicum and Bradyrhizobium elkanii . Nodulation assays of nodD1, nodD2, or nolA deletion mutants on the host plants Macroptilium atropurpureum (siratro) and Vigna unguiculata (cowpea) indicate that nolA is required for efficient nodulation, as nolA mutants exhibit a 6-day nodulation delay and reduced nodule numbers . The nolA phenotype was complemented by providing the nolA ORF in trans, indicating that the phenotype is due to the lack of the nolA ORF . nodD1 mutants displayed a 2-day nodulation delay, whereas nodD2 strains were indistinguishable from the wild type . Translational nodA-lacZ, nodD1-lacZ, nodD2-lacZ, and nolA-lacZ fusions were created . Expression of the nodA-lacZ fusion was induced by the addition of peanut, cowpea, and siratro seed exudates and by the addition of the isoflavonoids genistein and daidzein . In a nodD1 or nodD2 background, basal expression of the nodA-lacZ fusion increased two- to threefold . The level of expression of the nodD2-lacZ and nolA-lacZ fusions was low in the wild type but increased in nodD1, nodD2, and nodD1 nodD2 backgrounds independently of the addition of the inducer genistein . nolA was required for increased expression of the nodD2-lacZ fusion . These data suggest that a common factor is involved in the regulation of nodD2 and nolA, and they are also consistent with a model of nod gene expression in Bradyrhizobium (Arachis) sp . strain NC92 in which negative regulation is mediated by the products of the nodD1 and nodD2 genes.

J Virol, 1996 May, 70(5), 2842 - 51
Structure and function in the herpes simplex virus 1 RNA-binding protein U(s)11: mapping of the domain required for ribosomal and nucleolar association and RNA binding in vitro; Roller RJ et al.; The herpes simplex virus 1 US11 protein is an RNA-binding regulatory protein that specifically and stably associates with 60S ribosomal subunits and nucleoli and is incorporated into virions . We report that US11/ beta-galactosidase fusion protein expressed in bacteria bound to rRNA from the 60S subunit and not the 40S subunit . This binding reflects the specificity of ribosomal subunit association . Analyses of deletion mutants of the US11 gene showed that specific RNA binding activity, nucleolar localization, and association with 60S ribosomal subunits were found to map to the amino acid sequences of the carboxyl terminus of US11 protein, suggesting that these activities all reflect specific binding of US11 to large subunit rRNA . The carboxyl-terminal half of the protein consists of a regular tripeptide repeat of the sequence RXP and constitutes a completely novel RNA-binding domain . All of the mutant US11 proteins could be incorporated into virus particles, suggesting that the signal for virion incorporation either is at the amino-terminal four amino acids or is redundant in the protein.

J Infect Dis, 1996 May, 173(5), 1123 - 8
Passive and active immunotherapy for experimental pneumococcal pneumonia by polyvalent human immunoglobulin or F(ab')2 fragments administered intranasally; Ramisse F et al.; Experimental pneumococcal pneumonia in leukopenic BALB/c mice enabled evaluation of passive immunotherapy with human polyvalent intravenous immune globulin (IVIG) given intravenously or intranasally and with F(ab')2 fragments administered intranasally . For intravenous and intranasal IVIG, the respective effective doses were < 5 but > 0.5 mg/kg and < 250 but > 2.5 micrograms/kg . For F(ab')2 fragments, the effective dose was < 500 but > 2.5 micrograms/kg . Assessment of the acquired immune responses of passively protected mice and convalescing controls 3 weeks after primary infection showed that antibody responses to whole bacteria were serotype-specific in all mice . Mice protected with IVIG and F(ab')2 fragments had more antibodies to pneumolysin than did controls . In addition, treated mice acquired greater resistance to reinfection than untreated survivors . Thus, local passive immunotherapy may be an effective means of treating pneumococcal pneumonia and may promote acquired resistance to reinfection.

Am J Clin Nutr, 1996 May, 63(5), 836S - 41S
Genetic and molecular basis for copper toxicity; Harris ZL et al.; Recent studies resulted in the cloning of the genes responsible for Menkes syndrome and Wilson disease . Despite the distinct clinical phenotypes of these disorders, each gene encodes a highly homologous member of the cation-transport P-type ATPase family . The remarkable evolutionary conservation of these proteins in bacteria, yeast, plants, and mammals reveals a fundamental protein structure essential for copper export in all life forms . Characterization of a molecular defect in the rat homologue of the Wilson ATPase in the Long-Evans Cinnamon rat identifies an animal model of Wilson disease and will permit experimental analysis of the precise role of this ATPase in copper transport, the effects of specific inherited mutations on transport function, and the cellular and molecular mechanisms of tissue injury resulting from copper accumulation . Finally, recent molecular genetic analysis of a distinct group of patients with low serum ceruloplasmin and basal ganglia symptoms identified a series of mutations in the ceruloplasmin gene . The presence of these mutations in conjunction with the clinical and pathologic findings clarifies the essential biological role of this abundant copper protein in metal metabolism and identifies aceruloplasminemia as a novel autosomal recessive disorder of iron metabolism.

Infect Immun, 1996 May, 64(5), 1873 - 5
Absence of siderophore-like activity in Legionella pneumophila supernatants; Liles MR et al.; Conflicting reports have been given as to the existence of a Legionella pneumophila siderophore . Hence, we rigorously examined the reported siderophore-like activity using the chrome azurol S indicator . Although chrome azurol S reactivity was detected in supernatants, control experiments indicate that it was due to cysteine in the media . When bacteria were cultured in media without cysteine, no siderophores were detected.

Infect Immun, 1996 May, 64(5), 1858 - 61
Uptake and killing of Lyme disease and relapsing fever borreliae in the perfused rat liver and by isolated Kupffer cells; Sambri V et al.; In situ-perfused rat livers were infused with a single dose of 1.5 x 10(7) radiolabeled borreliae . Significant (P < 0.00005) differences in the liver uptake of the agents of Lyme borreliosis, Borrelia burgdorferi IRS, Borrelia afzelii VS461, and Borrelia garinii PBi, and that of the agents of relapsing fever, Borrelia hermsii, Borrelia parkeri, and Borrelia turicatae, were observed . The liver uptakes ranged between 65.9% for B . burgdorferi IRS and 40.5% for B . turicatae . Neither relapsing fever nor Lyme disease borreliae were recovered from infected livers when the livers were cultured in Barbour-Stoenner-Kelly II medium . The in vitro uptake of B . burgdorferi IRS by isolated rat Kupffer cells was rapid, and within 30 min of the infection, large intracellular aggregates of amorphous material were detectable by immunofluorescence with specific anti-B . burgdorferi antibody . The reculturing of B . burgdorferi IRS from Kupffer cells incubated for 24 h in RPMI medium before inoculation with bacteria was negative . The results obtained in this study indicated that borreliae are efficiently taken up and killed by rat hepatic macrophages in the absence of serum factors.

Infect Immun, 1996 May, 64(5), 1789 - 93
Intracellular survival and replication of Erysipelothrix rhusiopathiae within murine macrophages: failure of induction of the oxidative burst of macrophages; Shimoji Y et al.; We investigated the ability of a virulent wild-type parent strain and acapsular avirulent transposon mutants to enter and survive intracellularly within murine peritoneal macrophages . In the presence of normal or immune serum, the parent and mutant strains were both ingested; however, the number of ingested bacteria was three- to fourfold greater in the case of mutant strains than in the case of the parent strain . The parent strain, but not the mutant strains, survived and replicated intracellularly when ingested in the presence of normal serum, whereas both the parent and the mutant strains were readily killed when ingested in the presence of immune serum . To further investigate the mechanism by which the parent strain can survive and replicate within macrophages, we studied the oxidative burst response of macrophages to these strains by measuring chemiluminescence and intracellular reduction of Nitro Blue Tetrazolium dye . Challenge exposure of macrophages with either the parent strain preopsonized with immune serum or the mutant strains preopsonized with normal or immune serum induced a strong oxidative burst, whereas the level was very low when the parent strain was preopsonized with normal serum . Phagocytosis of either the parent strain, in the presence of immune serum, or the mutant strains, in the presence of normal or immune serum, by macrophages reduced large amounts of intracellular Nitro Blue Tetrazolium, whereas minimal amounts were reduced by the parent strain in the presence of normal serum . These results suggest that virulent E . rhusiopathiae can survive and subsequently replicate within murine macrophages when ingested in the presence of normal serum and that the reduced production of reactive oxidative metabolites by macrophages may, in part, be responsible for this occurrence.

Gastroenterology, 1996 May, 110(5), 1386 - 94
A mechanism by which Helicobacter pylori infection of the antrum contributes to the development of duodenal ulcer; Olbe L et al.; BACKGROUND & AIMS: Helicobacter pylori infection and duodenal ulcer disease are firmly correlated . However, the bacteria do mainly colonize the antrum, indicating an indirect pathogenic mechanism . The aim of this study was to test a concept claiming that H . pylori infection of the antrum selectively blocks normal inhibitory reflex pathways to gastrin and parietal cells . METHODS: The effect of antral distention was studied on gastric acid secretion stimulated by pentagastrin and on gastrin release stimulated by gastrin-releasing peptide in H . pylori-infected and noninfected patients with and without duodenal ulcer disease, as well as after eradication of the bacteria . RESULTS: The inhibitory effect on gastric acid secretion induced by antral distention was absent in H . pylori-infected patients irrespective of whether or not they had duodenal ulcer disease . The inhibitory mechanism was restituted in 8 of 10 patients within 9 months after successful eradication of H . pylori infection . Similar results were obtained in studies on gastrin release . CONCLUSIONS: H . pylori infection blocks normal, physiological inhibitory mechanisms from the antrum to both the gastrin cells and to the parietal cell region, resulting in increased gastrin release and impaired inhibition of gastric acid secretion, which will probably lead to an increased duodenal acid load as a general prerequisite for the development of duodenal ulcer disease.

Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 4331 - 5
Characterization of mouse angiogenin-related protein: implications for functional studies on angiogenin; Nobile V et al.; Angiogenin-related protein (Angrp), the putative product of a recently discovered mouse gene, shares 78% sequence identity with mouse angiogenin (Ang) . In the present study, the relationship of Angrp to Ang has been investigated by producing both proteins in bacteria and comparing their functional properties . We find that mouse Ang is potently angiogenic, but Angrp is not, even when assayed at relatively high doses . A deficiency in catalytic capacity, which is essential for the biological activity of Ang, does not appear to underlie Angrp's lack of angiogenicity . In fact, Angrp has somewhat greater ribonucleolytic activity toward tRNA and dinucleotide substrates than does Ang . Instead, an inability to bind cellular receptors is implicated since Angrp does not inhibit Ang-induced angiogenesis . Poor conservation of the Ang receptor recognition sequence 58-69 in Angrp most likely contributes to this defect . However, other substitutions must also influence receptor binding since an Angrp quadruple mutant that is identical to Ang in this segment still lacks both angiogenic activity and the capacity to inhibit Ang . The functional differences between Ang and Angrp, together with evidence presented herein that Angrp is regulated differently than Ang, suggest that the roles of the two proteins in vivo may be quite distinct.

J Virol Methods, 1996 Apr 26, 58(1-2), 187 - 92
Rapid screening of pseudorabies virus-specific cDNAs from a cDNA library; Ho TY et al.; In order to reduce the time and cost for screening of pseudorabies virus (PRV)-specific cDNAs, a rapid and inexpensive method was developed that involved subtractive hybridization of the plasmid, which contained cDNA fragment, to PRV genomic DNA which was bound to nylon membranes . Ninety percent of DNA background was subtracted successfully by this method and the eluted DNA in the form of plasmid could be used to transform bacteria directly . Applying this technique, 200 colonies were screened from a cDNA library containing 30000 colonies . Furthermore, 17 colonies containing PRV-specific cDNAs, including PRV43, UL42, gII, DNase, EP0, 11K, gX, and RSP40, were identified from the 200 colonies by colony hybridization, Southern hybridization, and DNA sequencing . Thus, the subtractive hybridization can be used to construct and successfully establish the PRV cDNA library from PRV-infected cells.

J Mol Biol, 1996 Apr 19, 257(5), 1031 - 41
Hyperthermostable surface layer protein tetrabrachion from the archaebacterium Staphylothermus marinus: evidence for the presence of a right-handed coiled coil derived from the primary structure; Peters J et al.; The scaffold of the surface layer covering the hyperthermophilic archaebacterium Staphylothermus marinus is formed by an extended filiform glycoprotein complex, tetrabrachion, which is anchored in the cell membrane at one end of a 70 nm stalk and branches at the other end into four arms of 24 nm length . The arms from a canopy-like meshwork by end-to-end contacts, enclosing a "quasi-periplasmic space" . The primary structure of the complex, obtained by an approach based entirely on the polymerase chain reaction, shows that the light and the heavy chains are encoded in this order in a single gene and are generated by internal proteolytic cleavage . One light chain associates with the N-terminal part of a heavy chain to form one of the four arms of the complex, comprising about 1000 residues . Following a glycine-rich linker of about ten residues, the C-terminal 500 residues of the four heavy chains converge to form a four-stranded parallel coiled coil, which ends in a transmembrane segment . The sequence of the coiled coil is exceptional in that the heptad repeat of hydrophobic residues typical for left-handed coiled coils shifts to an undecad repeat after an internal proline residue, indicating that the C-terminal part of the sequence forms a right-handed coiled coil . Such a periodicity has not been detected in coiled coils to date . The almost flawless pattern of aliphatic residues, mainly leucine and isoleucine, throughout the hydrophobic core of the stalk provide one explanation for its exceptional stability.

J Biol Chem, 1996 Apr 19, 271(16), 9764 - 70
Evidence for the direct interaction of the nifW gene product with the MoFe protein; Kim S et al.; The Azotobacter vinelandii nifW gene, under control of the nifH promoter, was subcloned into the broad host range multicopy plasmid pKT230 for overexpression in both wild-type and delta nifW strains of A . vinelandii . Unlike the parent delta nifW strain, which grows slowly relative to wild-type under N2-fixing conditions, both overproduction strains grow at the same rate, showing that the overexpressed nifW product is functional in vivo . The approximately 40-fold overexpressed protein was purified, and sequence analysis confirmed its identity . During purification it was observed that NifW in crude extracts ran above the predicted molecular weight on denaturing gels and that as the purification proceeded lower molecular weight forms appeared . Mass spectrometry and studies with protease inhibitors revealed that this abnormal behavior was due to proteolysis . Native molecular weight determinations demonstrate that NifW is a homomultimer, most likely a trimer . Native gel electrophoresis analysis shows that the behavior of wild-type and overexpressed NifW are identical and that when extracts are prepared anaerobically only the homomultimeric forms of NifW are observed . When extracts are exposed to oxygen, however, NifW becomes part of a very high molecular weight complex . Immunoprecipitation with NifW antibodies demonstrate that under those conditions NifW specifically associates with the MoFe protein . These data are consistent with a model whereby NifW is not involved in the initial assembly of an active MoFe protein but rather is part of a system design to protect the MoFe protein from O2 damage.

Structure, 1996 Apr 15, 4(4), 395 - 404
Crystal structure of a dimeric octaheme cytochrome c3 (M(r) 26,000) from Desulfovibrio desulfuricans Norway; Czjzek M et al.; BACKGROUND: The octaheme cytochrome C3 (M(r) 26,000; cc3) from Desulfovibrio desulfuricans Norway is a dimeric cytochrome made up of two identical subunits, each containing four heme groups . It is involved in the redox transfer chain of sulfate-reducing bacteria, which links the periplasmic oxidation of hydrogen to the cytoplasmic reduction of sulfate . The amino-acid sequence of cc3 shows similarities to that of the tetraheme cytochrome c3 (M(r) 13,000; c3) from the same bacteria . Structural analysis of cc3 forms a basis for understanding the precise roles of the multiheme-containing redox proteins and the reason for the presence of several different multiheme cytochromes in one bacterial strain . RESULTS: The crystal structure of cytochrome cc3 has been determined at 2.16 A resolution . The subunits display the c3 structural fold with significant amino-acid substitutions, relative to the tetraheme cytochromes c3, in the regions of the dimer interface . The identical subunits are related by a crystallographic twofold axis, with one heme of each subunit in close contact . The overall structure and the environments of the different heme groups are compared with those of the tetraheme cytochromes c3 . CONCLUSIONS: A common scheme for interactions between these types of cytochrome and their redox partners involves the interaction of a heme crevice, surrounded by positively charged lysine residues, with acidic residues surrounding the redox partner's functional group . Despite the relatively acidic character of cytochrome cc3, the crevice of one heme is surrounded by a high number of positively charged residues, in the same manner as has been reported for cytochromes c3 . The environment of this heme is formed by four flexible surface loops which are variable in length and orientation in the different c3-type cytochromes although the overall structural folds are very similar . It has been proposed that this region, adapted in topology and charge, is the interaction site for physiological partners and is also most likely to be the interaction site in the dimeric cytochrome cc3.

Eur J Biochem, 1996 Apr 15, 237(2), 468 - 75
Chemical structure of lipid A isolated from Comamonas testosteroni lipopolysaccharide; Iida T et al.; The chemical structure of lipid A of lipopolysaccharide isolated from Comamonas testosteroni was determined by quantitative analysis, methylation analysis, mass spectrometry and NMR spectroscopy . The lipid A backbone was found to consist of 6-O-(2-deoxy-2-amino-beta-D-glucopyranosyl)-2-deoxy-2-amino-alpha-D-g luc ose which was phosphorylated in positions 1 and 4' . Hydroxyl groups at positions 4 and 6' were unsubstituted, and position 6' of the non-reducing terminal residue was identified as the attachment site of the polysaccharide part . Liquid secondary-ion/mass spectrometry revealed a pseudomolecular ion at m/z 1572 {M-H}- as a major diphosphoryl lipid component carrying six acyl groups . Fatty acid distribution analysis and electrospray ionization/mass spectrometry of the lipid A showed that positions 2,2',3, and 3' of the sugar backbone were N-acylated or O-acylated by (R)-3-hydroxydecanoic acid, and that the hydroxyl groups of the amide-linked residues attached to positions 2 and 2' were further O-acylated by tetradecanoic and dodecanoic acids, respectively.

Eur J Biochem, 1996 Apr 15, 237(2), 406 - 13
One molecule of molybdopterin guanine dinucleotide is associated with each subunit of the heterodimeric Mo-Fe-S protein transhydroxylase of Pelobacter acidigallici as determined by SDS/PAGE and mass spectrometry; Reichenbecher W et al.; The molybdenum-containing iron-sulfur protein 1,2,3,5-tetrahydroxybenzene: 1,2,3-trihydroxybenzene hydroxyltransferase (transhydroxylase) of Pelobacter acidigallici was investigated by various techniques including mass spectrometry and electron paramagnetic resonance . Mass spectrometry confirmed that the 133-kDa protein is a heterodimer consisting of an alpha subunit (100.4 kDa) and a beta subunit (31.3 kDa) . The presence of a molybdenum cofactor was documented by fluorimetric analysis of the oxidized form A of molybdopterin . The enzyme contained 1.55 +/- 0.14 mol pterin and 0.92 +/- 0.25 mol molybdenum/mol enzyme (133 kDa) . Alkylation of the molybdenum cofactor with iodoacetamide formed di(carboxamidomethyl)-molybdopterin . Upon acid hydrolysis, 1.4 mol 5'GMP/mol enzyme (133 kDa) was released indicating that molybdenum is bound by a molybdopterin guanine dinucleotide . The alpha and beta subunits were separated by preparative gel electrophoresis . Both subunit fractions were free of molybdenum but contained equal amounts of a fluorescent form of the molybdenum cofactors . Mass spectrometry at various pH values revealed that an acid-labile cofactor was released from the large subunit and also from the small subunit . At X-band, 5-25 K, transhydroxylase (as isolated) showed minor EPR resonances with apparent g values around 4.3, 2.03 and, depending on the preparation, a further signal at g of approximately 1.98 . This signal was still detectable above 70 K and was attributed to a Mo(V) center . Upon addition of dithionite, a complex set of intense resonances appeared in the region g 2.08-1.88 . From their temperature dependence, three distinct sites could be identified: the Fe-S center I with gx,y,z at approximately 1.875, 1.942 and 2.087 (gav 1.968, detectable < 20 K); the Fe-S center II with gx,y,z at approximately 1.872, 1.955 and 2.051 (gav 1.959, detectable > 20 K); and the Mo(V) center consisting of a multiple signal around g 1.98 (detectable > 70 K).

