Microorganism

J Theor Biol, 1995 Aug 21, 175(4), 437 - 55
How much are homologous peptides homologous?
Incardona F, Morante S, Parisi V, Rossi GC.
A statistical analysis designed to deal with the problem of identifying homologous pairs of "short sequences of amino-acids" (= peptides) belonging to different proteins is presented . The conceptual novelty of the searching strategy proposed here lies in the fact that both the degree of homology of the two peptides of the pair (measured by a suitably defined affinity score) and the level of statistical significance of its occurrence are taken into account on equal footing . They are combined in a sort of "biological indicator", characterising each pair . Pairs for which the value of the biological indicator is larger than an appropriate threshold are taken as statistically significant and (putatively) biologically relevant . The method is employed in various test cases and proves to be reliable and efficient . In particular we have studied the cases in which the known existence of an auto-immune response has lead to the identification of homologous peptide pairs between human and viral or bacterial proteins . The detection efficiency of the algorithm in these cases turns out to be especially good when the most naive affinity table, the Identity matrix, is employed to measure the similarity of amino acidic pairs . In contrast, when the 250-PAM mutation matrix is used, the detection efficiency goes to zero.

Arch Biochem Biophys, 1995 Aug 20, 321(2), 526 - 30
Peroxisomal membrane protein PMP68 of mouse liver: cloning of a cDNA encompassing the nucleotide binding fold and epitope mapping of monoclonal antibodies to the expressed protein; Chen N et al.; We have isolated and sequenced a cDNA which encodes 376 amino acids toward the carboxy-terminus, and encompassing the putative nucleotide binding fold, of PMP68 (mouse liver peroxisomal integral membrane protein of 68 kDa) the major integral membrane protein of mouse liver peroxisomes . The protein sequence predicted from this cDNA shows 97.9% amino acid identity to this same region of rat liver PMP70, a member of the ATP-binding cassette protein superfamily (K . Kamijo, S . Taketani, S . Yokota, T . Osumi, and T . Hashimoto, 1990, J . Biol . Chem . 265, 4534-4540) . The section of the cDNA encoding the hydrophilic and putative cytoplasmic domain of PMP68 was expressed as a recombinant fusion protein in bacteria . Two monoclonal antibodies raised against this protein have been epitope-mapped to peptides generated by cyanogen bromide cleavage of the fusion protein . Antibody 1A4 recognizes a peptide whose sequence contains the first motif of the putative nucleotide binding fold of PMP68, and antibody 8F11 recognizes a carboxy-terminal peptide which includes the second motif of this nucleotide binding fold . These antibodies are expected to be useful in the elucidation of the biological function of this putative membrane transporter.

Gene, 1995 Aug 19, 161(2), 271 - 5
Cloning of the cDNA encoding human C/EBP gamma, a protein binding to the PRE-I enhancer element of the human interleukin-4 promoter; Davydov IV et al.; The positive regulatory element-I (PRE-I) is a strong enhancer element essential for expression of the human interleukin-4 (IL-4)-encoding gene . In order to identify transcription factors binding to PRE-I, we screened a cDNA expression library from human Jurkat T-cells . A cDNA encoding the human CCAAT/enhancer binding protein-gamma (hC/EBP gamma) was cloned . The deduced amino acid (aa) sequence of HC/EBP gamma contains 150 aa with high homology to mouse Ig/EBP-1 and rat C/EBP gamma . The mRNA of hC/EBP gamma is expressed at a high level in Jurkat T-cells in three forms generated via differential polyadenylation . DNA-binding experiments with recombinant protein produced in bacteria demonstrate that hC/EBP gamma binds to PRE-I, but not to unrelated DNA fragments . Our data also show that hC/EBP gamma may cooperate with Fos to bind PRE-I.

Gene, 1995 Aug 19, 161(2), 199 - 203
Isolation and characterization of the lysozyme-encoding gene from the silkworm Bombyx mori; Lee WJ et al.; We have isolated and characterized a Bombyx mori (Bm) cDNA encoding a lysozyme (Lyz) . A 90-bp DNA fragment was amplified by PCR using degenerate oligodeoxyribonucleotide primers derived from the known amino acid (aa) sequence of the Bm Lyz . These PCR fragments were used to screen a fat body cDNA library . A clone containing the complete lys cDNA (1294 bp) was isolated and completely sequenced . The deduced 137-aa sequence showed high homology with other chicken-type Lyz . Bm lys gene expression was constitutive in fat body, cuticular epidermal tissue and at a very low level in hemocytes . This gene expression was up-regulated in fat body, hemocytes and cuticular epidermal tissue following the injection of Gram+ bacteria.

Sci Total Environ, 1995 Aug 18, 170(1-2), 1 - 19
Collection of domestic waste . Review of occupational health problems and their possible causes; Poulsen OM et al.; During the last decade, a growing interest in recycling of domestic waste has emerged, and action plans to increase the recycling of domestic waste have been agreed by many governments . A common feature of these plans is the implementation of new systems and equipment for the collection of domestic waste which has been separated at source . However, only limited information exists on possible occupational health problems related to such new systems . Occupational accidents are very frequent among waste collectors . Based on current knowledge, it appears that the risk factors should be considered as an integrated entity, i.e . technical factors (poor accessibility to the waste, design of equipment) may act in concert with high working rate, visual fatigue due to poor illumination and perhaps muscle fatigue due to high work load . Musculoskeletal problems are also common among waste collectors . A good deal of knowledge has accumulated on mechanical load on the spine and energetic load on the cardio-pulmonary system in relation to the handling of waste bags, bins, domestic containers and large containers . However, epidemiologic studies with exposure classification based on field measurement are needed, both to further identify high risk work conditions and to provide a detailed basis for the establishment of occupational exposure limits for mechanical and energetic load particularly in relation to pulling, pushing and tilting of containers . In 1975, an excess risk for chronic bronchitis was reported for waste collectors in Geneva (Rufener-Press et al., 1975) and data from the Danish Registry of Occupational Accidents and Diseases also indicate an excess risk for pulmonary problems among waste collectors compared with the total work force . Surprisingly few measurements of potentially hazardous airborne exposures have been performed, and the causality of work-related pulmonary problems among waste collectors is unknown . Recent studies have indicated that implementation of some new waste collection systems may result in an increased risk of occupational health problems . High incidence rates of gastrointestinal problems, irritation of the eye and skin, and perhaps symptoms of organic dust toxic syndrome (influenza-like symptoms, cough, muscle pains, fever, fatigue, headache) have been reported among workers collecting the biodegradable fraction of domestic waste . The few data available on exposure to bio-aerosols and volatile compounds have indicated that these waste collectors may be simultaneously exposed to multiple agents such as dust containing bacteria, endotoxin, mould spores, glucans, volatile organic compounds, and diesel exhaust . Several studies have reported similar health problems as well as high incidence rates of pulmonary disease among workers at plants recycling domestic waste.(ABSTRACT TRUNCATED AT 400 WORDS)

Proc Natl Acad Sci U S A, 1995 Aug 15, 92(17), 8036 - 40
Mutation of the principal sigma factor causes loss of virulence in a strain of the Mycobacterium tuberculosis complex; Collins DM et al.; Tuberculosis continues to be responsible for the deaths of millions of people, yet the virulence factors of the causative pathogens remain unknown . Genetic complementation experiments with strains of the Mycobacterium tuberculosis complex have identified a gene from a virulent strain that restores virulence to an attenuated strain . The gene, designated rpoV, has a high degree of homology with principal transcription or sigma factors from other bacteria, particularly Mycobacterium smegmatis and Streptomyces griseus . The homologous rpoV gene of the attenuated strain has a point mutation causing an arginine-->histidine change in a domain known to interact with promoters . To our knowledge, association of loss of bacterial virulence with a mutation in the principal sigma factor has not been previously reported . The results indicate either that tuberculosis organisms have an alternative principal sigma factor that promotes virulence genes or, more probably, that this particular mutant principal sigma factor is unable to promote expression of one or more genes required for virulence . Study of genes and proteins differentially regulated by the mutant transcription factor should facilitate identification of further virulence factors.

FEBS Lett, 1995 Aug 14, 370(1-2), 15 - 8
Solubilization and purification of aldehyde-generating fatty acyl-CoA reductase from green alga Botryococcus braunii; Wang X et al.; Membrane-bound fatty acyl-CoA reductase from the green alga Botryococcus braunii has been solubilized from the microsomal preparation by 0.1% octyl beta-glucoside and purified to near homogeneity by Blue A agarose and palmitoyl-CoA agarose affinity column chromatography . The molecular mass of the enzyme was estimated by SDS-PAGE to be 35 kDa . The enzyme generates fatty aldehyde by reduction of fatty acyl-CoA with NADH as the reductant . The N-terminal amino acid sequence of this protein that represents the first eucaryotic aldehyde-generating reductase to be purified shows high homology with the N-terminus of fatty acid reductase from bacteria.

Genomics, 1995 Aug 10, 28(3), 450 - 61
Mammalian mitochondrial intermediate peptidase: structure/function analysis of a new homologue from Schizophyllum commune and relationship to thimet oligopeptidases; Isaya G et al.; Mitochondrial intermediate peptidase (MIP) is a component of the mitochondrial protein import machinery required for maturation of nuclear-encoded precursor proteins targeted to the mitochondrial matrix or inner membrane . We previously characterized this enzyme in rat (RMIP) and Saccharomyces cerevisiae (YMIP) and showed that MIP activity is essential for mitochondrial function in yeast . We have now defined the structure of a new MIP homologue (SMIP) from the basidiomycete fungus Schizophyllum commune . SMIP includes 4 exons of 523, 486, 660, and 629 bp separated by 3 short introns . The predicted SMIP, YMIP, and RMIP sequences share 31-37% identity and 54-57% similarity over 700 amino acids . When SMIP and RMIP were expressed in a yeast mip1 delta mutant, they were both able to rescue the respiratory-deficient phenotype caused by genetic inactivation of YMIP, indicating that the function of this enzyme is conserved in eukaryotes . Moreover, the MIP sequences show 20-24% identity and 40-47% similarity to a family of oligopeptidases from bacteria, yeast, and mammals . MIP and these proteins are characterized by a highly conserved motif, F-H-E-X-G-H-(X)2-H-(X)12-G-(X)5-D-(X)2-E-X-P-S-(X)3-E-X, centered around a zinc-binding site and appear to represent a new family of genes associated with proteolytic processing in the mitochondrial and cytosolic compartments.

J Theor Biol, 1995 Aug 7, 175(3), 325 - 53
Immune responses against multiple epitopes; Nowak MA et al.; The current understanding of antigenic escape dynamics is based on models with single epitopes . The usual idea is that a mutation which enables a pathogen (virus, bacteria, etc) to escape from a given immune response confers a selective advantage . The "escape mutant" may then increase in abundance until it induces a new specific response against itself . In this paper a new picture is developed, based on mathematical models of immune responses against several epitopes; the simplest such models can have very complicated dynamics, with some surprising features . The emergence of an escape mutant can shift the immunodominant response to another epitope . Even in the absence of mutations, antigenic oscillation is found, with distinct peaks of different virus variants and fluctuations in the size and specificity of the immune responses . The model also provides a general theory for immunodominance in the presence of antigenic variation . Immunodominance is determined by the immunogenicity and by the antigenic diversity of the competing epitopes . Antigenic oscillations and fluctuations in the cytotoxic T-lymphocyte response have been observed in infections with the human immunodeficiency virus (HIV) . Shifting the immune responses to weaker epitopes can represent a mechanism for disease progression based on evolutionary dynamics and antigenic diversity of the virus.

J Clin Periodontol, 1995 Aug, 22(8), 609 - 12
Dental treatment of Papillon-Lefèvre syndrome: 15-year follow-up; Tinanoff N et al.; A 9-year-old girl was initially treated for the periodontal component of Papillon-Lefevre syndrome by extraction of all patient's erupted teeth, after unsuccessful clinical treatment with two different antibiotics . Follow-up dental records at age 24 showed the patient to have generalized gingivitis and poor oral hygiene; however, no additional teeth were lost or mobile . Radiographically, the alveolar crests, lamina dura, and periodontal ligament spaces appeared normal for a subject with missing teeth . Initially, the patient had depressed polymorphonuclear leukocyte (PMN) chemotaxis and adherence, as well as evidence of periodontal infection with Actinobacillus actinomycetemcomitans, (A.a.) . The 6 and 15-year follow-ups showed normal PMN function and no detectable A.a . The improvement of the patient's PMN function was coincident with lack of detection of certain periodontopathic bacteria . If the PMN dysfunction of PLS is secondary to the infection, the reasons for the initiation of the disease still need to be clarified.