J Clin Invest, 1996 Apr 15, 97(8), 1890 - 9
Monocytic cell type-specific transcriptional induction of collagenase; Pierce RA et al.; Interstitial collagenase (MMP-1), a metalloproteinase produced by resident and inflammatory cells during connective tissue turnover, cleaves type I collagen fibrils . This catalytic event is rate limiting in remodeling of tissues rich in fibrillar collagen such as the skin and lungs . The regulation of collagenase expression is cell-type specific; bacterial LPS and zymosan, a yeast cell wall derivative, are potent inducers of collagenase expression in macrophages, but do not alter fibroblast collagenase expression . Since promoter elements controlling collagenase transcription in monocytic cells have not been previously defined, we sought to delineate responsive cis-acting elements of the collagenase promoter in transiently transfected human (U937) and murine (J774) monocytic cell lines . Deletion constructs containing as little as 72 bp of 5' -flanking sequence of the collagenase promoter were sufficient for LPS- or zymosan-mediated transcriptional induction, whereas phorbol inducibility exhibited an absolute requirement for upstream elements including the polyoma enhancer A-binding protein-3 site (-83 to -91) and TTCA sequence (-102 to -105) in both monocytic cells and fibroblasts . Mutagenesis of the activator protein-1 {AP-1} site at -72 abolished basal promoter activity and LPS/zymosan inducibility, while mutagenesis of an NF-kappaB-like site at -20 to -10 had no effect . Nuclear extracts from LPS- and zymosan-treated cells showed strong AP-1 activity by gel-shift analysis, and supershift analysis showed the AP-1 complexes contained specific members of both the jun and fos gene families . These data indicate that, in contrast to most LPS effects, AP-1, but not nuclear factor-kappaB, mediates LPS induction of collagenase transcription in macrophagelike cells . Furthermore, as compared to regulation by phorbol ester, collagenase induction in monocytic cells by cell wall derivatives of bacteria or yeast is largely independent of upstream promoter sequences.

Cancer Res, 1996 Apr 15, 56(8), 1746 - 50
Cloning and expression analysis of human bleomycin hydrolase, a cysteine proteinase involved in chemotherapy resistance; Ferrando AA et al.; A cDNA encoding human bleomycin hydrolase, a member of the cysteine proteinase family of proteins, has been cloned from a human brain cDNA library . The isolated cDNA contains an open reading frame coding for a polypeptide of 456 amino acids that contains all of the structural features characteristic of cysteine proteinases, including the cysteine, histidine, and asparagine residues that are essential for the catalytic properties of these enzymes . The deduced amino acid sequence for human bleomycin hydrolase shows 92, 40, and about 35% of identities with those determined for rabbit bleomycin hydrolase, yeast bleomycin hydrolase, and bacterial aminopeptidase C, respectively . Northern blot analysis of poly(A)+ RNAs isolated from a variety of human tissues demonstrated that human bleomycin hydrolase is expressed in all examined tissues, which is consistent with a putative role of this protein as a proteolytic enzyme involved in norman cellular protein degradation and turnover . Preliminary expression analysis of bleomycin hydrolase in different human tumors showed increased expression of the enzyme in a series of head and neck carcinomas when compared with paired adjacent normal mucosa . We also observed a variable degree of bleomycin hydrolase expression in different types of lymphoma, with low or undetectable levels in Hodgkin's disease samples and higher levels in Burkitt's lymphomas . These results are consistent with a proposed role for human bleomycin hydrolase in resistance of some tumor to bleomycin chemotherapy.

J Immunol, 1996 Apr 15, 156(8), 2809 - 18
Analysis of MHC class II presentation of particulate antigens of B lymphocytes; Vidard L et al.; To generate Ab responses to most protein Ags, B cells must first degrade proteins in endocytic compartments and then display antigenic peptides bound to MHC class II molecules . T helper lymphocytes recognize these complexes and stimulate the B cell to synthesize Ab . Although Ab play a key role in host defense against bacteria, it is believed that B cells are incapable of internalizing particulate Ags . However, we find that B lymphoblastoid cell lines and LPS-activated B lymphocytes can present particulate Ag up to 10(5)-fold more efficiently compared with soluble Ag . Moreover, particulate Ags are presented efficiently by unstimulated B cells when they bind to surface Ig . In comparison to B cells, macrophages in general presented particulate Ags 10- to 1000-fold more efficiently and could also present Ag from particles of a much wider range of sizes . We document by ultrastructural and immunofluorescence analysis that B lymphoblastoid cell lines bind and internalize these particles . The internalization and presentation of the particulate Ag is inhibited by cytochalasin B . In contrast, a similar morphologic analysis of normal lymphocytes demonstrated that while Ag beads are bound to the cell surface, they are internalized only rarely . These results suggest there may be both surface and intracellular pathways for the presentation of particulate Ags by B cells . Interestingly, for both macrophages and B cells, the epitopes generated from particulate and soluble Ags were not identical quantitatively or qualitatively, indicating that there are differences in how these forms of Ag are processed and presented.

J Biol Chem, 1996 Apr 12, 271(15), 8951 - 8
ERK3 is a constitutively nuclear protein kinase; Cheng M et al.; The ERK3 cDNA predicts a protein of 62,000 in size with a C-terminal domain that extends 180 amino acids beyond the conserved core of ERK family protein kinases . Immunoblotting with antibodies raised to recombinant protein and to peptides from the catalytic core and three regions of the C-terminal tail revealed that ERK3 is the expected size and is ubiquitously expressed in a variety of cell lines and tissues . ERK3, unlike the MAP kinases ERK1 and ERK2, is localized in the nucleus in exponentially growing, quiescent, and growth factor-stimulated cells . If the 180 amino acids at its C terminus are deleted, the resulting ERK3 fragment of 45 kDa is still found primarily in the nucleus, indicating that the C terminus is not required for its localization . Recombinant ERK3 expressed in mammalian cells or in bacteria is a protein kinase, as deduced from its capacity to autophosphorylate . Mutation of a conserved residue (Asp171) expected to be involved in catalysis eliminated autophosphorylation . Ser189 of ERK3, which corresponds to Thr183, one of the activating phosphorylation sites of ERK2, is autophosphorylated in vitro and phosphorylated in vivo . Despite marked similarities to ERK1 and ERK2, ERK3 does not phosphorylate typical MAP kinase substrates, indicating that it has distinct functions.

J Biol Chem, 1996 Apr 12, 271(15), 8675 - 81
Non-catalytic beta- and gamma-subunit isoforms of the 5'-AMP-activated protein kinase; Gao G et al.; The mammalian 5'-AMP-activated protein kinase (AMPK) is a heterotrimeric protein consisting of alpha-, beta-, and gamma-subunits . The alpha-subunit is the catalytic subunit and is related to the yeast Snf1p kinase . In this study, we report the cloning of full-length cDNAs for the non-catalytic beta- and gamma-subunits . The rat liver AMPK beta-subunit clone predicts a protein of 30,464 Da, which is related to the Sip1p, Sip2p, and Gal83p subfamily of yeast proteins that interact with Snf1p and are involved in glucose regulation of gene expression . The AMPK beta-subunit, when expressed in bacteria and in mammalian cells, migrates anomalously on SDS gels at an apparent molecular mass of 40 kDa . Rat and human liver AMPK gamma-subunit clones predict a protein of 37,577 Da (AMPK-gamma1), which is related to the yeast Snf4p protein that copurifies with Snf1p and to a larger family of other human AMPK gamma-isoforms . The mRNAs for both AMPK- beta and AMPK-gamma1 are widely expressed in rat tissues, consistent with a broad role for AMPK in cellular regulation . These data reveal a mammalian multisubunit protein kinase strikingly similar to the multisubunit glucose-sensing Snf1 kinase complex . The identification of isoform families for the AMPK subunits indicates the potential diversity of the roles of this highly conserved signaling system in nutrient regulation and utilization in mammalian cells.

Mol Gen Genet, 1996 Apr 10, 250(6), 655 - 64
The chloroplast chlL gene of the green alga Chlorella vulgaris C-27 contains a self-splicing group I intron; Kapoor M et al.; The chlL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants . The chloroplast genome of Chlorella vulgaris strain C-27 contains the first example of a split chlL gene, which is interrupted by 951 bp group I intron in the coding region . In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequence is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns . The 5' and 3' splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis . The absence of a significant ORF in the long P9 region (522 nt), separating the catalytic core from the 3' splice site, makes this intron different from the other known examples of group I introns . Guanosine-mediated attack at the 3' splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.

Cell Immunol, 1996 Apr 10, 169(1), 47 - 54
Regulation of the insect immune response: the effect of hemolin on cellular immune mechanisms; Lanz-Mendoza H et al.; Hemolin is a bacteria-inducible protein of the immunoglobulin superfamily identified in the silk moth Hyalophora cecropia . The role of this protein, in hemocyte aggregation and phagocytosis, was studied in vitro . Hemocyte aggregation, stimulated by phorbol myristate acetate or lipopolysaccharide (LPS), was prevented by hemolin in a dose-dependent fashion, but hemolin did not disrupt aggregates once they had been formed . Furthermore, hemolin was able to stimulate phagocytic activity in both hemocytes and hemocytic mbn-2 cells and this activity was enhanced by LPS . The enhanced phagocytosis produced by a combination of hemolin and LPS was prevented by the protein kinase C (PKC) inhibitors staurosporine and H-7, and PKC activity in hemocyte crude extracts was enhanced by hemolin and LPS, with the highest activity observed in the presence of both . Hemolin affected tyrosine phosphorylation of hemocyte proteins, enhancing the phosphorylation of two proteins of 20 and 30 kDa and preventing tyrosine phosphorylation of two proteins of 35 and 40 kDa . These results suggest that hemolin is involved in the regulation of the cellular immune responses via a pathway that includes PKC activation and protein tyrosine phosphorylation.

J Biol Chem, 1996 Apr 5, 271(14), 8365 - 72
A nuclear envelope-associated kinase phosphorylates arginine-serine motifs and modulates interactions between the lamin B receptor and other nuclear proteins; Nikolakaki E et al.; Previous studies have identified a subassembly of nuclear envelope proteins, termed "the LBR complex." This complex includes the lamin B receptor protein (LBR or p58), a kinase which phosphorylates LBR in a constitutive fashion (LBR kinase), the nuclear lamins A and B, an 18-kDa polypeptide (p18), and a 34-kDa protein (p34/p32) . The latter polypeptide has been shown to interact with the HIV-1 proteins Rev and Tat and with the splicing factor 2 (SF2) . Using recombinant proteins produced in bacteria and synthetic peptides representing different regions of LBR, we now show that the LBR kinase modifies specifically arginine-serine (RS) dipeptide motifs located at the nucleoplasmic, NH2-terminal domain of LBR and in members of the SR family of splicing factors . Furthermore, we show that the NH2-terminal domain of LBR binds to p34/p32, whereas a mutated domain lacking the RS region does not . Phosphorylation of LBR by the RS kinase completely abolishes binding of p34/p32, suggesting that this enzyme regulates interactions among the components of the LBR complex.

Mutat Res, 1996 Apr 2, 362(3), 227 - 36
Stability of microsatellites and minisatellites in Bloom syndrome, a human syndrome of genetic instability; Foucault F et al.; Bloom syndrome (BS) is a human cancer-prone genetic disorder essentially characterized by a generalized genetic instability including a high level of sister chromatid exchanges (SCEs) . Although mutator and hyper-Rec phenotypes of BS cells present analogies with those of bacteria and yeast defective in DNA mismatch repair, we report that (CA)(n) microsatellite alterations are undetectable in BS cells . Thus, our results suggest that the origin of BS mutator phenotype is not a major defect in DNA mismatch repair, allowing us to eliminate an attractive hypothesis for the pleiotropy of BS . We previously suggested that at least some of the intra-allelic rearrangements occurring in minisatellites could result from unequal SCEs . Although SCEs are abnormally frequent in BS cells, the present study failed to show any significant variation of the mutation rates of the two hypermutable minisatellites we analyzed . Thus, our results show that, in spite of an overall genetic instability, alterations in structural motifs known to be predisposed to instability by different mechanisms are undetectable in BS cells.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 3068 - 73
Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate; Schmidt A et al.; Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption . Its molecular target is still unknown . This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon . Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot hybridization that PTPepsilon is highly expressed in osteoclastic cells . A purified fusion protein of PTPepsilon expressed in bacteria was inhibited by ALN with an IC50 of 2 microM . Other PTP inhibitors--orthovanadate and phenylarsine oxide (PAO)-inhibited PTPepsilon with IC50 values of 0.3 microM and 18 microM, respectively . ALN and another bisphosphonate, etidronate, also inhibited the activities of other bacterially expressed PTPs such as PTPsigma and CD45 (also called leukocyte common antigen) . The PTP inhibitors ALN, orthovanadate, and PAO suppressed in vitro formation of multinucleated osteoclasts from osteoclast precursors and in vitro bone resorption by isolated rat osteoclasts (pit formation) with estimated IC50 values of 10 microM, 3 microM, and 0.05 microM, respectively . These findings suggest that tyrosine phosphatase activity plays an important role in osteoclast formation and function and is a putative molecular target of bisphosphonate action.

Biochem Mol Biol Int, 1996 Apr, 38(5), 937 - 55
A method to detect transmembrane helical segments at the nucleotide level; Lesnik T et al.; The analysis of base distributions at the three codon positions, in sequences coding for integral inner membrane proteins from Bacteria, reveal a global excess of thymine and a depletion of adenine, at codon position two . These genes were scanned using a sliding window, in which the average ratio of T to A at the second position of codons, TA(2) (converted to a logarithm, the LTA2 ratio) was computed . The profiles obtained reveal sharp and local peaks of the LTA2 ratio, which account for the high mean values of this parameter . For inner membrane proteins of known structure, the position and extent of these peaks correlate with the location of transmembrane alpha-helices . The prediction accuracy of the detection of such structures using the LTA2 ratio compares to that obtained using algorithms based on an hydrophobicity index of amino-acids . This new criterion presents several advantages, as it deals directly with the nucleotide sequence, does not rely on the values of empirical parameters and finds direct applications in molecular biology studies . Since the nucleotide sequences of transmembrane beta-strands do not give rise to peaks in the LTA2 ratio profile, the criterion appears to characterize exclusively alpha-helical segments in transmembrane proteins.

Endod Dent Traumatol, 1996 Apr, 12(2), 70 - 6
Scanning electron microscopic observations of apical root surfaces of teeth with apical periodontitis; Lomcali G et al.; The aim of this study was to observe apical root surfaces of teeth with chronic periapical lesions . Five premolars and three molars with radiographically demonstrable periapical lesions were extracted and fixed in 2.5% phosphate-buffered glutaraldehyde solution for 9 days . The apical 3-mm portion of 17 roots was removed and prepared for scanning electron microscope . Lacunar resorption zones were frequently observed on the root surfaces . Bacteria and yeast cells were detected in some of the lacunae . Periapical bacterial plaque with a smooth structure was present mostly around the main apical foramen . Cementum-like tissue deposits indicative of repair were seen adjacent to some resorption areas . Clastic cells tightly attached to crater-like depressions with finger-like projections were observed laterally on the specimens . Current research should be focused on new procedures to eliminate extraradicular microrganisms and periapical bacterial plaque in persistent periapical infections.

J Submicrosc Cytol Pathol, 1996 Apr, 28(2), 255 - 64
Electron microscopic study of the morphological changes of gastric mucous cell induced by Helicobacter pylori in human gastric ulcers; Ogata T et al.; Specimens from 8 cases of human gastric ulcers infected with H . pylori, 3 cases of gastric ulcers without H . pylori infection and mucosal specimens infected with H . pylori from 3 cases of early gastric cancers obtained at surgery were studied by transmission electron microscopy . In the surrounding epithelium of the ulcer, when present, the bacteria were preferentially located at the luminal side of the apical junctional complexes . This was accompanied by dome-like bulging of the apical cytoplasm, but the epithelial continuity was maintained . A consistent finding was the apocrine-like release of the apical cytoplasm into the lumen . In addition, there were cells with marked apical protrusions and cells with dissolution of mucous granules . Degenerative changes, such as cellular edema, vacuole formation and disruption of cell membrane were also observed . The cells which had shed their apical mucous area appeared to degenerate causing massive cell exfoliation and formation of denuded lamina propria . Similar changes of the surface mucous cells were observed in the mucosal specimens infected with H . pylori obtained from early gastric cancers, but such cell pathology was scarce in samples of the gastric ulcers without H . pylori infection . In some ulcers infected with H . pylori, the bacteria were also observed on the surface of the regenerating epithelial cells at the ulcer base . These findings suggest that H . pylori infection is an important factor in the development of gastric ulcers and in the prevention or delay in ulcer healing.

Oral Microbiol Immunol, 1996 Apr, 11(2), 103 - 8
Proteolytic artifacts in SDS-PAGE analysis of selected periodontal pathogens; Weidner MF et al.; The aim of the study was to examine whether proteolytic artifacts, which result in a loss and poor resolution of protein bands, occur during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis of cellular proteins from selected proteolytic (Porphyromonas gingivalis, Prevotella nigrescens and Treponema denticola) and non-proteolytic (Fusobacterium nucleatum) bacteria . Conditions to limit or prevent proteolysis were also investigated . Bacterial cells were incubated in solubilizing buffer (SDS+ beta mercaptoethanol) at room temperature for various periods of time before boiling . A control assay consisted of trichloroacetic acid-treated bacterial cells . Cellular proteins were separated by electrophoresis and stained with Coomassie blue . Proteolysis occurred very rapidly in the case of P . gingivalis (< 30 s), whereas a longer incubation time (> 1 h) was required to observe similar effects in P . nigrescens and T . denticola . No proteolysis was observed for F . nucleatum . In all cases, heat (100 degrees C) and low pH (< 4) treatments of bacterial cells could avoid production of proteolytic artifacts . Incorporation of specific protease inhibitors before solubilization of bacteria could also prevent proteolysis . More particularly, N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), iodoacetamide and diisopropylfluorophosphate (50 mM) were highly efficient for P . gingivalis, P . nigrescens and T . denticola, respectively . When outer membranes of P . gingivalis were prepared in the presence of TLCK, numerous additional protein brands, not seen in the absence of TLCK, were detected . The present study suggests that specific protease inhibitors, effective in preventing proteolysis, should be identified and added during cell fractionation and protein purification procedures.

J Inorg Biochem, 1996 Apr, 62(1), 67 - 73
Triorganosilicon (IV) compounds: synthesis, structural study, and biological activity; Singh D et al.; A few coordination compounds of silicon (IV) have been synthesized by the interaction of trimethyl- and triphenyl-chlorosilane with nitrogen-sulphur donor ligands . These compounds are monomeric, as indicated by molecular weight determination, and they behave as nonelectrolytes in dry DMF . From the electronic, infrared, 1H, and 13C NMR spectral results, it has been concluded that in these compounds, silicon is penta-coordinated in a trigonal bipyramidal environment . An assessment of biological activity of these compounds has shown that some of them are very active against P . mirabilis and others against S . viridans bacteria, while all of them show good fungicidal action against F . oxysporum, A . alternata, and A . niger.

Microbiology, 1996 Apr, 142 ( Pt 4), 867 - 72
Polysaccharide lyases from gellan-producing Sphingomonas spp; Sutherland IW et al.; A number of Sphingomonas strains capable of synthesizing the bacterial exopolysaccharide gellan and related polymers were shown to possess constitutive gellanase activity . In each case, the degradation of deacylated gellan was due to extracellular, eliminase-type enzymes (lyases) which cleave the sequence .. . beta-D-glucosyl 1,4-beta-D-glucuronosyl .. . in the tetrasaccharide repeat unit of the substrate polysaccharides . Deacetylated rhamsan was an alternative substrate but there was little or no action against most other polysaccharides with similar structures . Slight differences were found between the specificities of the lyases from different strains . Activities of gellan lyase preparations were generally low . As well as the extracellular 'gellanase' activity, all the bacteria possessed varying amounts of beta-D-glucosidase and beta-D-glucuronidase activities apparently located in the periplasm . The products from deacylated gellan and the chemically deacylated form of polysaccharide S194 (rhamsan gum), which is effectively a gentiobiosylated form of gellan, closely resembled those recently obtained by the authors from other, gellan-degrading, non-gellan-producing bacteria . The enzymes had negligible activity against the natural, acylated gellan and rhamsan polysaccharides from bacteria now designated as strains of Sphingomonas.