Eur J Gastroenterol Hepatol, 1995 Aug, 7 Suppl 1, S83 - 8
Differences in urease activity in live Helicobacter pylori cultured from patients with gastroduodenal diseases; Ito S et al.; AIM: To develop a reliable method for measuring urease activity in live bacteria, and to determine whether there are any differences in urease activity among the Helicobacter pylori strains involved in gastroduodenal disease . DESIGN: The stability of the method was examined in the first phase of the study, and in a second phase the mean urease activity in clinical isolates from different groups of patients was compared . MATERIALS AND METHODS: To assess the stability and reliability of the method, we assessed the relationship between bacterial proliferation and urease activity, the relationship between the number of bacteria and the optical density, and differences in urease activity among bacterial generations . Ten of the 3-day-old colonies in the third generation were suspended in phosphate-buffered saline, and urease activity was measured as 10(5) colony-forming units/ml bacteria . RESULTS: The assay system appeared to be effective, because the urease activity of live bacteria in the logarithmic growth period was constant, the number of bacteria and the optical density showed a linear correlation on a bilogarithmic graph and there was no significant difference in urease activity over three generations . With this method, urease activity varied from 0.192 to 80.42 mIU/10(5) colony-forming units of bacteria/ml . There was no significant difference in the mean urease activity of live bacteria from controls, gastric ulcer patients and duodenal ulcer patients . However, the mean urease activity in bacteria from cancer patients was significantly higher than that of controls or duodenal ulcer patients . CONCLUSIONS: H . pylori strains derived from cancer patients, which have relatively high levels of urease activity, might easily colonize the stomach and lead to much mucosal damage during the long course of H . pylori infection.

Mol Ecol, 1995 Aug, 4(4), 483 - 91
Molecular structure of the Frankia spp . nifD-K intergenic spacer and design of Frankia genus compatible primer; Nalin R et al.; The nifD-K intergenic spacer (IGS) of ArI3 and ACoN24d were found to have a length 265 and 199 nucleotides, respectively . They are markedly less conserved than the two neighbouring genes and have, in some instances, a repeated structure reminiscent of an insertion event . The repeated sequence and the IGSs have no detectable homology with sequences in DNA databanks . The IGS has a stem-loop structure with a low folding energy, lower than that between nifH and nifD . No convincing alignment of IGS sequences could be obtained among Frankia strains . Only between ACoN24d and ArI3, which belong to the same genomic species, was the alignment good enough to permit detection of a doubly repeated structure . No promoter could be detected in the IGSs . The putative nifK open reading frame (ORF) in Frankia strain ArI3 has a length of 1587 nucleotides, starting with a GTG codon, preceded by a ribosome binding site of a structure similar to that of nifH (GGAGGN7) . The codon usage was similar to that of previously sequenced Frankia genes with a strong bias toward G- and C-ending codons except in the case of glycine where GGT is frequent . Alignment of the three Frankia nifK sequences (EUN1f; ArI3 and ACoN24d) with those of other nitrogen-fixing bacteria permitted detection of a sequence conserved among the three Frankia strains but absent in the other sequences . A primer targeted to that region in combination with FGPD807-85 amplified the nifD-KIGS sequences of all Frankia strains (except the non-nitrogen-fixing Frankia strains CN3 and AgB1-9) and yet failed to amplify DNA of all other nitrogen-fixing bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

J Chemother, 1995 Aug, 7(4), 344 - 54
The place of tobramycin in lower respiratory tract infections (LRTI); Gialdroni Grassi G; The Author provides a review of clinical experience with tobramycin as therapy for lower respiratory tract infections, in comparison to other aminoglycosides, including the pharmacokinetics and toxicity, dwelling on oto- and nephrotoxicity . The article includes a discussion of various dosing regimens of the aminoglycosides, focussing on efficacy and toxicity arising from once-daily administration . The Author then provides a more detailed description of tobramycin's pharmacokinetics, indications for its use, and the possibilities of once-daily dosing, concluding that toxicity is favorably influenced by a single daily administration as well as efficacy, and that patient compliance and reduced hospital costs are other advantages of this regimen.

Biomaterials, 1995 Aug, 16(12), 917 - 20
Localized chronic suppurative bone infection as a sequel of peri-implantitis in a hydroxyapatite-coated dental implant; Piattelli A et al.; Plaque-induced lesions can produce peri-implant bone loss with ultimate implant loss . Although the peri-implant tissues seem to be more resistant than the periodontal ones to plaque and calculus, they can produce a more extensive spread of the infection to the deeper tissues around implants . The case of a 45-year-old female patient is presented in which, over a three year period, there was a progressive loss of peri-implant bone and the formation of a periapical radiolucency with an external fistula . The implant was removed and examined with the cutting-grinding system . Microscopy examination showed that most of the hydroxyapatite (HA) was still adherent to the metal . There was a detachment in the area of the HA-titanium interface . The implant surface was almost completely covered by bacteria . Bacteria were also present in the bone medullary spaces surrounding the implant . The infection of the periodontal tissues had progressed into the alveolar bone, thus producing a localized bone infection . The cause of the implant failure is probably related to a defective connection of the abutment or to overloading of the implant due to the presence of interlocks in the prosthetic restoration.

Clin Infect Dis, 1995 Aug, 21(2), 341 - 4
Zaldaride maleate (a new calmodulin antagonist) versus loperamide in the treatment of traveler's diarrhea: randomized, placebo-controlled trial; Okhuysen PC et al.; The present study was undertaken to compare the efficacy of a new calmodulin antagonist, zaldaride maleate, with that of placebo or loperamide in persons with traveler's diarrhea . One hundred seventy-nine patients were randomized to receive loperamide (4 mg followed by 2 mg after each unformed stool), zaldaride maleate (20 mg four times per day), or placebo . During the initial 48 hours of therapy, zaldaride maleate decreased the number of unformed stools by 30% and the duration of illness by 23% when compared with placebo . Loperamide was superior to both zaldaride maleate and placebo during the initial hours of treatment . However, after 48 hours of treatment, loperamide and zaldaride maleate were equally efficacious, decreasing by > 50% the number of unformed stools passed in a 24-hour interval (P, not significant), and were both superior when compared with placebo (P < .0001 and P = .0048, respectively) . The apparent superiority of loperamide early in the course of therapy appeared to be related to a loading-dose effect and not to any differences in antidiarrheal properties.

Hybridoma, 1995 Aug, 14(4), 347 - 54
Generation and characterization of monoclonal antibodies specific for members of the mammalian 70-kDa heat shock protein family; Green JM et al.; The 70-kDa heat shock proteins (hsp70) are a highly conserved, abundant, and ubiquitous family of proteins expressed by all organisms from bacteria to humans . It is well established that hsp70 family members function as molecular chaperones and aid in the intracellular folding of newly synthesized or denatured proteins . Current evidence suggests an emerging role for hsp70 family members in immune responses and in clinically important responses to stress and tissue damage . Here we report the generation and characterization of several MAbs to hsp70 family members . Immune responses to this highly conserved family were induced in mice by immunization with synthetic peptides that contain regions of the mouse mitochondrial hsp70 coupled to a potent helper T cell epitope derived from tetanus toxoid . The resulting MAbs include ones specific for the human and mouse mitochondrial hsp70 and others that show cross-reactivity among the family members and recognize the mitochondrial hsp70, the endoplasmic reticulum resident hsp70, Bip/grp78, the constitutively expressed cytosolic hsp70, hsc70, and the heat-induced member, hsp70 . Significantly, these MAbs are effective in Western blotting, in immunoprecipitation, and in immunofluorescence, and thus should find applications in the purification and detection of members of this important family.

Protein Sci, 1995 Aug, 4(8), 1608 - 17
Multidomain organization of eukaryotic guanine nucleotide exchange translation initiation factor eIF-2B subunits revealed by analysis of conserved sequence motifs; Koonin EV; Computer-assisted analysis of amino acid sequences using methods for database screening with individual sequences and with multiple alignment blocks reveals a complex multidomain organization of yeast proteins GCD6 and GCD1, and mammalian homolog of GCD6-subunits of the eukaryotic translation initiation factor eIF-2B involved in GDP/GTP exchange on eIF-2 . It is shown that these proteins contain a putative nucleotide-binding domain related to a variety of nucleotidyltransferases, most of which are involved in nucleoside diphosphate-sugar formation in bacteria . Three conserved motifs, one of which appears to be a variant of the phosphate-binding site (P-loop) and another that may be considered a specific version of the Mg(2+)-binding site of NTP-utilizing enzymes, were identified in the nucleotidyltransferase-related domain . Together with the third unique motif adjacent to the the P-loop, these motifs comprise the signature of a new superfamily of nucleotide-binding domains . A domain consisting of hexapeptide amino acid repeats with a periodic distribution of bulky hydrophobic residues (isoleucine patch), which previously have been identified in bacterial acetyltransferases, is located toward the C-terminus from the nucleotidyltransferase-related domain . Finally, at the very C-termini of GCD6, eIF-2B epsilon, and two other eukaryotic translation initiation factors, eIF-4 gamma and eIF-5, there is a previously undetected, conserved domain . It is hypothesized that the nucleotidyltransferase-related domain is directly involved in the GDP/GTP exchange, whereas the C-terminal conserved domain may be involved in the interaction of eIF-2B, eIF-4 gamma, and eIF-5 with eIF-2.

Protein Sci, 1995 Aug, 4(8), 1577 - 86
The distribution of alpha-helix propensity along the polypeptide chain is not conserved in proteins from the same family; Munoz V et al.; We address the question of whether the distribution of secondary structure propensities of the residues along the polypeptide chain (denominated here as secondary structure profiles) is conserved in proteins throughout evolution, for the particular case of alpha-helices . We have analyzed by CD the conformation of peptides corresponding to the five alpha-helices of two alpha/beta parallel proteins (ComA and Ara) . The large alpha-helical population of peptide ComA-4 detected by CD in aqueous solution has been confirmed by NMR . These proteins are members of the CheY and P21-ras families, respectively, which have been studied previously in the same way (Munoz V, Jimenez MA, Rico M, Serrano L, 1995, J Mol Biol 245:275-296) . Comparison of the helical content of equivalent peptides reveals that protein alpha-helix propensity profiles are not conserved . Some equivalent peptides show very different helical populations in solution and this is especially evident in very divergent proteins (ComA and CheY) . However, all the peptides analyzed so far adopted an important population of helical conformations in the presence of 30% trifluoroethanol, indicating that there could be a conserved minimal requirement for helical propensity.

J Vet Med Sci, 1995 Aug, 57(4), 777 - 9
An outbreak of goose parvovirus infection in Japan; Takehara K et al.; In a Muscovy duck breeding-growing farm in Aomori prefecture, most of ducklings hatched during spring in 1994 died within two-week-old . The mortality was nearly 100% . In most cases, birds died without clinical signs and some with leg weakness . By serological and virological tests, the outbreak was identified as a goose parvovirus infection . In pathological test, however, no typical manifestations of goose parvovirus infections (hepatitis and intranuclear inclusion bodies in hepatic cells) were detected.

Ostomy Wound Manage, 1995 Aug, 41(7A Suppl), 37S - 44S; discussion 45S
Moist wound healing: the clinical perspective; Kerstein MD; Wound dressings can have a profound effect on the repair process and patient quality of life . Topical treatment modalities have been found to influence reepithelialization, granulation tissue formation, the degradation of fibrin and necrotic tissue, the incidence of wound infections, the pH of the wound as well as airborne dispersal of bacteria, pain and cost of care . Clinical studies have shown that, compared to conventional gauze, most moisture-retentive dressings will reduce time to healing, the rate of wound infections, patient pain and the cost of care . However, clinical studies have also revealed differences among some of the moisture-retentive dressings available today . As our knowledge about the effect of wound care interventions continues to expand, treatment decisions should be made based on the results of controlled clinical studies.

J Mol Evol, 1995 Aug, 41(2), 211 - 23
Molecular phylogeny of the Homoptera: a paraphyletic taxon; von Dohlen CD et al.; Homoptera and Heteroptera comprise a large insect assemblage, the Hemiptera . Many of the plant sap-sucking Homoptera possess unusual and complex life histories and depend on maternally inherited, intracellular bacteria to supplement their nutritionally deficient diets . Presumably in connection with their diet and lifestyles, the morphology of many Homoptera has become greatly reduced, leading to major controversies regarding the phylogenetic affiliations of homopteran superfamilies . The most fundamental question concerns whether the Homoptera as a whole are monophyletic . Recent studies based on morphology have argued that the Homoptera Sternorrhyncha (Aphidoidea, Coccoidea, Psylloidea, Aleyrodoidea) is a sister group to a group comprising the Homoptera Auchenorrhyncha (Fulgoroidea, Cicadoidea, Cercopoidea, Cicadelloidea) and the Heteroptera, making the Homoptera paraphyletic . We sequenced the 5' 580-680 base pairs of small-subunit (18S) ribosomal DNA from a selection of Homoptera, Hemiptera, and their putative outgroups, the Thysanoptera and Psocoptera, to apply molecular characters to the problem of Homoptera phylogeny . Parsimony, distance, maximum-likelihood, and bootstrap methods were used to construct trees from sequence data and assess support for the topologies produced . Molecular data corroborate current views of relationships within the Sternorrhyncha and Auchenorrhyncha based on morphology and strongly support the hypothesis of homopteran paraphyly as stated above . In addition, it was found that Homoptera Sternorrhyncha have extra, GC-rich sequence concentrated in a variable region of the 18S rDNA, which indicates that some unique evolutionary processes are occurring in this lineage.

Plant Physiol, 1995 Aug, 108(4), 1461 - 9
The genomic region of rbcLS in Synechococcus sp . PCC 7942 contains genes involved in the ability to grow under low CO2 concentration and in chlorophyll biosynthesis; Ronen-Tarazi M et al.; Several genes involved in the ability of Synechococcus sp . PCC 7942 to grow under different CO2 concentrations were mapped in the genomic region of rbcLS (the operon encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase) . Insertion of a cartridge encoding kanamycin resistance within open reading frame (ORF) 78, designated ccmJ, located 7 kb upstream of rbcLS, resulted in a kanamycin-resistant, high-CO2-requiring mutant, M3, which does not contain normal carboxysomes . ccmJ shows significant homology to csoS1 encoding a carboxysomal shell polypeptide in Thiobacillus neopolitanus . Analysis of the polypeptide pattern of a carboxysome-enriched fraction indicated several differences between the wild type and the mutant . The amount of the ribulose-1,5-bisphosphate carboxylase/oxygenase subunits was considerably smaller in the carboxysomal fraction of the mutant when compared to the wild type . On the basis of the sequence analyses, ORF286 and ORF466, located downstream of ccmJ, were identified as chlL and chlN, respectively, which are involved in chlorophyll biosynthesis in the dark.