Mol Gen Mikrobiol Virusol, 1996 Apr-Jun, (2), 32 - 9
{Cloning proviral DNA sequences of the human T-cell leukemia virus (HTLV-1) from the genome of cultured MT-4 cells}; Susloparov MA et al.; MT-4 cell line is a continuous strain of human T lymphocytes expressing defective noninfective subviral HTLV-1 particles . A fragment of sequence encoding the p24 protein and gene for envelope protein (env) have been obtained from genomic DNA of this culture by polymerase chain reaction . Both HTLV-1 fragments were cloned in bacterial vectors, and the nucleotide sequence of these regions was determined . The cloned DNA fragment encoding the p24 has only four point nucleotide exchanges . Analysis of the env gene structure revealed that the sequence had several amino acid exchanges and two deletions (13 bp and 70 bp).

FEBS Lett, 1996 Apr 1, 383(3), 191 - 5
Rapid estimation of relative amide proton exchange rates of 15 N-labelled proteins by a straightforward water selective NOESY-HSQC experiment; Bockmann A et al.; A straightforward heteronuclear pseudo-3D NOESY-HSQC pulse sequence using radiation damping to selectively invert magnetization at the water frequency was developed to estimate the amide proton exchange rates in 15N-labelled proteins . The peak intensities in the resultant 2D spectrum allow a direct classification of amide proton exchange rates according to short (ms), intermediate (ms to s) or long (> or = s) residence times . This method was successfully used for the analysis of amide proton exchange rates in the 15N-labelled FruR DNA-binding domain and pertinent information about its dynamics was obtained.

Appl Environ Microbiol, 1996 Apr, 62(4), 1467 - 70
Homology between genes for aromatic hydrocarbon degradation in surface and deep-subsurface Sphingomonas strains; Kim E et al.; The cloned genes for aromatic hydrocarbon degradation from Sphingomonas yanoikuyae B1 were utilized in Southern hybridization experiments with Sphingomonas strains from the surface and deep-subsurface environments . One hybridization pattern was obtained with BamHI-digested genomic DNAs for two surface strains, while a differing pattern was seen for five deep-subsurface strains . The cross-hybridizing genes were located in the chromosomes of the surface strains and on plasmids in the deep-subsurface strains.

Boll Chim Farm, 1996 Apr, 135(4), 232 - 5
Aminoazoles in heterocyclic synthesis . Synthesis of some new pyrimidobenzimidazoles and pyrazolonopyridines as well as triazolo-, tetrazolo- and pyrazolo-pyrimidines of pharmaceutical interest; Kandeel EM et al.; The reactivity of 3-cinnamoylindole, 3-cinnamoylantipyrine and 1-(3-methyl-1-phenyl-5-pyrazolon-4 yl)-4-antipyrin-4-yl)-prop-2-ene-1- one(la-c) towards 2-aminobenzimidazole, 3-amino-1,2,4-triazole, 5-aminotetrazole monohydrate, 5-amino-3-phenylpyrazole and 3-amino-1-phenyl-2-pyrazolin-5-one to give the fused heterocycles 2a-c, 4a,b, 5a,b, 8a,b, and 10a-c has been investigated . Structures of the synthesized compounds were confirmed by both the analytical and the spectral data (IR, UV and 1H NMR).

Zhonghua Hu Li Za Zhi, 1996 Apr, 31(4), 193 - 6
{Air analysis of the doctor and nurse offices in tall hospital buildings}; Li MY et al.; O2, PO2, CO2, total number of bacteria were detected in doctor-nurse offices which are at the same side on the 6 and 7th floor in the first affiliated hospital of China Medical University . The results showed that the detected O2 and PO2 in doctor-nurse office were conformed to the normal range of country's indoor standard . The average value of bacteria detection was above the standard Which is less than 500/m3 in ordinary ward given by the Ministry of Health of China . The average CO2 detection was below 0.07%, but sometimes above 0.07% during the 8:30 detection in the morning, total number of bacteria was a little higher too, which suggested that more attention are needed.

J Clin Microbiol, 1996 Apr, 34(4), 778 - 84
Immunomagnetic separation and solid-phase detection of Bordetella pertussis; Stark M et al.; In the present study, novel solid-phase methods were used for both sample preparation and PCR detection of Bordetella pertussis . The sample preparation was performed by immunomagnetic separation with paramagnetic beads coated with polyclonal antibodies directed toward the surface antigens of the bacteria . The precoated immunobeads were directly used on nasopharyngeal aspirates to capture the bacteria on the solid support and were subsequently transferred to the PCR tube with no further manipulations . The region encompassing the pertussis toxin promoter was analyzed to allow direct discrimination between the three major Bordetella species (B . pertussis, B . parapertussis, and B . bronchiseptica) . The resulting amplicons were captured on a second magnetic solid phase, allowing detection and restriction analysis of the target sequence . A colorimetric detection system based on a DNA binding fusion protein enabled the use of standardized enzyme-linked immunosorbent format tests both for the detection of Bordetella spp . and for species evaluation . When the optimized system was evaluated on 55 clinical aspirate samples, 21 of 22 (95%) culture-positive samples were positive by the system that we developed . In addition, two samples were positive by the PCR-based assay, while the culture assay was negative . The implications of these results are discussed.

J Immunol, 1996 Apr 1, 156(7), 2608 - 15
Stress-induced enhancement of antigen-specific cell-mediated immunity; Dhabhar FS et al.; The studies described here demonstrate that the activation of the physiologic stress response systems of the body can enhance immune function in vivo . This enhancement is observed as a large and long lasting increase in allergic contact sensitivity or delayed-type hypersensitivity, an immune reaction which involves an Ag-specific, cell-mediated immune response . In contrast, acute stress has no effect on the course of irritant contact sensitivity, an immune reaction that does not involve an Ag-specific memory response . A comparison of infiltrating leukocyte numbers in sections of inflamed skin from unstressed and stressed animals shows that stress induces a significant and persistent increase in numbers of leukocytes at the site of the delayed-type hypersensitivity reaction . These results demonstrate that a relatively mild behavioral manipulation can enhance an important class of immune responses that mediate harmful (allergic dermatitis) as well as beneficial (resistance to certain viruses, bacteria, and tumors) aspects of immune function . The implications that these studies have for clinical, diagnostic, and experimental manipulations involving cell-mediated immune function are discussed.

Semin Arthritis Rheum, 1996 Apr, 25(5), 318 - 36
Immunopathogenesis and spectrum of infections in systemic lupus erythematosus; Iliopoulos AG et al.; OBJECTIVE . This study reviews the pertinent immunopathologic mechanisms that contribute to increased susceptibility to infection in patients with systemic lupus erythematosus (SLE) and the spectrum of infections in patients with SLE . It assesses the impact on patient care, morbidity, and mortality . METHODS . The authors performed an extensive search of the literature through MEDLINE, using appropriate keywords and manual search of bibliography cited by retrieved papers . RESULTS . Infection is a leading cause of morbidity and mortality in SLE and may change the natural course of the disease . Patients with SLE display numerous cellular and humoral abnormalities that are expressed in a heterogeneous pattern and contribute in varying degrees to susceptibility to infectious agents . The use of corticosteroid and immunosuppressive drugs is only partially responsible for the high infection rate in lupus patients . Common as well as opportunistic pathogens are frequently encountered among patients with SLE . The susceptibility that SLE patients exhibit to certain bacteria is now better recognized, and a broadening spectrum of pathogens is being reported . The immunization of patients against influenza and pneumococcus and the administration of prophylactic therapy against Pneumocystis carinii infection in certain patients have recently been proposed . CONCLUSION . Disease activity and treatment are responsible for the extensive defects of the immune defense system in lupus patients that increase their susceptibility to an extensive range of infections . Infection imposes a serious burden on lupus patients and on the caring physician.

Infusionsther Transfusionsmed, 1996 Apr, 23(2), 71 - 5
Decrease of fibronectin following repeated infusion of highly substituted hydroxyethyl starch; Treib J et al.; OBJECTIVE . Fibronectin (Fn) plays an important part in unspecific defense mechanisms because of its ability to mediate the binding of foreign-body particles, bacteria, collagen, and other macromolecules to phagocytising cells of the reticulo-endothelial system (RES) . The aim of the present study was to examine the effect of a 10-day hemodilution therapy on Fn concentration in humans . DESIGN . The patients were randomized and treated with either 10% hydroxyethyl starch (HES) 200/0.62 or 6% HES 200/0.62 . SETTING . Neurology department of an university clinic . PATIENTS . We examined 12 patients with cerebrovascular perfusion disturbances . INTERVENTIONS . The Fn concentration was determined using simple radial immunodiffusion . RESULTS . The Fn concentration dropped significantly in all 12 patients (p < 0.01) in a dose-dependent manner beyond the dilution effect . 10% HES 200/0.62 caused a Fn decrease from 26.6 +/- 9.2 to 10.0 +/- 2.2 mg/dl (-62.2%), 6% HES reduced Fn from 25.5 +/- 9.9 to 15.0 +/- 3.2 mg/dl (-41.1%) . In one patient there was a continuous decrease of Fn from 41.0 down to only 6.4 mg/dl . CONCLUSION . According to the results of animal experiments, the decrease of Fn seems to indicate depression of the RES . Besides its defense function, Fn probably plays a role in embryogenesis, wound healing, and blood clotting . Therefore, we assume that the drug-induced reduction of Fn possibly has clinical relevance.

Zoolog Sci, 1996 Apr, 13(2), 319 - 23
A characteristic difference among GroEL homologs from intracellular symbionts of closely-interrelated species of aphid; Komaki K et al.; Nucleotide sequences encoding GroEL homologs of intracellular symbionts in three closely interrelated aphids were compared with one another and that for GroEL . It was suggested that in these proteins a particular position is highly susceptible to amino acid substitution, through which the GroEL homologs of symbionts seemed to have acquired a unique function on top of the activity as molecular chaperone . This may represent a rare example of non-neutral evolution of molecule under the positive selection pressure.