FEMS Microbiol Lett, 1995 Aug 1, 130(2-3), 129 - 35
A mutation that decreases the efficiency of plasmid R1 replication leads to the activation of parD, a killer stability system of the plasmid; Ruiz-Echevarria MJ et al.; The silent parD (kis/kid) stability operon of plasmid R1 is normally repressed by the co-ordinated action of the Kis and Kid proteins . In this report it is shown that a mutation in repA, the gene of the plasmid replication protein, that reduces two-fold the copy number of the plasmid, leads to the derepression of the parD system . This derepression can be prevented by a suppressor mutation in copB, a copy number control gene of plasmid R1, that increases the efficiency of replication of the repA mutant . Derepression of the wild-type parD system leads to high plasmid stability . These data show the activation of a plasmid stability operon by a mutation that reduces the efficiency of wild-type plasmid replication.

J Leukoc Biol, 1995 Aug, 58(2), 137 - 50
Associations between the neuroendocrine and immune systems; Weigent DA et al.; Organisms respond to infection with complex adaptations involving bidirectional communication between the immune and neuroendocrine systems . The idea of intercellular communication between the neuroendocrine and immune systems via common signal molecules has provided a conceptual framework for such crosstalk . The studies to date show that cells of the immune system contain receptors for neuroendocrine hormones and can also be considered a source of pituitary and hypothalamic peptides . The structure and pattern of synthesis of these peptides by leukocytes appear similar to neuroendocrine hormones, although some differences exist . Once secreted, these peptide hormones may function as endogenous regulators of the immune system as well as conveyors of information from the immune to the neuroendocrine system . The plasma hormone concentrations contributed by lymphocytes usually do not reach the levels required when the pituitary gland is the source, but because immune cells are mobile, they have the potential to locally deposit the hormone at the target site . Likewise, other studies show that cells of the neuroendocrine system contain receptors for cytokines and can also be considered a source of cytokines, particularly interleukin-1 (IL-1) and IL-6 . In the pituitary IL-1 beta coexists with thyroid stimulating hormone in a subpopulation of thyrotropes, suggesting it may have a role as a pituitary paracrine factor . The cytokines, including IL-1, IL-2, IL-6, interferon-gamma, and tumor necrosis factor, exert profound effects on hypothalamic pituitary axes . It is our hypothesis that the relay of information to the neuroendocrine system represents a sensory function for the immune system wherein leukocytes recognize stimuli that are not recognizable by the central and peripheral nervous systems (i.e., bacteria, tumors, viruses, and antigens) . The recognition of such noncognitive stimuli by immunocytes is then converted into information and a physiological change occurs . Future studies into the physiological role that cytokines and neuroendocrine hormones have in these systems will be of considerable interest for both immunologists and endocrinologists.

J Bacteriol, 1995 Aug, 177(16), 4765 - 71
Minimal requirements of the Streptomyces lividans 66 oriC region and its transcriptional and translational activities; Zakrzewska-Czerwinska J et al.; Deletion analysis of a previously constructed minichromosome revealed that a stretch of DNA which is longer than 623 bp but shorter than 837 bp is required for autonomous replication of the Streptomyces lividans chromosome . Each of the dnaA and dnaN genes flanking the oriC region is individually transcribed from two promoters . Within the intergenic, nontranslatable region between the dnaA and dnaN genes, five main transcripts and several less abundant transcripts of various lengths as well as one of the promoters were identified . The introduction of additional DnaA boxes in S . lividans led to a significant increase in dnaA gene transcripts and to an enhanced level of the DnaA (73-kDa) protein . In summary, the data suggest that dnaA gene transcription is autoregulated and that initiation of the S . lividans chromosome is tightly controlled.

Exp Cell Res, 1995 Aug, 219(2), 709 - 16
G protein function during biomembrane fusion in Dictyostelium: presence and importance of a G alpha s subunit during fertilization and phagocytosis; Browning DD et al.; Previous work has shown that GTPase function is essential for fertilization and cannibalistic phagocytosis during the sexual development of Dictyostelium discoideum . In this work, the importance of heterotrimeric G proteins during these events was established further using aluminum fluoride which inhibited both fertilization and cannibalistic phagocytosis, as well as the phagocytosis of bacteria by vegetative amoebae . Using distinct immune sera directed against the amino terminus and the carboxy terminus of mammalian G alpha s, we have provided unique evidence for a G alpha s subunit of approximately 55 kDa in D . discoideum (referred to as dG alpha s) . Furthermore, this protein localizes to the membranes of fusing cells as well as to both vegetative and zygote giant cells, indicating that it might function during fertilization as well as during both vegetative (i.e., bacterial) and cannibalistic (i.e., amoebal) phagocytosis . During its down-regulation in nonphagocytic cells new isozymes of dG alpha s appear, suggesting that it may be posttranslationally modified . Having identified a putitive G alpha s homologue, this work has set the stage for further investigations into its function in Dictyostelium.

Biochem J, 1995 Aug 1, 309 ( Pt 3), 715 - 9
The cloning and sequence of the C isoform of PtdIns4P 5-kinase; Divecha N et al.; In this study we describe the purification and sequencing of the C isoform of platelet PtdIns4P 5-kinase . Subsequently a cDNA was isolated from a human circulating-leucocyte library, which when sequenced was shown to contain all of the peptides identified in the purified protein . In addition, expression of this cDNA in bacteria led to the production of a protein which was recognized by specific monoclonal antibodies raised to the bovine brain enzyme {Brooksbank, Hutchings, Butcher, Irvine and Divecha (1993) Biochem . J . 291, 77-82} and also led to the appearance of PtdIns4P 5-kinase activity in the bacterial lysates . Interestingly, the cDNA showed no similarity to any of the previously cloned inositide kinases . A search of the DNA databases showed that two proteins from Saccharomyces cerevisiae shared close similarity to this enzyme, one of which, the mss4 gene product, has been implicated in the yeast inositol lipid pathway . These data suggest that the PtdIns4P 5-kinases are a new family of inositide kinases unrelated to the previously cloned phosphoinositide 3/4-kinases.

Arch Biochem Biophys, 1995 Aug 1, 321(1), 191 - 8
Characterization, subcellular localization, and developmental regulation of a cysteine proteinase from Dictyostelium discoideum; Mehta DP et al.; Previous studies showed that vegetative cells of Dictyostelium discoideum make a cysteine proteinase called proteinase-1, which contains multiple residues of GlcNAc-1-P linked directly to peptidyl serines . As a prelude to understanding the function of this novel carbohydrate modification, we purified and extensively characterized this proteinase in terms of its enzymatic activity, subcellular localization, and developmental regulation . The purified enzyme has an apparent molecular weight of 38 kDa in heat-denatured, reducing SDS/PAGE and 55 kDa under nonreducing conditions . Native gel electrophoresis and isoelectric focusing revealed two protein bands with equal activity and having pI values of 2.5 and 2.6 . Even more complex patterns are found in non-heat-denatured SDS/PAGE gels . However, partial amino acid sequencing of the purified protein gave predominantly a single sequence . The enzyme is inhibited by trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, Na-p-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, and leupeptin, has a pH optimum of 5.0, and cofractionates with lysosomal enzymes in bacterially grown cells . It appears to comprise about 90% of the total cysteine proteinase activity in cells at a time when the cells have just finished clearing the bacterial lawn . Prior to this point and after the onset of development, its level is 2- to 20-fold lower . This remarkably fine regulation parallels the developmental regulation of other cysteine proteinases in Dictyostelium . Based on these results it appears that proteinase-1 may be primarily used for specialized proteolysis just before the onset of development rather than for simply digesting the bacteria for food.

Proc Natl Acad Sci U S A, 1995 Aug 1, 92(16), 7148 - 52
Sequence analysis of a mannitol dehydrogenase cDNA from plants reveals a function for the pathogenesis-related protein ELI3; Williamson JD et al.; Mannitol is the most abundant sugar alcohol in nature, occurring in bacteria, fungi, lichens, and many species of vascular plants . Celery (Apium graveolens L.), a plant that forms mannitol photosynthetically, has high photosynthetic rates thought to results from intrinsic differences in the biosynthesis of hexitols vs . sugars . Celery also exhibits high salt tolerance due to the function of mannitol as an osmoprotectant . A mannitol catabolic enzyme that oxidizes mannitol to mannose (mannitol dehydrogenase, MTD) has been identified . In celery plants, MTD activity and tissue mannitol concentration are inversely related . MTD provides the initial step by which translocated mannitol is committed to central metabolism and, by regulating mannitol pool size, is important in regulating salt tolerance at the cellular level . We have now isolated, sequenced, and characterized a Mtd cDNA from celery . Analyses showed that Mtd RNA was more abundant in cells grown on mannitol and less abundant in salt-stressed cells . A protein database search revealed that the previously described ELI3 pathogenesis-related proteins from parsley and Arabidopsis are MTDs . Treatment of celery cells with salicylic acid resulted in increased MTD activity and RNA . Increased MTD activity results in an increased ability to utilize mannitol . Among other effects, this may provide an additional source of carbon and energy for response to pathogen attack . These responses of the primary enzyme controlling mannitol pool size reflect the importance of mannitol metabolism in plant responses to divergent types of environmental stress.

J Bacteriol, 1995 Aug, 177(15), 4549 - 52
Regulation of the glnBA operon of Rhodobacter capsulatus; Borghese R et al.; In a recent report identifying the promoters of the Rhodobacter capsulatus glnBA operon, it was suggested that an internal promoter upstream of the glnA gene probably resulted in different levels of glnBA and glnA transcripts (D . Foster-Hartnett and R . G . Kranz, J . Bacteriol . 176:5171-5176, 1994) . Therefore, to investigate the regulation, we constructed and examined the expression of a number of translational fusions in R . capsulatus glnBA . The results support a role for posttranscriptional regulation.

Biochemistry, 1995 Aug 1, 34(30), 9809 - 18
RuvB protein-mediated ATP hydrolysis: functional asymmetry in the RuvB hexamer; Marrione PE et al.; A survey of RuvB protein-mediated ATP hydrolysis yields the following observations . (1) The RuvB protein exhibits a DNA-independent ATPase activity with a turnover number (based on a RuvB monomer) approaching 6 min-1 and a Km of 154 microM . Single-stranded DNA and linear duplex DNA have small but significant effects on this activity . (2) At ATP concentrations near the Km, the ATPase activity is attenuated after approximately 60 turnovers/RuvB monomer . The attenuation does not reflect inhibition by ADP . Addition of ATP to 3 mM triggers an immediate resumption of ATP hydrolysis . The attention is enhanced somewhat by ssDNA and reduced somewhat by linear dsDNA . (3) ATP hydrolysis is dramatically stimulated by circular dsDNA, reinforcing the notion that RuvB translocates along the DNA in a reaction coupled to ATP hydrolysis . The kcat increases by at least 2-4-fold on circular duplexes depending on conditions, and the inactivation of RuvB at ATP concentrations near the Km does not occur . The ATPase activity on circular dsDNA also exhibits a partial substrate inhibition by ATP . (4) Optimal ATP hydrolysis requires approximately 1 DNA circle/RuvB hexamer, suggesting that multiple RuvB hexamers on a circle have an inhibitory effect on the ATPase activity . (5) With or without any of these DNA cofactors, a burst of ATP hydrolysis is observed under pre-steady-state conditions equivalent to 1 ATP per 3-3.3 RuvB monomers (2 ATP/hexamer) . The substrate inhibition and burst results suggest the presence of nonequivalent ATP hydrolytic sites in a RuvB hexamer . The attenuation of ATPase activity observed under some conditions may also be a manifestation of nonequivalent ATP hydrolytic sites.

Infect Immun, 1995 Aug, 63(8), 3069 - 72
pH and calcium dependence of hemolysis due to Rickettsia prowazekii: comparison with phospholipase activity; Ojcius DM et al.; Rickettsia prowazekii invades nucleated cells through phagocytosis and subsequently proliferates in the cytoplasm of the host cell . Hemolysis and a phospholipase A2 (PLA2) activity at neutral pHs have previously been reported; even though the phagosomal environment is most likely acidic . We here show that R . prowazekii and R . typhi also lyse erythrocytes at mildly acidic pHs, compatible with an early phagosomal compartment . For R . prowazekii, hemolysis at an acidic pH but not a neutral pH is enhanced by Ca2+, raising the possibility that more than one membranolytic factor may be produced by the rickettsiae . The rickettsiae alone display PLA2 activity, implying that the enzyme is of bacterial rather than erythrocyte or host cell origin . Moreover, the PLA2 activity requires divalent cations (Ca2+ or Mg2+), and, as with many extracellular PLA2s from other species, it has a preference for acidic over neutral phospholipids . The pH dependence of PLA2 is similar to that of the hemolysis without Ca2+, but in the presence of the hemolysis buffers (which contain Mg2+), there is no calcium-induced enhancement at acidic pHs . Thus, these rickettsiae are endowed with a membranolytic activity that could contribute to the escape of the bacteria from early phagosomal compartments, and it is likely that multiple toxins may be used for membrane lysis.

Infect Immun, 1995 Aug, 63(8), 3048 - 53
Oral immunization of pigs with viable or inactivated Actinobacillus pleuropneumoniae serotype 9 induces pulmonary and systemic antibodies and protects against homologous aerosol challenge; Hensel A et al.; A dose-defined aerosol infection of pigs was used to study the immunogenic and protective potentials of oral immunization with dead or live Actinobacillus pleuropneumoniae serotype 9 reference strain CVI 13261 against an aerogenic challenge . Pigs were vaccinated with a single dose of 10(11) CFU of viable (n = 8) or inactivated (n = 8) A . pleuropneumoniae given orally in a gelatin capsule . After 3 weeks, vaccinated pigs and nonvaccinated controls were challenged aerogenically with a dose of 10(8) CFU of A . pleuropneumoniae CVI 13261 . The protective efficacy of oral immunization was evaluated by clinical and postmortem examinations . Bronchoalveolar lavage in pigs was performed during the experiment to obtain lavage samples for assessment of local antibodies . Isotype-specific antibody responses in sera and in bronchoalveolar lavage fluids were determined by enzyme-linked immunosorbent assays based on whole-cell antigen . Oral immunization did not induce clinical side effects . After aerosol challenge, two animals of both vaccinated groups (25% in each case) showed a moderate fever for 2 days, whereas all four pigs (100%) of the nonvaccinated control group developed severe fever . In contrast to the controls, which developed severe pleuropneumonia, the vaccinated pigs had only mild pulmonary lesions . Three weeks after challenge, 13 of 16 vaccinated pigs (81%) were found to be free of pathomorphological changes of the lungs . From two of these pigs immunized with live bacteria we were able to reisolate A . pleuropneumoniae . A significant systemic and pulmonary increase in the concentrations of immunoglobulin A (IgA), IgM, and IgG antibodies reactive with A . pleuropneumoniae was detectable after aerosol challenge in both vaccinated groups . Immunization with viable bacteria was found to induce significantly higher concentrations of each Ig isotype in bronchoalveolar lavage fluids and sera than immunization with inactivated A . pleuropneumoniae . These serological findings were not reflected in the reduction in clinical disease after challenge in comparison to the case for the pigs vaccinated with inactivated bacteria . We concluded that a single oral administration of A . pleuropneumoniae provides partial clinical protection against aerosol challenge infection in the respiratory tract.

Infect Immun, 1995 Aug, 63(8), 2892 - 8
Fine specificity of the genetically controlled immune response to native and recombinant gp15/400 (polyprotein allergen) of Brugia malayi; Allen JE et al.; Polyprotein allergens are a family of structurally homologous molecules from parasitic nematodes which induce specific immunoglobulin E in infected individuals . We show here that both H-2 and non-H-2 factors determine the ability of mice to generate T- and B-cell responses to the filarial polyprotein allergen (Brugia malayi gp15/400) . Further, H-2 and non-H-2 genes can complement one another to overcome nonresponsiveness to this molecule . However, these genetic restrictions govern only responses to the native glycoprotein and all strains of mice respond equivalently when immunized with a recombinant polypeptide . Overlapping fragments of gp15/400 were constructed to compare the T-cell and antibody responses to native versus recombinant gp15/400 in responder (BALB/c H-2d) and nonresponder (B10.D2 H-2d, CBA H-2k, and BALB.K H-2k) strains . BALB/c mice generated T-cell responses to the same fragment (positions 89 to 133 and 1 to 21) whether immunized with native or recombinant material, although the antibody responses differed in fine specificity, H-2k mice, unresponsive to the native molecule, generated T cells responsive to the centrally located peptide (positions 57 to 100) only when immunized with the recombinant . Antibody responses in H-2k mice were directed at the peptide (positions 11 to 67) which is glycosylated in the native molecule . Our findings suggest that recognition of gp15/400 is affected by modifications that occur in the parasite but are absent when the molecule is produced in bacteria . This study provides a detailed evaluation of the immune response to an important nematode antigen as a start to the unraveling of the complex interaction of these multicellular parasites with mammalian hosts.

J Virol, 1995 Aug, 69(8), 4717 - 26
Stimulation of basal transcription from the mouse mammary tumor virus promoter by Oct proteins; Kim MH et al.; The steroid hormone-inducible promoter of mouse mammary tumor virus (MMTV) contains three overlapping sequences related to the consensus octamer motif ATGCAAAT . Basal promoter activity in the absence of hormone induction from a template in which all three octamer elements were mutated was decreased by two-to threefold in in vitro transcription assays . Oct-1 protein purified from HeLa cell nuclear extracts, as well as recombinant Oct-1 expressed in bacteria, recognized MMTV octamer-related sequences, as shown by DNase I footprinting . Furthermore, rabbit polyclonal antiserum directed against recombinant Oct-1 completely inhibited the formation of specific complexes between MMTV octamer-related sequences and proteins present in nuclear extracts of HeLa cells, indicating that Oct-1 is the major protein in HeLa nuclear extracts that recognizes octamer-related sequences in the MMTV promoter . In addition, depletion of Oct-1 from the nuclear extract by using Oct-1-specific antiserum or a sequence-specific DNA affinity resin decreased in vitro transcription from the wild-type MMTV promoter to a level identical to that obtained from a promoter in which all three octamer-related sequences were mutated . Addition of purified HeLa Oct-1 or recombinant Oct-1 to the depleted extract selectively increased transcription from the wild-type relative to the mutated promoter, demonstrating that Oct-1 transcription factor stimulates basal transcription from the MMTV promoter . A similar effect was observed when purified recombinant Oct-2 was added to the Oct-1-depleted extract, suggesting that Oct-2 may play an important role in MMTV transcription in B cells.

Curr Microbiol, 1995 Aug, 31(2), 134 - 7
Genetic relationships among strains of Xylella fastidiosa from RAPD-PCR data; Pooler MR et al.; Genetic relationships among 11 Xylella fastidiosa strains isolated from mulberry, almond, ragweed, grape, plum, elm, and citrus were determined by random amplified polymorphic DNA (RAPD) . Twenty-two 10-base primers amplified a total of 77 discrete polymorphic bands . Phenetic analysis based on a similarity matrix corresponded well with previous reports on X . fastidiosa RFLP-based similarity relationships, indicating that RAPD-PCR amplification products can be used as a reliable indicator of genetic distance in X . fastidiosa . Cladistic analysis suggests the existence of five groups of X . fastidiosa: the citrus group, the plum-elm group, the grape-ragweed group, the almond group, and the mulberry group.

Nippon Kyobu Geka Gakkai Zasshi, 1995 Aug, 43(8), 1203 - 7
{A surgical case of infective endocarditis accompanied with immune complex glomerulonephritis}; Ozaki T et al.; Infective endocarditis (IE) is often accompanied with renal disease . Recently it is thought that immune complex is formed by infecting agent of IE deposit to glomerulus and this contributes to glomerulonephritis . The case was 59-year-old man . He admitted with symptoms of proteinuria, hematuria and fever and diagnosed aortic regurgitation with vegetative change of aortic valve with echocardiogram . After admission he had stroke . Renal function was poor and circulating immune complex (CIC) value was high . Str . viridans was defined by blood culture examination . Aortic valve replacement was done because of no normalization of infective response and of renal function . Bacteria was not detected from the vegetation of valve but detected in the vegetation of transitional zone to mitral valve . Postoperative course was uneventful and renal function became normal with normalization of infective response . Anti C3d antibody value became normal and he discharged 50th postoperative day . In case of ineffective medical treatment the early removal of infected valve will make a successful result.

Endod Dent Traumatol, 1995 Aug, 11(4), 172 - 6
Pulpal reaction to a dental adhesive in deep human cavities; Torstenson B; In the last years several dental adhesives have been developed . They are supposed to chemically adhere to dentin and a liner to protect the pulp is not used . The aim of this study was to compare the short-term pulpal reaction, in an intra-toothpair study, between a dental adhesive, Scotchbond 2, and a lining system, Tubulitec, in combination with P-50 in surface-sealed cavities . Deep buccal cavities in 16 human pairs of premolars, 32 teeth, were restored in vivo with a light cured composite resin, P-50 . To minimize bacterial contamination all cavities were treated with a cleanser, Tubulicid, and the cavities were surface-sealed with temporary cement, Coltosol . One tooth in each pair, the test, was treated with Scotchprep Dentin Primer and Scotchbond 2 Light Cure Dental Adhesive . In the other tooth in the pair, the control, Tubulitec Primer and Liner were used . The teeth were extracted after 6-14 days . The sections were evaluated for degree of inflammation and the presence of bacteria . Irrespective of treatment of dentin the majority of teeth, 23, including one pulpal exposure, revealed no inflammation or a few inflammatory cells . In four test teeth, including one pulpal exposure, and two controls, growth of bacteria was found on the cavity walls and slight or moderate inflammation was seen in the corresponding pulps . In one test and two control teeth slight inflammation was seen but no bacteria could be detected . In the absence of bacteria Scotchbond 2 did not seem to irritate the pulp.(ABSTRACT TRUNCATED AT 250 WORDS)

Curr Biol, 1995 Aug 1, 5(8), 869 - 72
Photosynthesis . Regulation by redox signalling; Allen JF et al.; Photosynthesis is light-driven redox chemistry . Molecular redox signalling, the coupling of gene expression to electron transfer, is now implicated in the adaptation of photosynthesis to variation in light quality and quantity.

Tuber Lung Dis, 1995 Aug, 76(4), 330 - 5
Identification of Mycobacterium intracellulare by a polymerase chain reaction using species-specific primers; Yamazaki T et al.; SETTING: The polymerase chain reaction (PCR) is a rapid and specific method used to amplify a certain DNA fragment . It is applicable to rapid diagnosis of mycobacterial infections . By use of species-specific primers, it is possible to identify mycobacteria by PCR . In this study, a newly constructed primer was tested for specificity for Mycobacterium intracellulare in the PCR . OBJECTIVE: M . intracellulare is one of the most frequently found bacteria in opportunistic infection in AIDS, and rapid identification of this species is important . The purpose of this study was to construct a primer specific to this species as a suitable tool for identification . DESIGN: PCR products of M . tuberculosis and M . intracellulare, obtained by using the primers YNP-1 and YNP-2, were sequenced and compared . They showed a difference in the base sequences . A sequence unique to M . intracellulare was used as the primer specific to this species . Various mycobacterial and non-mycobacterial DNAs were used as the primer specific to this species . Various mycobacterial and non-mycobacterial DNAs were used as the template to evaluate the specificity of the newly constructed primers, YNP-7 and YNP-8 . Sputum samples were also examined by PCR using the primers . RESULTS: In total 25 species of culture mycobacterial and non-mycobacterial strains and 76 sputum samples were tested by PCR . Only M . intracellulare DNA was amplified with PCR using the primers YNP-7/8 . CONCLUSION: The specificity of the newly constructed primers for M . intracellulare was confirmed.

J Am Soc Nephrol, 1995 Aug, 6(2), 207 - 13
Intracellular acidification mediates the inhibitory effect of peritoneal dialysate on peritoneal macrophages; Douvdevani A et al.; Commercial peritoneal dialysis solution (CDS) is known to have a detrimental effect on the capacity of peritoneal macrophages (PM phi) to kill bacteria and produce acute phase cytokines . This cytotoxic effect is largely caused by the low pH of CDS . Because the cytoplasmic pH (pHi) is an important determinant of cellular function, the effect of CDS on the pHi of PM phi from continuous ambulatory peritoneal dialysis patients was studied . The pHi of PM phi was measured fluorometrically in N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered salt solution (HBSS) or CDS at pH values of 5.3, 6.5, and 7.0, values that represent the pH existing in dialysate during the first 30 min of dwell time . For any given pH of the experimental medium, the pHi was always more acidic in CDS than in HBSS . When PM phi were incubated with a lactate-containing HBSS, a cellular acidification was observed that was similar to that attained by exposure to CDS at the same pH . This supports the hypothesis that the decrease in pHi was due to the influx of lactic acid from the CDS into the PM phi . In order to demonstrate a causal association between the CDS-induced cellular acidification and a defect in phagocytosis and cytokine production, these functions were studied after pHi clamping by means of K+/nigericin . It was found that clamping pHi to values below 6.5 led to a markedly reduced tumor necrosis factor-alpha production and phagocytosis . However, at values of pHi > 6.5, these functions were normal . (ABSTRACT TRUNCATED AT 250 WORDS)

Kekkaku, 1995 Aug, 70(8), 467 - 72
{Reproducibility of MTD system for detection of Mycobacterium tuberculosis: a cooperative study among six laboratories}; Abe C et al.; The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD) is a rapid test for the detection of Mycobacterium tuberculosis, utilizing the rRNA amplification method . For assessing the reliability and reproducibility of the method, a co-operative blind study was conducted among 6 laboratories . Materials for test were sputum and water samples containing known numbers of Mycobacterium bovis BCG or Mycobacterium avium, and samples without bacteria . From three of 6 laboratories, false-positive results were reported for bacteria negative samples, however, the ratio was below 10%; 8.3% (3/36 samples), 5.6% (2/36), and 2.8% (1/36), respectively . It indicates the indispensability of negative controls for sample pretreatment and RNA extraction stages in the routine MTD test . In every laboratory, all the samples with 10(2) BCG in water and 10(4) BCG in sputum were found to be MTD positive . For the sputum samples with 10(2) BCG, positive results with the ratio above 80% were reported from 4 laboratories . These results indicate that the MTD test based on rRNA amplification method is quite useful for the rapid diagnosis of M . tuberculosis infection.