J Vet Med Sci, 1996 Apr, 58(4), 297 - 303
Immunohistological evaluation on respiratory lesions of pigs intranasally inoculated with Actinobacillus pleuropneumoniae serotype 1; Ajito T et al.; Nine-week-old pigs were inoculated intranasally with 6.10 X 10(3) (group 10(3)), 10(5) (group 10(5)) and 10(7) (group 10(7)) colony-forming unit of Actinobacillus pleuropneumoniae (App) serotype 1 designated HA-337 strain, respectively . One pig in group 10(5) and 2 pigs in group 10(7) died with dispnea and hemorrhagic pleuropneumonia within 20 to 48 hr post inoculation (PI) . All pigs necropsied on 7 days in groups 10(5) and 10(7) had focal fibrous pleuropneumonia . Histologically, pulmonary lesions were classified into three stages; peracute, acute and subacute . Fatal cases in group 10(7) had peracute lesion composed of severe edema, hemorrhage and necrobiosis of alveoli, with mononuclear cells infiltration in the dilated interlobulus . The fatal case in group 10(5) had acute pulmonary lesion composed of focal or linear infiltration of round and fusiform cells that frequently showed swirling pattern in alveoli . The surviving cases in group 10(5) and 10(7) had subacute lesion composed of multifocal pulmonary necrosis surrounded by fibrous tissue . The swirling pattern was clearly seen in demarcation zone . Immunohistochemically, App antigens scattered as intact bacteria in alveoli, dilated interlobular septa and pleura, and lymph vessels in peracute and acute lesions . Areas of necrosis were also stained weakly . Although no antigen was detected in cytoplasm of macrophages and infiltrated cells in peracute lesions, App antigen was detected as positively stained mass in cytoplasm of some macrophages in acute lesions . In subacute lesions, App antigens were recognized as intact bacteria in necrotic areas and among the swirling pattern cells of demarcation zone . Macrophages had App antigens as a large mass of pigment in the cytoplasm in area of fibrosis.

Zentralbl Bakteriol, 1996 Apr, 283(4), 485 - 91
Invasive ability of C . jejuni/coli isolates from children with diarrhea and the effect of iron-regulated proteins; Schwartz D et al.; The invasive ability of C . jejuni/coli strains isolated from children with diarrhea was studied using an in vitro HEp-2 cell invasion assay . The ratio between the number of intracellular bacteria and the number of bacteria in the inoculum was determined (invasion index) . It was found that under anaerobic conditions, there was a significant decrease in the invasion index as compared to standard conditions (5% CO2) . Of 11 strains tested, seven were determined as invasive on the basis of invasion indexes within the range of 0.0002-0.01 . In a previous study {D . Schwartz et al., Zbl . Bakt . 280, 338-347 (1994)}, it was found, that most of the C . jejuni/coli isolates tested produced an outer membrane protein when grown under conditions of iron depletion (IRP) . The IRP were detected in eight of the nine strains tested in the present study (five invasive and three non-invasive strains) . In one non-invasive strain, IRP was not detected . When kept under conditions of iron depletion, one of the invasive strains exhibited a significant increase in invasive capacity . The results suggest that iron depletion seems to stimulate the invasion capacity of C . jejuni/coli in vitro.

Vet Microbiol, 1996 Apr, 49(3-4), 249 - 55
Replication of Australian porcine isolates of Ileal symbiont intracellularis in tissue culture; Collins AM et al.; Ileal symbiont intracellularis (ISI) isolated from Australian cases of PIA and PHE was replicated in the rat ileum enterocyte cell line IEC 18 . The number of ISI within cells varied, as did the number of ISI infected within the monolayer . At 24 h post infection a large number of cells were infected with approximately 100 ISI per cell . At the termination of infection, fewer IEC 18 cells were infected but ISI had replicated to fill the cell cytoplasmic space . Numerous foci of infected cells were visible in the monolayer, containing as many as 15 densely infected cells . Division of ISI infected cells indicated the transmission of ISI in the cytoplasm to daughter cells . This suggests that the replication of ISI in culture appears to be reasonably cell dependent . No cytopathic effects were observed in the infected cultures.

Aliment Pharmacol Ther, 1996 Apr, 10 Suppl 1, 73 - 7
Role of vacA and the cagA locus of Helicobacter pylori in human disease; Blaser MJ; Helicobacter pylori are 'slow' bacteria that may cause disease decades after acquisition . Bacterial pathogenesis often involves features, including conserved genes, shared by many different species . As such, despite its unique niche in the human body, the pathogenesis of H . pylori infection most likely shares mechanisms with other bacteria . In this paper, two genes, vacA and cagA, which appear unique to H . pylori and which may reflect the particular requirements of H . pylori for long-term residence in the human stomach will be discussed . At present the function of these genes for H . pylori is not known yet other characteristics have been defined.

Curr Opin Struct Biol, 1996 Apr, 6(2), 142 - 50
Circular assemblies; Antson AA et al.; During 1994 and 1995, the structures of the serum amyloid P component, the bacterial chaperonin GroEL, the 20S proteasome, the bacterial light-harvesting complexes and the tryptophan operon RNA-binding attenuation protein have been determined . These structures all form circular assemblies in which the individual subunits are related by rotational symmetry . In most cases the circular organization generates a new biophysical property and a specific biological function which have presumably been selected by evolution.

Oncology (Huntingt), 1996 Apr, 10(4), 599 - 606, 611-2; discussion 615-6
The outpatient management of febrile neutropenia in cancer patients; Freifeld AG et al.; Treatment of fever and neutropenia in cancer patients has been recognized for 30 years as a medical emergency, requiring prompt in-hospital evaluation and institution of broad-spectrum intravenous (i.v.) antibiotics . This action was deemed necessary due to the high frequency of life-threatening infections in febrile neutropenic patients, with no way to distinguish patients who are infected from those who are not . In recent years, it has become clear that not all neutropenic cancer patients are at the same level of risk for developing severe infections or life-threatening complications during neutropenia . Those who are at low risk may be candidates for treatment outside the hospital setting, either with i.v . regimens or potent oral antibiotics . The identification of low-risk febrile neutropenic patients and the specific outpatient approaches that have been tested to date are discussed . Outpatient management of fever during neutropenia could obviously be much less costly than standard inpatient care and could improve quality of life for low-risk patients undergoing cancer therapy.

J Periodontol, 1996 Apr, 67(4), 433 - 42
Host defensive functions in a family manifesting early-onset periodontitis; Arai H et al.; Family case studies help us identify host risk factors in periodontal disease . In this study we examine a family consisting of a mother (40 years old, with rapidly progressive periodontitis), her elder daughter (14 years old, with localized juvenile periodontitis), and younger daughter (13 years old, with simple gingivitis) . We examined 1) the peripheral neutrophil functions (chemotactic migration, phagocytosis, superoxide production); 2) lymphocyte functions (proliferative activity and cytokine productivity of T cells, immunoglobulin {Ig} M productivity of B cells when stimulated with pokeweed mitogen); 3) phenotypic analyses of peripheral lymphocyte subpopulations; 4) serum IgG antibody titers against periodontopathic bacteria; and 5) serological type of HLA class II . All the subjects exhibited high T4/T8 ratios due to high percentage of CD4-positive cells, showed high IgG titers to Actinobacillus actinomycetemcomitans, and had a HLA DQw1 in common . The mother showed a slight deficiency of neutrophil chemotactic migration to N-formyl methyonyl leucyl phenylalanin (fMLP), raised interleukin-2 productivity of T cell, and high levels of IgG titers to Porphyromonus gingivalis and Fusobacterium nucleatum . Both daughters showed weak T cell proliferative response to anti-CD3 monoclonal antibody and low IgM productivity . Low lymphocyte responsiveness may be involved in the pathogenesis of periodontal disease of these daughters; therefore, the lymphocyte dysfunctions shown should be considered in relation to the progression of periodontal disease.

Trends Biochem Sci, 1996 Apr, 21(4), 129 - 33
The ethylene signal transduction pathway in Arabidopsis: an emerging paradigm?
Chang C.
In the plant Arabidopsis, ethylene signaling involves at least two putative receptors, both of which resemble the "two-component' regulators known almost exclusively in bacteria . Downstream in the pathway is a putative serine/threonine protein kinase related to the animal Raf protein kinases . This novel combination of signaling proteins has parallels with the postulated osmolarity-response pathway in yeast.

J Nat Prod, 1996 Apr, 59(4), 348 - 54
Isolation of 20 glycosides from the starfish Henricia downeyae, collected in the Gulf of Mexico; Palagiano E et al.; Thirteen new (1-13) and seven known (14-20) steroid glycosides were isolated from Henricia downeyae, collected from the offshore waters of the northern Gulf of Mexico . Ethanolic extracts of these starfish caused growth inhibition in bacteria and fungi, potent antifouling activity against barnacle and bryozoan larvae, and feeding deterrent activity against a marine fish . The known compounds are typical glycosides found in several species of the family Echinasteridae, i.e., Echinaster sp . and Henricia laeviuscola . One of the new compounds belongs to this group, whereas the remaining 12 new compounds represent a novel series of steroid glycosides which have aglycons with structural similarities to the "asterosaponins" . They possess a delta 9(11) 3 beta, 6 alpha-dihydroxysteroidal aglycon with 23-oxo or 22,23-epoxy functionalities and often a 20-hydroxyl group in the side chain . The sulfate is located at C-6 and the saccharide moiety at C-3, in contrast with the asterosaponins which have the sulfate at C-3 and the oligosaccaride moiety at C-6 . All the new compounds contain a glucuronic acid unit, which is uncommon among steroid glycosides from echinoderms . The structures of the new compounds, isolated in amounts ranging from 3.4 to 0.9 mg, were determined by interpretation of their spectral data and by comparison with spectral data of known compounds.

S Afr Med J, 1996 Apr, 86(4), 365 - 8
Assessment and 2-year follow-up of some factors associated with severity of respiratory infections in early childhood; Wesley AG et al.; OBJECTIVE: To assess the effect of some factors on the severity of acute respiratory infection (ARI) in children . DESIGN: In a case control study, children with pneumonia were matched with controls who had upper respiratory infection . They were compared in respect of nutrition, household crowding and smoke pollution, and the presence of current viral respiratory infection . Both cohorts were followed up for 18-24 months to determine if there was a difference in subsequent respiratory sequelae . SETTING: Primary health care-based cohorts of peri-urban township children . PARTICIPANTS: Forty-eight children < 3 years of age with pneumonia (index cases) were matched by age and presentation time with controls who suffered only from upper respiratory infection . All came from underprivileged communities . Index cases were selected as they presented and the study was conducted between February 1988 and June 1991 . MAIN OUTCOME MEASURE: Any difference between index cases and controls in respect of the four factors listed under 'Design' . Follow-up home visits determined whether subsequent sequelae of the two grades of ARI were different . RESULTS: The presence of current viral infection at entry to the study was evident in 21 of those with pneumonia and 12 controls (difference between groups = 19.15%, 95% confidence intervals 0.25 - 38.05, P = 0.052) . Overcrowding in the home was comparable . Index homes were occupied by a mean of 3.57 (SD 1.54) children and 5.26 (SD 4.84) adults, control homes by 3.51 (SD 1.80) children and 4.36 (SD 2.02) adults . Occupancy of the room in which the child slept was also not significantly different: index group mean 4.23 (SD 1.55) and controls 4.02 (SD 1.38) (mean difference 0.21, 95% Cl 0.378 - 0.798, P = 0.485) . Correlation of bedroom crowding with young age (< 1 year) or weight-for-age centiles was not significant in either cohort (r < 0.3 in all) . The prevalence of viral infection was not increased by degree of crowding in either group (P = 0.636) . Domestic smoke pollution was similar: cigarette smoking occurred in 75% of index homes and 69% of control homes . Wood or coal fires were used in 19% of index and 14% of control homes . The nutritional status of both groups proved to be similar . Fifteen per cent of index children and 12% of controls had weight-for-age centiles < or = 10th centile (difference = 3.26%, 95% Cl -10.72 - 17.24, P = 0.649) . Two-year home follow-up visits were completed in 75% of the index and 69% of the control group . The balance were followed up for 18 months . There was no difference between index and control children in the recurrence of respiratory symptoms (P = 0.664) or need to visit a health facility (P = 0.302) . CONCLUSIONS: Factors shown elsewhere to contribute to the acquisition or severity of ARI could not be demonstrated as important in this study . The children with pneumonia and their matched controls with upper respiratory infections came from equally overcrowded and smoke-filled homes, had comparable nutritional status which was not markedly poor, and had an equal incidence of current viral infection . Subsequent ill-health was not found to be greater in the pneumonia group.

Trop Med Int Health, 1996 Apr, 1(2), 243 - 50
Necrotizing and suppurative lymphadenitis in Leishmania major infections; Gaafar A et al.; The pathology of lymph nodes and subcutaneous nodules in 6 patients with cutaneous leishmaniasis (Oriental sore) due to Leishmania major is described in this paper . In 3 patients enlarged epitrochlear lymph nodes were found to be associated with primary skin lesions in the forearm . The lymph node in one patient showed a necrotizing granulomatous reaction that simulated tuberculous lymphadenitis . Leishmania parasites were, however, found in sections of the node, and staining for mycobacteria was negative . The second patient presented with an abscess and a discharging sinus in the epitrochlear region . Parasites were found in smears of the pus and cultures for bacteria were negative . The lesion healed with antimonial therapy . In the third patient the lesion resembled cat-scratch disease and showed stellate abscesses and granulomas . Leishmania parasites were also identified in the sections . Sections of a subcutaneous nodule from the fourth patient showed a necrotizing granuloma . The lesion healed spontaneously and the patient became leishmanin-positive . In two other patients fine needle aspiration of the subcutaneous nodules showed parasites, granuloma and necrosis . We concluded that L . major disseminates from the primary cutaneous lesion via the lymphatics to the subcutaneous tissues and the regional lymph nodes . The subcutaneous nodules and lymphadenopathy may persist long after the primary lesion had healed . The primary lesion is sometimes inconspicuous . Necrotizing and suppurative lymphadenitis due to L . major have to be distinguished from other causes of necrosis and suppuration such as tuberculosis and cat-scratch disease.

J Lab Clin Med, 1996 Apr, 127(4), 333 - 9
Recombinant ribosomal P2 protein can unmask anti-ribosomal P autoantibodies from healthy adults; Pan ZJ et al.; Autoantibodies to ribosomal P proteins (anti-P) are detected almost exclusively in the serum samples from patients with systemic lupus erythematosus when conventional enzyme-linked immunosorbent assay and immunoblotting techniques are used . Anti-P are not detected in serum samples from healthy adults by these techniques . By treating serum from healthy adults with ribosome-coated beads, we unexpectedly unmasked anti-P in virtually all individuals . This unmasking of anti-P occurs by the displacement of an antibody inhibitor from anti-P . We wanted to determine whether anti-P from healthy adults could also be unmasked by treatment of their serum or plasma with isolated ribosomal P proteins . Recombinant human ribosomal P2 protein was produced in bacteria as a TrpE fusion protein, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and isolated as strips of membranes corresponding to the size of the P2 fusion protein . Serum or plasma from six healthy adults and three patients with systemic lupus erythematosus were incubated with these strips overnight rather than for 2 hours, as is done in conventional immunoblots . Acid eluates were obtained from the strips and analyzed for antibody activity by immunoblot . Eluates from healthy adults and patients contained antibodies reactive with recombinant ribosomal P2 protein . They also reacted with the three ribosomal P proteins in purified rabbit ribosomes . Their anti-P activity was completely inhibited by a peptide corresponding to the immunodominant linear epitope of the ribosomal P proteins . The antibodies in the eluate were immunoglobulin G . We conclude that anti-P autoantibodies from healthy adults can be unmasked by affinity purification on denatured, recombinant ribosomal P proteins and that antigen excess is sufficient for inhibitor displacement.

Biol Bull, 1996 Apr, 190(2), 195 - 202
Vertical transmission of chemoautotrophic symbionts in the bivalve Solemya velum (Bivalvia: Protobranchia); Krueger DM et al.; Adults of the bivalve species Solemya velum live in symbiosis with chemoautotrophic bacteria in specialized gill bacteriocytes . The bacteria play an essential nutritional role in the mature association, fixing CO2 via the Calvin cycle with energy obtained through the oxidation of reduced sulfur compounds . To understand how the continuity of this partnership is maintained between host generations, we investigated the mode of symbiont transfer in S . velum . A diagnostic assay using the polymerase chain reaction and primers specific for the S . velum symbiont ribulose-1,5-bisphosphate carboxylase (RubisCO) gene consistently detected bacterial sequence in female gonad tissue, suggesting the presence of symbiont cells in host ovaries and a vertical mode of symbiont transmission from mother to offspring . Furthermore, intracellular bacteria were present in the developing gills of juveniles that had not yet hatched from the gelatinous capsule in which larval development occurs (11 days after fertilization) . By 64 days postfertilization, the typical adult gill ultrastructure of alternating bacteriocytes and symbiont-free-intercalary cells was apparent . Knowledge about the mode of symbiont transfer in S . velum allows further study into the dynamics of host-symbiont interactions in chemoautotrophic associations.

J Bacteriol, 1996 Apr, 178(8), 2328 - 33
The proton/electron ration of the menaquinone-dependent electron transport from dihydrogen to tetrachloroethene in "Dehalobacter restrictus"; Schumacher W et al.; In the anaerobic respiration chain of "Dehalobacter restrictus," dihydrogen functioned as the electron donor and tetrachloroethene (PCE) functioned as the electron acceptor . The hydrogenase faced the periplasm, and the PCE reductase faced the cytoplasmic side of the membrane . Both activities were associated with the cytoplasmic membrane . UV spectroscopy showed that membrane-bound menaquinone (MQ) was reduced by oxidation of H2 and reoxidized by reduction of PCE, indicating that MQ functions as an electron mediator . Fast proton liberation (t1/2 = 6 +/- 2 s) during electron transport from H2 to PCE and to trichloroethene (TCE) after addition of either PCE or TCE to H2-saturated cells resulted in an extrapolated H+/e- ratio of 1.25 +/- 0.2 . This ratio indicated that besides the formation of protons upon oxidation of H2, vectorial translocation of protons from the inside to the outside could also occur . Proton liberation was inhibited by carbonylcyanide m-chlorophenylhydrazone (CCCP), 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO), and CuCl2 . Fast proton liberation with an H+/e- ratio of 0.65 +/- 0.1 was obtained after addition of the MQ analog 2,3-dimethyl-1,4-naphthoquinone (DMN) as an oxidant pulse . This acidification was also inhibited by CCCP, HOQNO, and CuCl2 . Oxidation of reduced DMN by PCE was not associated with fast acidification . The results with DMN indicate that the consumption and release of protons associated with redox reactions of MQ during electron transfer from H2 to PCE both occurred at the cytoplasmic side of the membrane . The PCE reductase was photoreversibly inactivated by 1-iodopropane, indicating that a corrinoid was involved in the PCE reduction.

Eur J Immunol, 1996 Apr, 26(4), 934 - 8
Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12; Duchmann R et al.; There is now increasing evidence that hyperresponsiveness towards intestinal flora is a crucial event in the pathogenesis of inflammatory bowel disease (IBD) . In support of this hypothesis, we recently described in humans that tolerance exists towards indigenous intestinal flora but is broken in active IBD lesions . In the present study, we have attempted to transfer this model into mice from different genetic backgrounds (BALB/c, SJL/J, C3H/HeJ) . We found that mononuclear cells from spleen, small bowel and large bowel of mice do not proliferate, i.e . are tolerant when exposed to bacterial sonicates derived from autologous intestine (BsA) but do proliferate, i.e . are immune when exposed to bacterial sonicates derived from the heterologous intestine of syngenic littermates (BsH) . Furthermore, we demonstrate that both local and systemic tolerance to BsA is broken in a murine model of chronic intestinal inflammation induced by the hapten reagent 2, 4, 6-trinitrobenzene sulfonic acid (TNBS), which mimics several important characteristics of Crohn's disease . Tolerance to BsA was restored and TNBS-induced colitis was abrogated in mice systemically treated with interleukin (IL)-10 or antibodies to IL-12 . Treatment specifically restored tolerance to BsA, but did not suppress proliferation to BsH . In summary, we here report a new model for the study of immunity and tolerance towards bacterial products . Our data suggest that tolerance to BsA is an important protective mechanism and that restoration of tolerance intestinal flora by IL-10 and antibodies to IL-12 may be of potential therapeutic utility in patients with inflammatory bowel disease.

Endocrinology, 1996 Apr, 137(4), 1497 - 500
Localization of binding sites in the central nervous system for leptin (OB protein) in normal, obese (ob/ob), and diabetic (db/db) C57BL/6J mice; Malik KF et al.; Leptin (OB protein) fused to the FLAG epitope and a kinase recognition site was expressed in bacteria, immunopurified, and phosphorylated using {gamma-(33)P} ATP . The resulting probe was used to characterize the distribution of leptin binding sites within brain sections of normal, ob/ob, and db/db C57BL/6J male mice . Leptin binding sites were found in leptomeninges and choroid plexus . Leptin binding within the choroid plexus is slightly elevated in ob/ob mice when compared to normal males (p<0.05) . Binding of leptin by the choroid plexus of db/db male mice is lower than in normal males (p<0.05), but normally distributed . Based on the association and dissociation rates of leptin binding on tissue sections, we estimate the K(D) of the choroid plexus site at 0.25X10(-9) M . From our results, we hypothesize that the binding of leptin to its site may cause the release or transport of uncharacterized factor(s) into the cerebral spinal fluid (CSF) to affect neuronal populations controlling feeding and metabolism.