J Clin Microbiol, 1995 Aug, 33(8), 2162 - 5
Immunomagnetic separation and PCR for detection of Helicobacter pylori in water and stool specimens; Enroth H et al.; The detection of Helicobacter pylori in clinical and environmental samples by PCR sometimes requires removal of polymerase inhibitors . We have used a magnetic immunoseparation technique as pre-PCR treatment to facilitate direct detection of H . pylori in stool and water specimens . Rabbit hyperimmune antiserum was produced and magnetic beads were coated with purified immunoglobulin G, which reacted with and bound to both coccoid and rod-shaped forms of H . pylori . When PCR was applied for the detection of H . pylori from cultured samples, the number of organisms that was required for positive scores varied significantly . For a 3-day culture of H . pylori, samples containing 10(2) bacteria per ml are needed for a positive score; for a 6-day culture, samples containing 10(4) bacteria per ml are needed; and for a 10-day culture, samples containing 10(6) bacteria per ml are needed . These results indicate that the coccoid forms of H . pylori may have a different antigenicity and DNA content and are therefore more difficult to detect by immunomagnetic separation and PCR than the rod-shaped forms . Spiked samples with the addition of feces, spiked water samples, and a patient stool specimen were all scored positive with this technique.

Microbiology, 1995 Aug, 141 ( Pt 8), 1947 - 55
Detection of novel marine methanotrophs using phylogenetic and functional gene probes after methane enrichment; Holmes AJ et al.; A major limitation of rRNA-targeted group-specific probes is that they may cross-react with organisms of other physiological, or even phylogenetic groups when applied to environmental samples containing unknown sequences . We have exploited the restricted physiology of methane-oxidizing bacteria to assess the specificity and efficiency of probes for this physiological type which target the 16S rRNA or genes involved in methanotroph physiology . Seawater samples were enriched for methanotrophs by addition of methane and essential nutrients . The changes in composition of the bacterial population were monitored by analysis of 16S rRNA gene libraries . Methanotroph group-specific probes failed to give a signal with samples from these enrichments even though a methanol dehydrogenase structural gene was detected . A 16S rDNA sequence that was abundant only after methane addition was recovered and found to show a close phylogenetic relationship to Methylomonas . Organisms containing this sequence were observed in enrichments by in situ hybridization . The combination of enrichment on methane and screening with the broad specificity methanol dehydrogenase probe allowed detection of novel methanotrophs that were not detected with the original suite of methanotroph group-specific probes.

J Bacteriol, 1995 Aug, 177(16), 4730 - 41
The replication of an IncL/M plasmid is subject to antisense control; Athanasopoulos V et al.; A 2,385-bp sequence that contains the information for the autonomous replication of the IncL/M plasmid pMU604 was characterized . Genetic analyses revealed that the replicon specifies at least four structural genes, designated repA, repB, repC, and rnaI . The repA gene encodes a protein with a molecular weight of 40,861 which probably functions as an initiator for replication . The functions of the proteins of the repB and repC genes are unclear; however, mutations in the start codon of repB reduced the expression of both repB and repA, indicating that these two genes are translationally coupled . The rnal gene encodes a small antisense RNA of about 75 to 77 bases and is responsible for the incompatibility phenotype, thus implicating its role as the main copy number determinant . RNAI exerts its effect in trans to repress the expression of repA at the posttranscriptional level . Furthermore, two complementary sequences of 8 bases, with the potential to interact and form a putative pseudoknot structure, were identified in the leader region of the repA mRNA . Base-pairing between the two complementary sequences was shown to be critical for efficient repA expression . A model for the regulation of pMU604 replication involving both translational coupling and pseudoknot formation is proposed.

J Cell Biol, 1995 Aug, 130(3), 733 - 44
Functional analysis of posttranslational cleavage products of the neuron-glia cell adhesion molecule, Ng-CAM; Burgoon MP et al.; Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth . In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng-CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains) . To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria . Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135 . In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand . Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F135 fragment of the molecule . The results also indicated that proteolytic cleavage of Ng-CAM200 is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F135 on L cells (and presumably on neurons) . In contrast to the cell binding results, the F80 but not the F135 fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the FnIII repeats of F80 . The observations that a protein corresponding to F135 contains the cell aggregation sites whereas one corresponding to the F80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration.

Shock, 1995 Aug, 4(2), 117 - 20
A comparison of survival at different degrees of hemorrhagic shock in germ-free and germ-bearing rats; Ferraro FJ et al.; We have previously reported superior survival after one level of hemorrhagic shock in germ-free (GF) rats compared with germ-bearing (GB) rats . The objective of this study was to determine the effect of the GF state on survival at different degrees of hemorrhagic shock . GF and GB rats were bled to a mean arterial blood pressure of 30 mmHg . Shock was terminated after 10, 20, 40, or 80% of the maximum shed blood volume was reabsorbed spontaneously . Both shock time and time to decompensation were significantly longer in GF rats (p < .05) . Comparative survival was greater for GF rats at most levels of shock (p < .01) . This superiority in survival was greatest at moderate shock levels and decreased at severe shock levels . There may be several reasons for the increased tolerance of GF animals to hemorrhagic shock such as metabolic or immunologic variations . It is hard to avoid the fact, however, that the most notable difference between the GF and GB rat is the presence or absence of bacteria.

Curr Opin Neurobiol, 1995 Aug, 5(4), 443 - 8
Genetic approaches to mechanosensory transduction; Kernan M et al.; Genetic approaches in several organisms provide the means of solving a previously intractable problem: characterizing the molecular foundations of the mechanical senses . In nematode mechanosensory cells, members of a novel class of epithelial ion channel subunits have been implicated as components of a mechanically gated channel . In insect mechanosensory bristles, mutations specifically defective in mechanoreceptor potentials have been identified . And in bacteria, a stretch-activated channel has been molecularly characterized for the first time . Although mechanosensitivity can be a property of an isolated channel, sensory transduction in eukaryotic mechanosensory cells probably requires the interaction of several membrane and cytoskeletal components.

Arch Oral Biol, 1995 Aug, 40(8), 699 - 705
The flow rate and electrolyte composition of whole saliva elicited by the use of sucrose-containing and sugar-free chewing-gums; Dawes C et al.; On two occasions, 12 adults collected unstimulated saliva and then eight samples of saliva over a 20-min period while chewing 3 g of either Wrigley's Spearmint sucrose-containing gum (SCG) or sugar-free gum (SFG) at 70 chews/min . The flow rates peaked initially, then fell with duration of stimulation . With the SFG they were slightly but significantly higher than with the SCG after 4 min of chewing . The sum of the concentrations of cations minus the sum of the concentrations of anions was not significantly different from zero for saliva elicited by the SCG . However, for unstimulated saliva and that elicited by SFG, there was a slight positive anion balance . A second series of saliva collections with SCG and SFG was made by the same 12 participants and these samples were analysed for lactate . For these collections the flow rates with SCG were not significantly less than with the SFG . The lactate concentration in saliva elicited by SCG peaked at 1.82 mmol/l in samples collected over 8-15 min, whereas samples of saliva elicited by SFG had a mean lactate concentration of 0.21 mmol/l . Of the lactate formed during the metabolism of sucrose by the oral bacteria, only 2% or less appeared to be derived from the metabolism of micro-organisms free in saliva, the balance presumably being formed in dental plaque and entering the saliva by diffusion . All saliva samples were supersaturated with respect to hydroxyapatite but stimulated saliva was significantly more supersaturated than unstimulated saliva.(ABSTRACT TRUNCATED AT 250 WORDS)

Vet Microbiol, 1995 Aug, 45(4), 339 - 50
Infection of cultured rat enterocytes by Ileal symbiont intracellularis depends on host cell function and actin polymerisation; Lawson GH et al.; The mechanisms of entry of Ileal symbiont intracellularis into IEC-18 rat enterocyte cells and subsequent bacterial proliferation were examined in centrifuge-assisted and static infections . Live, oxygen or neomycin damaged, and formalin killed bacteria, each rapidly entered viable cells . Live or damaged bacteria did not enter cells nor proliferate within cells after static infection of cells cooled to 5 degrees C . Infection of cells was greatly reduced at 20 degrees or 32 degrees compared to infection at 37 degrees C . Centrifuge-assisted infection was also reduced by chilling the cells . Cytochalasin D but not B inhibited the entry process indicating an actin-dependent infection, although other pathways may also be involved in centrifuge-assisted infections . Drugs capable of modifying cell membrane charge, heparin receptors or trypsin-labile proteins were all inactive in preventing or enhancing infection . We therefore conclude that infection of enterocytes by IS intracellularis is dependent on host cell activity and actin polymerization, but is independent of bacterial viability.

Scand J Gastroenterol, 1995 Aug, 30(8), 745 - 51
Dyspeptic symptoms and gastric emptying of solids in patients with functional dyspepsia . Role of Helicobacter pylori infection; Caballero-Plasencia AM et al.; BACKGROUND: Our aim was to investigate the relation between dyspeptic symptoms, gastric emptying of digestible and indigestible solids, and Helicobacter pylori infection in patients with functional dyspepsia . METHODS: We used isotopic labeling and radiologic techniques to study gastric emptying of a solid meal and of 10 radiopaque indigestible solids in 50 healthy volunteers and 50 patients with functional dyspepsia . In addition, we determined the presence of seven symptoms of dyspepsia and added the score for each symptom to obtain an index of dyspepsia for each patient . RESULTS: Seventy-eight per cent of our dyspeptic patients had gastroparesis to a solid meal, and 68% to indigestible solids . We found no apparent relation between gastroparesis or H . pylori infection and dyspeptic symptoms separately or as an index of dyspepsia . Moreover, the presence of the bacteria was not related to gastroparesis to a solid meal or to indigestible solids . CONCLUSIONS: We conclude that neither symptoms of dyspepsia nor H . pylori appears to be related to gastroparesis to solids . H . pylori infection is not related to dyspeptic symptoms.

Poult Sci, 1995 Aug, 74(8), 1381 - 7
Film oxygen transmission rate effects on ground chicken meat quality; Dawson PL et al.; Effects of the packaging film oxygen transmission rate (OTR) on the odor, color, aerobic plate count, and cooked volatile compounds in ground chicken leg meat were evaluated over a 14-d refrigerated storage (4 C) period . Freshly desinewed and ground chicken leg meat was packaged under minimal vacuum in five films of different OTR . Films ranged from 30 to 12,000 mL oxygen/m2 per 24 h . Odor scores of meat in packages opened after 10 d were lower for the lowest OTR film than films with higher OTR . Aerobic plate counts increased at a faster rate in the higher OTR films, and cooked meat volatile profiles showed little variation due to OTR film type . Specific products will require different packaging to optimize shelf-life quality . For ground chicken leg meat, an intermediate OTR film is best to maintain a majority of the quality attributes during refrigerated storage.

Mol Cell Probes, 1995 Aug, 9(4), 277 - 82
Development of an efficient PCR method for toxin typing of Actinobacillus pleuropneumoniae strains; Frey J et al.; A method has been developed which allows the determination of the activator, the structural and the secretion genes of the three toxins ApxI, ApxII and ApxIII in Actinobacillus pleuropneumoniae in only two PCR reactions . The oligonucleotide primers were designed to amplify a significant part of the activator and structural genes apxICA, apxIICA and apxIIICA together in a single PCR reaction giving amplification products which differ in length, in order to be clearly separated by agarose gel electrophoresis . Variations in the apxIA and apxIIIA genes which were found in different serotypes were taken into account in the design of the primers to give a uniform amplification product for both variants of the apxIA and the apxIIIA genes . The secretion genes apxIBD and apxIIIBD are also detected in a single PCR reaction containing two pairs of oligonucleotide primers which yield two differently sized fragments to differentiate between apxIBD and apxIIIBD genes . The reference strains of A . pleuropneumoniae serotypes 1-12 and 104 field strains representing all serotypes obtained from various laboratories worldwide were analysed for their content of apx genes . The two PCR reactions give toxin gene patterns which are characteristic for different groups of serotypes in A . pleuropneumoniae and allow the rapid differentiation of five toxin type groups, group 1 including serotypes 1, 5a, 5b, 9 and 11, group 2 including serotypes 2, 4, 6, 8, group 3 with serotype 3, group 4 with serotype 7 and 12 and group 5 with serotype 10 . The method enhances and facilitates differentiation of A . pleuropneumoniae strains for diagnostics and epidemiology and allows the detection of serotypes with atypical toxin patterns.

J Biotechnol, 1995 Jul 31, 41(2-3), 81 - 90
The potentials of the in vitro protein biosynthesis system; Stiege W et al.; Recent developments indicate that with the in vitro protein biosynthesis system a new technology emerges, which will find in the future its application in biotechnology . Up to date the best system described produces 2 mg of proteins per 0.5 ml after 100 h . The potentials of the in vitro protein biosynthesis system include not only the production of proteins, but especially the synthesis of proteins, which are toxic to living cells, or proteins with unnaturally modified or isotope-labelled amino acids in specific positions . Among the advantages of the in vitro system, when compared to current cloning techniques, are the purity of the proteins synthesized and their superior biological activities . Thus, the application of this technology will be manifold ranging from the production of proteins with improved or even new characteristics to the potentials of improving the methods of protein design and structural characterization.

Int J Cancer, 1995 Jul 28, 62(3), 276 - 82
Characterization of cell lines established from human hepatocellular carcinoma; Park JG et al.; We characterized 8 human hepatocellular-carcinoma cell lines established from the primary tumors of Korean patients . All lines showed substrate adherence and one line from anaplastic tumor also grew as floating aggregates . Most cultured cells maintained many morphological characteristics of the original tumors from which they were derived . Doubling times varied from 34 to 72 hr . All lines showed relatively high viability and were not contaminated with Mycoplasma or bacteria . All lines showed aneuploidy and were proven to be unique by DNA fingerprinting analysis . Hepatitis-B-virus (HBV) DNA was integrated in the genomes of all lines . Two of the cell lines (SNU-354, SNU-368) showed expression of HBV and HBVx (HBx) transcripts . SNU-354 strongly expressed albumin, and SNU-368 expressed transferrin and insulin-like growth factor II . No lines produced alpha-fetoprotein at the RNA and protein level . These cell lines represent useful tools for in vitro studies related to hepatocellular carcinoma.

Biochemistry, 1995 Jul 25, 34(29), 9617 - 24
Stable photobleaching of P840 in Chlorobium reaction center preparations: presence of the 42-kDa bacteriochlorophyll a protein and a 17-kDa polypeptide; Hager-Braun C et al.; Simple procedures for the anaerobic preparation of photoactive and stable P840 reaction centers from Chlorobium tepidum and Chlorobium limicola in good yield are presented and quantitated . The subunit composition was tested by cosedimentation in sucrose density gradients . For C . limicola, it minimally comprises four subunits: the P840 reaction center protein PscA, the BChla antenna protein FMO, the FeS protein PscB with centers A and B, and a positively charged 17-kDa protein denoted PscD . The preparation from Chlorobium tepidum additionally contained PscC, a cytochrome c-551 . The BChla absorption peak of the purified complexes was at 810 nm, with a shoulder at 835 nm . The ratio of the shoulder to the peak was 0.25, which corresponds to 1 reaction center per 70 BChla molecules if a uniform extinction coefficient of BChla is assumed . However, bleaching at 610 nm in continuous light corresponded up to 1 photoactive reaction center per 50 BChla molecules . Therefore, either the extinction coefficient of BChla in the reaction center is overestimated or the one for photobleaching is underestimated . In any case, the major portion of the reaction center was photoactive in the preparations . A P840 reaction center subcomplex, lacking PscD and deficient in FMO and PscB, but retaining the cytochrome c subunit, was obtained as a side product . It was photoinactive and had an absorption peak at 814 nm and a 835/814 absorbance ratio of 0.42 . FMO and PscB show the tendency to form a complementary subcomplex . FMO and PscD are apparently required to stabilize the photoactive reaction center, while the cytochrome c subunit is not.

Biochemistry, 1995 Jul 25, 34(29), 9451 - 8
Characterization of the interaction between PQQ and heme c in the quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni; de Jong GA et al.; Quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni (QH-EDH) contains two cofactors, 2,7,9-tricarboxy-1H-pyrrolo{2,3-f}quinoline-4,5-dione (PQQ) and heme c . Since previous studies on the kinetics of this enzyme suggested that both participate in electron transfer, spectroscopic investigations were performed of the oxidized and reduced holo- and apoenzyme (without PQQ but with heme c) to reveal the nature of the interaction between the two redox centers . From this it appears that the properties of the heme in the enzyme are affected by the presence of PQQ, as judged from the shift of the maxima in the ultraviolet/visible absorption spectra of the heme moiety in both reduced and oxidized QH-EDH and the 60-mV increase of the heme midpoint redox potential caused by PQQ addition . Also 1H-NMR spectroscopy was indicative for interaction since binding of PQQ induced shifts in the resonances of the methyl groups of the porphyrin ring in the oxidized form of the apoenzyme and a shift in the methionine heme ligand resonance of the reduced form of the apoenzyme . On the other hand, resonance Raman spectra of the heme in the different enzyme forms were nearly similar . These results suggest that a major effect of PQQ binding to apo-QH-EDH is a rotation of the methionine ligand of heme c . Since no intermediate 1H-NMR spectra were observed upon titration of apoenzyme with PQQ, apparently no exchange occurs of PQQ between (oxidized) holo- and apoenzyme at the NMR time scale and at that of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc R Soc Lond B Biol Sci, 1995 Jul 22, 261(1360), 55 - 63
Evolution and phylogeny of Wolbachia: reproductive parasites of arthropods; Werren JH et al.; Wolbachia are cytoplasmically inherited bacteria found in reproductive tissues of many arthropod species . These bacteria are associated with reproductive alterations in their hosts, including parthenogenesis, reproductive incompatibility and feminization . A fine-scale phylogenetic analysis was done using DNA sequences from ftsZ, a rapidly evolving bacterial cell-cycle gene . ftsZ sequences were determined for 38 different Wolbachia strains from 31 different species of insects and one isopod . The following results were found: (i) there are two major division of Wolbachia (A and B) which diverged 58-67 millions years before present based upon synonymous substitution rates; (ii) a general concordance is found between the ftsZ and 16S rDNA phylogenies, indicating that these represent bacterial strain (rather than simply gene) phylogenies; however, a possible example of recombination between A and B division bacteria may have occurred in the feminizing Wolbachia present in an isopod; (iii) extensive horizontal transmission of Wolbachia has occurred between insect taxa, including different insect orders; one strain in particular (designated Adm) shows extensive recent horizontal transmission; (iv) there is an association between the Wolbachia found in a parasitic wasp (Nasonia) and its fly host (Protocalliphora), suggesting exchange of bacteria between these species; (v) parthenogenesis induction has evolved several times among the Wolbachia; and (vi) some insects harbour infections with more than one Wolbachia strain, even within individual insects.

Proc Natl Acad Sci U S A, 1995 Jul 18, 92(15), 6842 - 6
Augmented DNA-binding activity of p53 protein encoded by a carboxyl-terminal alternatively spliced mRNA is blocked by p53 protein encoded by the regularly spliced form; Wolkowicz R et al.; DNA-binding activity of the wild-type p53 is central to its function in vivo . However, recombinant or in vitro translated wild-type p53 proteins, unless modified, are poor DNA binders . The fact that the in vitro produced protein gains DNA-binding activity upon modification at the C terminus raises the possibility that similar mechanisms may exist in the cell . Data presented here show that a C-terminal alternatively spliced wild-type p53 (ASp53) mRNA expressed by bacteria or transcribed in vitro codes for a p53 protein that efficiently binds DNA . Our results support the conclusion that the augmented DNA binding activity of an ASp53 protein is probably due to attenuation of the negative effect residing at the C terminus of the wild-type p53 protein encoded by the regularly spliced mRNA (RSp53) rather than acquisition of additional functionality by the alternatively spliced C' terminus . In addition, we found that ASp53 forms a complex with the non-DNA-binding RSp53, which in turn blocks the DNA-binding activity of ASp53 . Interaction between these two wild-type p53 proteins may underline a mechanism that controls the activity of the wild-type p53 protein in the cell.

Proc Natl Acad Sci U S A, 1995 Jul 18, 92(15), 6808 - 12
MEKK1 phosphorylates MEK1 and MEK2 but does not cause activation of mitogen-activated protein kinase; Xu S et al.; A constitutively active fragment of rat MEK kinase 1 (MEKK1) consisting of only its catalytic domain (MEKK-C) expressed in bacteria quantitatively activates recombinant mitogen-activated protein (MAP) kinase/extracellular signal-regulated protein kinase (ERK) kinases 1 and 2 (MEK1 and MEK2) in vitro . Activation of MEK1 by MEKK-C is accompanied by phosphorylation of S218 and S222, which are also phosphorylated by the protein kinases c-Mos and Raf-1 . MEKK1 has been implicated in regulation of a parallel but distinct cascade that leads to phosphorylation of N-terminal sites on c-Jun; thus, its role in the MAP kinase pathway has been questioned . However, in addition to its capacity to phosphorylate MEK1 in vitro, MEKK-C interacts with MEK1 in the two-hybrid system, and expression of mouse MEKK1 or MEKK-C in mammalian cells causes constitutive activation of both MEK1 and MEK2 . Neither cotransfected nor endogenous ERK2 is highly activated by MEKK1 compared to its stimulation by epidermal growth factor in spite of significant activation of endogenous MEK . Thus, other as yet undefined mechanisms may be involved in determining information flow through the MAP kinase and related pathways.

EMBO J, 1995 Jul 17, 14(14), 3452 - 60
The role of the GrpE homologue, Mge1p, in mediating protein import and protein folding in mitochondria; Westermann B et al.; Mge1p, a mitochondrial GrpE homologue, has recently been identified in the yeast Saccharomyces cerevisiae and a role for this protein in precursor import has been reported . To dissect the molecular mechanism of Mge1p function, conditional mge1 mutants were constructed . Cells harbouring mutant mge1 accumulated precursor proteins at restrictive temperature . Both kinetics and efficiency of import were reduced in mitochondria isolated from strains possessing mutant mge1 . Binding of mitochondrial-Hsp70 (mt-Hsp70) to incoming precursor proteins was abolished at restrictive temperature . Nucleotide-dependent dissociation of mt-Hsp70 from the import component MIM44 was reduced in mitochondria from mutant mge1 strains . Furthermore, at restrictive temperature an increase of incompletely folded, newly imported protein and enhanced protein aggregation was observed in mitochondria isolated from the mutant strains . We conclude that Mge1p exerts an essential function in import and folding of proteins by controlling the nucleotide-dependent binding of mt-Hsp70 to substrate proteins and the association of mt-Hsp70 with MIM44.

Mech Ageing Dev, 1995 Jul 14, 81(2-3), 97 - 106
Decreased capacity of aged mice to produce interferon-gamma in Legionella pneumophila infection; Fujio H et al.; We investigated the difference in natural resistance to Legionella pneumophila infection between aged (18-20-month-old) and young (3-month-old) mice of ddY strain . Aged mice were more susceptible to the bacterial infection than young mice; 50% lethal doses of L . pneumophila for aged and young mice were 2.2 x 10(7) and 8.5 x 10(7) colony forming units (CFU), respectively, after intraperitoneal injection of the bacteria . The bacterial burden in the livers was larger in aged than young mice after a challenge with a sublethal dose of L . pneumophila . However, peritoneal macrophages of aged mice paradoxically had a greater capacity to kill intracellular L . pneumophila than those of young mice . Interferon-gamma (IFN-gamma) production from naive spleen cells was compared after an in vitro stimulation with formalin-killed L . pneumophila . Spleen cells of aged mice produced significantly less IFN-gamma than those of young mice . When anti-murine IFN-gamma monoclonal antibody was administered before the bacterial infection, the subsequent bacterial burden in the livers significantly increased in young but not in aged mice . These data suggest that, in aged mice, IFN-gamma production is depressed at an early phase of L . pneumophila infection and it renders aged mice more susceptible to the infection.

Curr Opin Ophthalmol, 1995 Aug, 6(4), 86 - 94
Basic science and applications of in vivo microscopy; Mathers WD et al.; Confocal microscopy creates a scanned image from a point light source and point detection or a scanning slit to remove scattered light and improve optical resolution . This also results in optical sectioning of tissues . These capabilities can be employed to image structures in the human cornea, in vivo, both for research and for the diagnosis and treatment of human disease . Optical sections, when recombined, can lead to three-dimensional reconstructions from which very useful information is obtained . Investigators have found keratocyte density decreases from anterior to posterior in the stroma of the rabbit cornea . Surface epithelial desquamation can also be studied and the effects of contact lens use can be demonstrated . The instrument is also useful for diagnosing and guiding therapy for some human diseases such as Acanthamoeba keratitis . Colonies of bacteria may also be observed and treatment evaluated in patients with infectious crystalline keratopathy . Confocal microscopy can also image the retina.

J Biol Chem, 1995 Jul 7, 270(27), 16409 - 14
Identification of tuberin, the tuberous sclerosis-2 product . Tuberin possesses specific Rap1GAP activity; Wienecke R et al.; Tuberous sclerosis (TSC) is a human genetic syndrome characterized by the development of benign tumors in a variety of tissues, as well as rare malignancies . Two different genetic loci have been implicated in TSC; one of these loci, the tuberous sclerosis-2 gene (TSC2), encodes an open reading frame with a putative protein product of 1784 amino acids . The putative TSC2 product (tuberin) contains a region of limited homology to the catalytic domain of Rap1GAP . We have generated antisera against the N-terminal and C-terminal portions of tuberin, and these antisera specifically recognize a 180-kDa protein in immunoprecipitation and immunoblotting analyses . A wide variety of human cell lines express the 180-kDa tuberin protein, and subcellular fractionation revealed that most tuberin is found in a membrane/particulate (100,000 x g) fraction . Immunoprecipitates of native tuberin contain an activity that specifically stimulates the intrinsic GTPase activity of Rap1a . These results were confirmed in assays with a C-terminal fragment of tuberin, expressed in bacteria or Sf9 cells . Tuberin did not stimulate the GTPase activity of Rap2, Ha-Ras, Rac, or Rho . These results suggest that the loss of tuberin leads to constitutive activation of Rap1 in tumors of patients with tuberous sclerosis.