Endocrinology, 1996 Apr, 137(4), 1395 - 401
Regulation of estrogen sulfotransferase in human endometrial adenocarcinoma cells by progesterone; Falany JL et al.; During the secretory phase of the human menstrual cycle, the endometrium is minimally responsive to the estrogens secreted from the ovaries . Conjugation of beta-estradiol (E2) with sulfate is thought to be an important mechanism in the regulation of the levels of active E2 in endometrial tissue . Estrogen sulfation is reportedly increased during the secretory phase in response to the high levels of progesterone secreted by the ovaries . Estrogen sulfotransferase (hEST), a distinct form of human cytosolic sulfotransferase (ST) with an affinity for E2 and estrone at low nanomolar concentrations, has recently been cloned and expressed in mammalian cells and in bacteria (J Steroid Biochem Mol Biol 52:529, 1995) . At least two other forms of human cytosolic ST, dehydroepiandrosterone ST (hDHEA-ST) and the phenol-sulfating form of phenol-ST (hP-PST), also conjugate estrogens but at micromolar concentrations . This report describes the specific induction of hEST in human Ishikawa endometrial adenocarcinoma cells by progesterone as a model for the increases in estrogen sulfation observed in women during the secretory phase of the menstrual cycle . Treatment of Ishikawa cells with 10 microns progesterone for 48 h resulted in a 7-fold increase in the sulfation of 20 nM E2 . The sulfation of selective substrates for human dehydroepiandrosterone sulfotransferase (hDHEA-ST) and the two forms of phenol sulfotransferase (hP-PST, hM-PST) were not affected by treatment with progesterone . The levels of immunoreactive hEST and hEST mRNA in the Ishikawa cells were both increased by progesterone, whereas the levels of immunoreactive hDHEA-ST, hP-PST, and hM-PST were not altered . hEST activity was not induced by treatment of Ishikawa cells with varying concentrations of E2, testosterone, or cortisol . The induction of hEST by progesterone was inhibited by RU-486, indicating that progesterone is acting via the progesterone receptor . These results indicate that progesterone is capable of specifically inducing hEST and estrogen sulfation in human Ishikawa adenocarcinoma cells and suggest a mechanism for increasing estrogen sulfation in the endometrium during the secretory phase of the menstrual cycle.

Ophthalmology, 1996 Apr, 103(4), 650 - 6
Bleb-related endophthalmitis after trabeculectomy with mitomycin C; Higginbotham EJ et al.; PURPOSE: To determine whether filtering blebs resulting from adjunctive use of mitomycin C (MMC) leads to an increased risk of endophthalmitis . METHODS: The authors retrospectively reviewed the records of 232 consecutive trabeculectomies performed at the W . K . Kellogg Eye Center with adjunctive use of MMC from May 1990 through June 1993 . Data obtained from the records included patient age, sex, race, type of glaucoma, site of filtration surgery, concentration and duration of exposure to MMC, presence of early or late bleb leakage, and the occurrence of endophthalmitis . RESULTS: Three patients were lost to follow-up less than 1 month after surgery . A total of 229 eyes of 192 patients (11 women and 82 men) were included in the study . Mean follow-up of patients remaining free of infection was 18.5 +/- 10.8 months (range, 1-44 months) . The overall incidence of bleb-related endophthalmitis was 2.6% . Endophthalmitis developed in 8% of patients (4 or 50) in whom an inferior approach was used and in 1.1% (2 or 179) in whom a superior approach was used (P = 0.02, Fisher's exact test) . The estimated odds ratio for the development of endophthalmitis after trabeculectomy with adjunctive MMC for inferior versus superior filtration sites was 7.7 . CONCLUSION: Short-term follow-up of trabeculectomies performed with adjunctive use of MMC demonstrates an overall incidence of endophthalmitis comparable to filtrationprocedures performed with 5-fluorouracil or without antifibrotic agents . However, inferior trabeculectomy performed with adjunctive MMC carries a significantly increased risk of bleb-related endophthalmitis compared with filters performed superiorly.

Nucleic Acids Res, 1996 Apr 1, 24(7), 1252 - 9
Comparative analysis of ribonuclease P RNA structure in Archaea; Haas ES et al.; Although the structure of the catalytic RNA component of ribonuclease P has been well characterized in Bacteria, it has been little studied in other organisms, such as the Archaea . We have determined the sequences encoding RNase P RNA in eight euryarchaeal species: Halococcus morrhuae, Natronobacterium gregoryi, Halobacterium cutirubrum, Halobacteriurn trapanicum, Methanobacterium thermoautotrophicum strains deltaH and Marburg, Methanothermus fervidus and Thermococcus celer strain AL-1 . On the basis of these and previously available sequences from Sulfolobus acidocaldarius, Haloferax volcanii and Methanosarcina barkeri the secondary structure of RNase P RNA in Archaea has been analyzed by phylogenetic comparative analysis . The archaeal RNAs are similar in both primary and secondary structure to bacterial RNase P RNAs, but unlike their bacterial counterparts these archaeal RNase P RNAs are not by themselves catalytically proficient in vitro.

J Leukoc Biol, 1996 Apr, 59(4), 505 - 11
Immunoregulation by interleukin-12; Trinchieri G et al.; Interleukin-12 (IL-12) is a heterodimeric cytokine produced primarily by antigen-presenting cells (monocytes, macrophages, dendritic cells, and B cells) . Its production is stimulated by bacteria, bacterial products, and intracellular parasites and enhanced by priming with granulocyte-macrophage colony-stimulating factor (CM-CSF) and interferon-gamma (IFN-gamma) or inhibited by IL-10 . The major biological activity of IL-12 is on T and natural killer (NK) cells in which it increases cytokine production, proliferation, and cytotoxicity . Its production occurs several hours after exposure to infections agents, which induces a rapid production of IFN-gamma by NK and later by T cells . This IFN-gamma potentiates antigen-presenting cell functions important in clearing infections agents (phagocytosis, oxidative burst, and production of nitrous oxide) and also increases further production of IL-12 . IL-12 has been clearly demonstrated to be important in the generation of CD4 and CD8 type 1 T cells both in vivo and in vitro . Our data reveals that IL-12 primes naive T cells for high IFN-gamma and IL-10 production, whereas IL-4 is required for IL-4 priming, thus suggesting that these genes and possibly others are independently regulated . IL-12 is therefore involved in the skewing of cytokine production toward a type 1 and has been implicated in being involved in selective mechanisms of established T cells . It is now becoming clear that the IL-12 acts as both a proinflammatory cytokine and an immunomodulator and therefore bridges the innate and adaptive immune responses.

J Bacteriol, 1996 Apr, 178(7), 1928 - 35
Increased production of colicin E1 in stationary phase; Eraso JM et al.; The synthesis of colicin E1 is known to be regulated by the SOS response, anaerobiosis, and catabolite repression . The expression of cea-lacZ fusions was also found to be stimulated when cells reached stationary phase . This increase in expression was determined to be due to depletion of nutrients from the medium, since the addition of fresh medium reversed the effect . Expression of the fusion increased when cells were starved in 10 mM MgSO4 and when they were grown in conditioned medium in which cells had been grown previously . The stimulation of expression occurred when the cea-lacZ fusion was present in single-copy as well as in multicopy plasmids . Finally, the data were consistent with this increase being independent of the SOS response, anaerobiosis, catabolite repression, and integration host factor as well as the stationary-phase regulators encoded by rpoS and lrp.

J Bacteriol, 1996 Apr, 178(7), 1908 - 13
Physical map of the genome of Planctomyces limnophilus, a representative of the phylogenetically distinct planctomycete lineage; Ward-Rainey N et al.; A physical map of the chromosome of Planctomyces limnophilus DSM 3776T was constructed by pulsed-field gel electrophoresis techniques . A total of 32 cleavage sites for the rare-cutting restriction endonucleases PacI, PmeI, and SwaI were located on the chromosome, which was shown to be circular and approximately 5.2 Mbp in size . An extrachromosomal element was detected but was found not to be cleaved by any of the enzymes used in the analysis of the chromosome . The order of the fragments on the chromosome was determined by hybridization of excised, labelled restriction fragments to Southern blots of pulsed-field gel electrophoresis-separated restriction digests . Seven genetic markers, rrs, rrl, atpD, tuf, gyrB, rpoD, and dnaK, on the chromosome were located by hybridization . Probes for all genetic markers were obtained by PCR . For five of these markers, probes were constructed by PCR with degenerate primers targeting conserved sequences . The arrangement of the genetic markers was compared with that found in other bacteria.

J Bacteriol, 1996 Apr, 178(7), 1858 - 65
Biochemical and molecular characterization of the extracellular esterase from Streptomyces diastatochromogenes; Tesch C et al.; An esterase of Streptomyces diastatochromogenes was purified to homogeneity from culture filtrate . The purified enzyme had a molecular mass of 30,862 +/- 5.8 Da, as determined by electrospray mass spectrometry . The esterase-encoding gene was cloned on a 5.1-kb MboI fragment from S . diastatochromogenes genomic DNA into Streptomyces lividans TK23 by using plasmid vector pIJ702 . Nucleotide sequence analysis predicted a 978-bp open reading frame, estA, encoding a protein of 326 amino acids, a potential ribosome binding site, and a putative 35- or 36-residue signal peptide for secretion in S . lividans or S . diastatochromogenes, respectively . The transcriptional initiation site was mapped 29 nucleotides upstream from the predicted translational start codon of estA in S . diastatochromogenes . The protein sequence deduced from the estA gene was similar to that of the esterase from the plant pathogen Streptomyces scabies . Both enzymes lacked the conserved motif GXSXG carrying the active-site serine of hydrolytic enzymes . A serine modified by {1,3-3H}diisopropyl fluorophosphate was located at position 11 of the mature enzyme in the sequence GDSYT . This finding and results obtained by site-directed mutagenesis studies indicate that serine 11 may be the active-site nucleophile.

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