Nature, 1995 Jul 6, 376(6535), 57 - 8
Isolation of a hyperthermophilic archaeum predicted by in situ RNA analysis; Huber R et al.; A variety of hyperthermophilic bacteria and archaea have been isolated from high-temperature environments by plating and serial dilutions . However, these techniques allow only the small percentage of organisms able to form colonies, or those that are predominant within environmental samples, to be obtained in pure culture . Recently, in situ 16S ribosomal RNA analyses of samples from the Obsidian hot pool at Yellowstone National Park, Wyoming, revealed a variety of archaeal sequences, which were all different from those of previously isolated species . This suggests substantial diversity of archaea with so far unknown morphological, physiological and biochemical features, which may play an important part within high-temperature ecosystems . Here we describe a procedure to obtain pure cultures of unknown organisms harbouring specific 16S rRNA sequences identified previously within the environment . It combines visual recognition of single cells by phylogenetic staining and cloning by 'optical tweezers' . Our result validates polymerase chain reaction data on the existence of large archael communities.

Gene, 1995 Jul 4, 160(1), 7 - 16
Evidence for an evolutionary relationship among type-II restriction endonucleases; Jeltsch A et al.; Type-II restriction-modification (R-M) systems comprise two enzymes, a DNA methyltransferase (MTase) and a restriction endonuclease (ENase), each of which specifically interact with the same 4-8 bp sequence . All type-II MTases share several amino acid (aa) sequence motifs, which makes an evolutionary relatedness among these enzymes probable . The type-II ENases, in contrast, except for some homologous isoschizomers, do not share significant aa sequence similarity . Therefore, ENases in general have been considered unrelated . Here we show that in addition to the analysis of the genotype (aa sequence), a comparison of the phenotype (recognition sequence) of these enzymes can provide independent information regarding evolutionary relationships, and thereby, help to analyze the significance of weak aa sequence similarities . Multistep Monte-Carlo analyses were employed to demonstrate that the recognition sequences of those ENases, which were found to be related by a progressive multiple aa sequence alignment, are more similar to each other than would be expected by chance . This analysis supports the notion that not only type-II MTases, but also type-II ENases did not arise independently in evolution, but rather evolved from one or a few primordial DNA-modifying and DNA-cleaving enzymes, respectively.

Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6389 - 93
Evolution of single and double Wolbachia symbioses during speciation in the Drosophila simulans complex; Rousset F et al.; Maternally inherited bacteria of the genus Wolbachia are responsible for the early death of embryos in crosses between uninfected females and infected males in several insect species . This phenomenon, known as cytoplasmic incompatibility, also occurs between strains infected by different symbionts in some species, including Drosophila simulans . Wolbachia was found in two species closely related to D . simulans, Drosophila mauritiana, and Drosophila sechellia, and shown to cause incompatibility in the latter species but not in D . mauritiana . Comparison of bacterial and mtDNA history clarifies the origins of bacterial and incompatibility polymorphisms in D . simulans . Infection in D . mauritiana is probably the result of introgression of an infected D . simulans cytoplasm . Some D . simulans and D . sechellia cytoplasmic lineages harbor two bacteria as a consequence of a double infection which probably occurred in a common ancestor . The descendant symbionts in each species are associated with similar incompatibility relationships, which suggests that little variation of incompatibility types has occurred within maternal lineages beyond that related to the density of symbionts in their hosts.

J Gen Virol, 1995 Jul, 76 ( Pt 7), 1729 - 36
In vitro cleavage of hepatitis C virus polyprotein substrates by purified recombinant NS3 protease; D'Souza ED et al.; The non-structural protein NS3 of hepatitis C virus has been expressed in bacteria as a polyhistidine fusion protein which can be produced in a soluble form and easily purified by affinity chromatography . Using an in vitro transcription and translation system we have been able to demonstrate that this protein can proteolytically process substrate molecules derived from the non-structural region of the polyprotein . Using this assay system we have been able to optimize basic biochemical characteristics of the purified enzyme . Parallel experiments show that the full-length NS3 protein also possesses ATPase activity, indicating the bifunctional nature of the protein . In contrast, purified NS3 in which the predicted catalytic serine has been mutated loses protease but retains ATPase activity.

Pediatr Pathol Lab Med, 1995 Jul-Aug, 15(4), 547 - 53
Conjunctival biopsy in patients with Kawasaki disease; Burns JC et al.; Kawasaki disease (KD) is an acute vasculitis of infants and young children that is associated with bilateral nonexudative conjunctivitis during the acute illness . Epidemiologic evidence has suggested an infectious cause but the etiology of KD remains unknown . We examined conjunctival biopsy specimens from seven patients with typical KD to characterize the pathologic changes during the acute disease . Light microscopic examination revealed nonspecific, mild inflammatory changes that included vascular dilatation, infiltration with scattered lymphocytes, increased numbers of plasma cells in the conjunctival stroma, and increased prominence of goblet cells in the epithelium . No pathogens were identified by special stains for bacteria and rickettsiae, nor were viral particles seen by electron microscopy . We conclude that the conjunctivitis of acute KD is characterized by vascular dilatation with a mild mononuclear cell response with no pathognomonic features . The conjunctiva can be readily sampled in these patients and biopsy may prove useful in selected patients to exclude other clinical entities in the differential diagnosis.

Int J Syst Bacteriol, 1995 Jul, 45(3), 467 - 71
Moraxella caprae sp . nov., a new member of the classical Moraxellae with very close affinity to Moraxella bovis; Kodjo A et al.; Eight phenotypically homogeneous Moraxella-like strains were isolated from the nasal flora of healthy goats . Total genomic DNA-DNA hybridization, DNA base composition determination, and genetic transformation studies were performed to determine the relationships of these bacteria to the classical moraxellae . The eight new isolates exhibited very high levels of genetic affinity to Moraxella bovis, as shown by quantitative and qualitative genetic transformation data, and exhibited high DNA-DNA relative binding ratios to each other (63% or more) but lower levels of DNA homology with all of the other species investigated, including the closely related classical moraxellae . Our results, combined with the general morphologic and phenotypic profiles of these organisms, indicate that they should be classified with the classical moraxellae, and we propose the name Moraxella caprae for them . Strain 8897 (= CCUG 33296 {corrected} = NCTC 12877) is the type strain of M . caprae.

Int J Syst Bacteriol, 1995 Jul, 45(3), 429 - 35
Confirmation of the species Prevotella intermedia and Prevotella nigrescens; Frandsen EV et al.; The elevation of the two genotypes of Prevotella intermedia to species rank as P . intermedia and Prevotella nigrescens has increased the need for reliable differentiation between the two taxa . In this study, 53 strains, including strains whose species affiliations were known as well as fresh dental plaque isolates, were subjected to a multilocus enzyme electrophoretic analysis, DNA analyses in which we used whole genomic DNA, rRNA sequences, and an oligonucleotide specific for the former P . intermedia genotype II (P . nigrescens) as probes, and a sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of soluble cellular proteins . All of these tests consistently separated the strains into the same two distinct groups corresponding to P . intermedia and P . nigrescens, confirming that the two species constitute two distinct populations of bacteria . Each of the tests used independently provided reliable identification to the species level . A previously reported heterogeneity in the pattern of human immunoglobulin A1 (IgA1) degradation was not confirmed . No differences between species were observed . All of the strains induced total degradation of IgA1 within 48 h, a property that may be a virulence factor in periodontal disease development . The enzymes responsible for IgA1 degradation were not inactivated by the physiological proteinase inhibitors alpha 2-macroglobulin and alpha 1-proteinase inhibitor.

Ital J Gastroenterol, 1995 Jul-Aug, 27(6), 285 - 90
Tissue staining for Helicobacter pylori in intestinal metaplasia: correlation with its extension and histochemical subtypes; Testoni P et al.; The role played by Helicobacter pylori (Hp) infection in the occurrence of non-cardial gastric adenocarcinoma is suggestive but still debated . This study aimed to evaluate: a) the prevalence of Helicobacter-like organisms in antral bioptic specimens of 291 patients with chronic gastritis with antral atrophy and different subtypes of intestinal metaplasia (IM); b) the presence of a possible different positive tissue staining for the bacteria in the complete and incomplete intestinal metaplasia . Of the 291 patients, 222 cases (76.3%) showed type I IM, 28 cases (9.6%) type II IM and 41 cases (14.1%) type III IM . Helicobacter-like organisms were found in 42.9% of cases and positive tissue staining rate appeared to be inversely related to the extension of IM (58.7% in IM extended in less than 30% of specimens, 30.2% in IM extended between 30% and 60%, 2.7% in IM exceeding 60% of the biopsed area) . The inverse correlation between lower positive tissue staining for Helicobacter-like organisms and greater extension of IM was statistically significant (p < 0.001) . Incomplete metaplasia appeared to be unrelated to age and associated with a lower positive tissue staining for Helicobacter-like organisms; among patients with type I metaplasia, 118/222 showed Hp-positive bioptic specimens, vs 7/69 of types II and III (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Avian Dis, 1995 Jul-Sep, 39(3), 606 - 16
Detection of Mycoplasma gallisepticum, M . synoviae, and M . iowae by multi-species polymerase chain reaction and restriction fragment length polymorphism; Garcia M et al.; A single set of oligonucleotide primers was designed from known 16S ribosomal RNA (rRNA) sequences of Mycoplasma gallisepticum (MG), M . synoviae (MS), and M . iowae (MI) . This set of primers selectively amplifies a 780-base-pair DNA fragment within the 16S rRNA gene of MG, MS, and MI but does not amplify other avian mycoplasmas or other bacteria . The detection limit of the multi-species polymerase chain reaction (PCR) was approximately 100 mycoplasma (MG, MS, MI) colony-forming units per PCR reaction . The PCR product was differentiated by restriction fragment length polymorphism with the restriction enzymes HpaI, HpaII, and MboI . Preliminary results from field samples suggest that this technique could be a useful and rapid diagnostic test for the detection of these three pathogenic poultry mycoplasmas.

Minerva Ginecol, 1995 Jul-Aug, 47(7-8), 327 - 9
{Dysspermia due to inflammation . The evaluation of sperm cultures}; Piacentino R et al.; The study evaluates 160 cases of positive spermioculture taken from 522 sterile individuals examined by the authors at the Couple Sterility Outpatient unit in Department A of the Institute of Gynecology and Obstetrics at Turin University during the period between January 1984 and December 1993 . The germs responsible for infection were assayed in order to evaluate the strains which showed the highest incidence every year . Whereas there was no significant change in the absolute number of cases of sterility over the period, the number of cases caused by infection increased significantly during the second five-year period . It was found that the germs predominantly implicated in the genesis of male sterility formed part of the so-called mixed flora group, responsible in women for syndromes of often asymptomatic bacterial vaginosis which are not identified and consequently not treated.

J Biolumin Chemilumin, 1995 Jul-Aug, 10(4), 239 - 45
A comparative study of PCR product detection and quantitation by electro-chemiluminescence and fluorescence; Yu H et al.; Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)3(2+))-end-labelled primers . In this way, biotin for capture and Ru(bpy)3(2+) for detection are directly incorporated into the PCR product obviating subsequent probe hybridization . PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)3(2+)-labelled primers amplified a 1 kilobase region . Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection . ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity . Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels . However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining . The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.

Mol Cell Biol, 1995 Jul, 15(7), 3813 - 22
Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system; Hollenberg SM et al.; With modified two-hybrid technology, we have isolated a member of a new family of basic helix-loop-helix (bHLH) transcription factors . Thing1 (Th1) was identified in a screen of a mouse embryo cDNA library as a partner for the Drosophila E protein daughterless . RNA in situ hybridization and reverse transcriptase-PCR demonstrate a stage- and tissue-specific distribution for the expression of Th1 . Although tissue specific, the expression pattern of Th1 is fairly complex . During development, Th1 mRNA is widely expressed in extraembryonic tissues, portions of the heart, autonomic ganglia, the gut, and pharyngeal arches . At embryonic day 7.5 (E7.5), extraembryonic derivatives show robust Th1 expression . By E8.5, expression in the embryonic heart becomes detectable . During the next 2 days of development, the signal also includes gut and pharyngeal arches . Predominant expression at E13.5 is in neural crest derivatives, especially the autonomic nervous system and adrenal medulla . Expression of Th1 persists in the adult, in which it is localized to the smooth muscle cells of the gut . In vitro, Th1 protein recognizes a set of DNA sites that are more degenerate than has been determined for other bHLH factors, indicating a reduced binding specificity . Transient transfection of NIH 3T3 cells with GAL4-Th1 fusions reveals a repression activity mediated by the Th1 bHLH domain . In combination, these properties define Th1 as a new bHLH protein with a unique set of properties.

J Virol, 1995 Jul, 69(7), 4364 - 72
Amino-terminal domains of the bovine papillomavirus type 1 E1 and E2 proteins participate in complex formation; Benson JD et al.; Interaction between the E1 and E2 papillomavirus proteins appear to play an important role in viral DNA replication, although the exact domains of each protein involved in this interaction have not been identified . Using bovine papillomavirus type 1 (BPV-1) as a model for examining interactions between E1 and E2, we have used the two-hybrid and glutathione S-transferase (GST) fusion systems to map domains of BPV-1 E1 and E2 that interact in vivo and in vitro . In the two-hybrid system experiments, portions of BPV-1 E2 were expressed in Saccharomyces cerevisiae as LexA fusion proteins, which were tested for interaction with various domains of BPV-1 E1 . These assays indicated that domains sufficient for E1-E2 interaction are present within the amino-terminal 250 amino acids of E1 and within the first 91 amino acids of E2 . Interestingly, a LexA fusion protein that included amino acid residues 53 to 161 of BPV E2 demonstrated transcriptional activation in this system . In vitro binding assays using combinations of BPV-1 E1-GST fusion proteins and BPV-1 E2 expressed by in vitro translation confirmed the observations from the yeast system; a GST fusion protein containing the first 222 amino acids of BPV-1 E1 bound specifically to full-length BPV-1 E2 in vitro . Furthermore, E1(1-222)-GST bound to forms of E2 deleted of the carboxy-terminal DNA binding-dimerization domain, suggesting that E2 dimerization is not required for this interaction . Finally, in vitro interaction between E1-GST and E2 was observed at 22 degrees C but not at 4 degrees C.

Nat Genet, 1995 Jul, 10(3), 307 - 12
Cloning of the galactokinase cDNA and identification of mutations in two families with cataracts; Stambolian D et al.; Galactokinase is an essential enzyme for the metabolism of galactose and its deficiency causes congenital cataracts during infancy and presenile cataracts in the adult population . We have cloned the human galactokinase cDNA, which maps to chromosome 17q24, and show that the isolated cDNA expresses galactokinase activity in bacteria and mammalian cells . We also describe two different mutations in this gene in unrelated families with galactokinase deficiency and cataracts . The availability of the cloned galactokinase gene provides an important reference to identify mutations in patients with galactokinase deficiency and cataracts.

Protein Sci, 1995 Jul, 4(7), 1346 - 55
Simple models for the analysis of binding protein-dependent transport systems; Shilton BH et al.; Mathematical modeling was used to evaluate experimental data for bacterial binding protein-dependent transport systems . Two simple models were considered in which ligand-free periplasmic binding protein interacts with the membrane-bound components of transport . In one, this interaction was viewed as a competition with the ligand-bound binding protein, whereas in the other, it was considered to be a consequence of the complexes formed during the transport process itself . Two sets of kinetic parameters were derived for each model that fit the available experimental results for the maltose system . By contrast, a model that omitted the interaction of ligand-free binding protein did not fit the experimental data . Some applications of the successful models for the interpretation of existing mutant data are illustrated, as well as the possibilities of using mutant data to test the original models and sets of kinetic parameters . Practical suggestions are given for further experimental design.

J Clin Microbiol, 1995 Jul, 33(7), 1966 - 7
Errors arising from incorrect orientation of E test strips; Hamilton-Miller JM et al.; Major errors arise if E test strips are placed upside down . Asymmetric zones, or no zone at all, may result . MICs indicated by upside-down tests were almost always considerably higher than true values . This situation differs markedly from that for conventional testing, where orientation of disks is not important.

J Clin Microbiol, 1995 Jul, 33(7), 1896 - 8
Comparison of three methods for culturing throat swabs from cystic fibrosis patients; Hoppe JE et al.; In patients with cystic fibrosis who do not produce sputum, deep throat swabs are cultured for potential respiratory pathogens . Usually these swabs are directly streaked onto selective agar media . In a study of 50 pediatric cystic fibrosis patients, we compared this traditional method using rayon swabs with two methods having quantitative modifications: calcium alginate swabs eluted in Ringer's lactate and rayon swabs eluted in normal saline . The eluates were then processed quantitatively (three-step dilution series) . The yield of potential pathogens was significantly higher with the two quantitative methods . Overall, the combination of alginate with Ringer's lactate was superior to the combination of rayon with saline, although only some of these differences achieved statistical significance.

J Clin Microbiol, 1995 Jul, 33(7), 1842 - 6
Determination of MICs for Mycobacterium avium-M . intracellulare complex in liquid medium by a colorimetric method; Gomez-Flores R et al.; We investigated the potential of a rapid colorimetric microassay based on the reduction of dimethylthiazol-diphenyltetrazolium bromide (MTT) for determining the growth of Mycobacterium avium-M . intracellulare complex (MAC) and MICs of clofazimine, resorcinomycin A, and the quinolone PD 127391 against MAC . The reduction of MTT was directly proportional to the number of viable bacteria . A comparison of the MTT reduction test with the {3H}glycerol uptake assay showed the former to possess higher analytical sensitivity for detecting MAC growth in microtiter plates . The MIT reduction test avoids the use of radioisotopes and costly material and equipment; it is reliable, reproducible, and convenient for rapid routine susceptibility testing of MAC.

Chemosphere, 1995 Jul, 31(2), 2637 - 59
Assessment of structurally related chemicals: toxicity and ecotoxicity of acrylic acid and acrylic acid alkyl esters (acrylates), methacrylic acid and methacrylic acid alkyl esters (methacrylates); Greim H et al.; BUA compiled the available data on toxicity and ecotoxicity for several acrylic and methacrylic acid esters and their corresponding acids . A comparison of these data revealed a qualitative similarity in the toxicological and ecotoxicological properties of the compounds considered . The data indicate that methacrylates are less reactive than the corresponding acrylates.

Appl Microbiol Biotechnol, 1995 Jul, 43(3), 545 - 50
Glyphosate-degrading isolates from environmental samples: occurrence and pathways of degradation; Dick RE et al.; The metabolism of the organophosphonate herbicide glyphosate was investigated in 163 environmental bacterial strains, obtained by a variety of isolation strategies from sites with or without prior exposure to the compound . Isolates able to use glyphosate as sole phosphorus source were more common at a treated site, but much less abundant than those capable of using the glyphosate metabolite aminomethylphosphonic acid (AMPA) . Nevertheless, all 26 strains found to metabolise the herbicide did so via an initial cleavage of its carbon-phosphorus bond to yield sarcosine; no evidence for its metabolism or co-metabolism to AMPA was obtained.

J Magn Reson B, 1995 Jul, 108(1), 12 - 21
Improved excitation pulse bandwidths using shaped pulses, with application to heteronuclear half filters in macromolecular NMR; Hyre DE et al.; The advantageous use of sinc-shaped pulses in heteronuclear half filters is explored for studying biological macromolecules . The typical square, or hard, pulse used in half-filter pulse sequences for heteronuclear excitation results in suboptimal suppression of unwanted resonances due to incomplete inversion of spins . The novel use of short-duration shaped pulses applied at high power achieves more uniform excitation profiles over the extended frequency ranges often needed for heteronuclear filtering . This approach is used in the development of a double-tuned omega 1, omega 2-double-half-filtered, double-quantum-filtered COSY experiment . The efficiency of this experiment incorporating sinc pulses compares favorably with that obtained with square pulses in a mixture of 13C-labeled and unlabeled amino acids . Sinc-pulse-filtered spectra of the 24 kDa methionine repressor protein dimer MetJ, uniformly 13C-labeled expect at two unlabeled methionine residues, were also obtained to demonstrate the utility of this approach in biomacromolecular studies.

J Eukaryot Microbiol, 1995 Jul-Aug, 42(4), 388 - 91
Culture and phylogenetic characterization of Trichomitus trypanoides Duboscq & Grassè 1924, n . comb.: a trichomonad flagellate isolated from the hindgut of the termite Reticulitermes santonensis Feytaud; Berchtold M et al.; A trichomonad flagellate strain R1 was isolated from the hindgut contents of the termite Reticulitermes santonensis Feytaud . The flagellate was cultivated at 28 degrees C in anaerobic medium containing yeast extract, minerals and vitamins . The isolate fed on living bacteria . It showed the typical morphological and ultrastructural features of the trichomonads, closely resembling Trichomitus trypanoides . In order to determine its phylogenetic position the small subunit ribosomal DNA (SSU rDNA) of the flagellate was amplified in vitro using the polymerase chain reaction (PCR), cloned in a plasmid vector and sequenced . Comparison of the obtained sequence with so far available SSU rRNA/rDNA sequences showed strongest similarity (89%) to the sequence of Tritrichomonas foetus . The phylogenetic analysis with parsimony and distance matrix methods placed Trichomitus trypanoides strain R1 near by the root of the phylogenetically so far analyzed eukaryotic organisms . This confirms that termites harbour hindgut symbionts, which originate from very early evolved eukaryotes.

Biochem J, 1995 Jul 1, 309 ( Pt 1), 215 - 20
Human ciliary neurotrophic factor: a structure-function analysis; Kruttgen A et al.; Ciliary neurotrophic factor (CNTF) promotes survival in vitro and in vivo of several neuronal cell types including sensory and motor neurons . The primary structure of CNTF suggests it to be a cytosolic protein with strong similarity to the alpha-helical cytokine family which is characterized by a bundle of four anti-parallel helices . CNTF exerts its activity via complexation with CNTF receptor (CNTF-R) . This complex consists of a CNTF-binding protein (CNTF-R) and two proteins important for signal transduction {gp130 and leukaemia inhibitory factor receptor (LIF-R)} . We have shortened the cDNA coding for CNTF at both the 5' and the 3' end and expressed the truncated proteins in bacteria . Biological activities of the protein preparations were determined by their ability to induce proliferation of BAF/3 cells that were stably transfected with CNTF-R, gp130 and LIF-R cDNAs . CNTF proteins with 14 amino acid residues removed from the N-terminus were biologically active whereas the removal of 23 amino acids resulted in an inactive protein . In addition, 18 amino acid residues could be removed from the C-terminus of the CNTF protein without apparent loss of bioactivity, but further truncation at the C-terminus yielded biologically inactive proteins . The introduction of two point mutations into the CNTF protein at a site that presumably interacts with one of the two signal-transducing proteins resulted in a CNTF mutant with no measurable bioactivity . In addition, a model of the three-dimensional structure of human CNTF was constructed using the recently established structural co-ordinates of the related cytokine, granulocyte colony-stimulating factor . CD spectra of CNTF together with our mutational analysis and our three-dimensional model fully support the view that CNTF belongs to the family of alpha-helical cytokines . It is expected that our results will facilitate the rational design of CNTF mutants with agonistic or antagonistic properties.

Comp Biochem Physiol A Physiol, 1995 Jul, 111(3), 429 - 32
Local stimulatory effect of short-chain fatty acids on the mucus release from the hindgut mucosa of rats (Rattus norvegicus); Sakata T et al.; Short chain fatty acids such as acetic, propionic and butyric acids produced by hindgut bacteria affect various intestinal functions . However, the effect of short-chain fatty acids on mucus release from the hindgut mucosa was not clear . This study tested if these acids stimulate mucus release from hindgut mucosa and if the effect of these acids is local or requires a systemic mediation . Approximately 2 ml of either a physiologic mixture of acetic, propionic and butyric acids (100, 20 and 60 mmol/l, pH adjusted to 6.1) or 180 mmol/l sodium chloride (control solution, pH adjusted to 6.1) was infused into each permanently isolated cecum of anesthetized rats (Rattus norvegicus) through the ileal stoma by hydrostatic pressure . The cecum and distal colon were removed 1 hr after the infusion . The number of mucin containing cells per crypt section was counted on histological sections of these segments . The number of mucin containing cells was smaller in the cecum (but not in the distal colon) of rats infused in short-chain fatty acids compared with that of control rats . These results indicated that short-chain fatty acids locally stimulated mucus release from hindgut mucosa.

Arthritis Rheum, 1995 Jul, 38(7), 1005 - 13
Amplification of plasmid and chromosome Chlamydia DNA in synovial fluid of patients with reactive arthritis and undifferentiated seronegative oligoarthropathies; Bas S et al.; OBJECTIVE . To investigate the hypothesis that whole bacteria might be found in the joints of patients with Chlamydia-associated reactive arthritis . METHODS . The presence of 2 plasmid- and 2 chromosome-specific sequences of Chlamydia DNA was investigated by amplification with the polymerase chain reaction, in synovial fluid (SF) samples from 71 patients with various arthropathies . RESULTS . Chlamydia DNA was found in SF samples from 22 patients . CONCLUSION . Whole chlamydiae are likely present in the SF of patients with Chlamydia-associated reactive arthritis.

Mutat Res, 1995 Jul, 329(2), 173 - 81
Gamma-ray mutagenesis measurement in mammalian cells; Puck TT et al.; Application of conventional in vitro mutagenesis testing has so far failed to result in marked reduction of the total incidence of cancer . At least part of the reason may lie in the frequent use of a cell target too small to yield adequate sensitivity, and in failure to take into account the effects of cell killing in the assessment of mutagenic action . A single theoretical analysis fits the results of experimental data on gamma-irradiation applied to single marker gene testing in bacteria and to cytogenetic analysis of irradiated mammalian cells, and permits determination of the mean mutagenic dose, DoM, without complication due to cell killing . Cytogenetic monitoring of human lymphocytes which can detect mutagenic effects of gamma-radiation down to doses of < 0.1 Gy (10 rad) will also furnish an estimate of repair effectiveness at these low levels and may well be a useful tool in a program for prevention of cancer and other genetic disease.

Lab Invest, 1995 Jul, 73(1), 96 - 102
Persistence of Brucella abortus in the livers of T cell-deficient nude mice; Cheville NF et al.; BACKGROUND: Brucella abortus is sequestered in lymphoid tissues, bone, and liver during chronic asymptomatic brucellosis of cattle and humans . The sites of cellular infection, cytopathology of infected cells, and mechanisms of bacterial recrudescence have not been identified . A laboratory model is needed for the study of chronic brucellosis . EXPERIMENTAL DESIGN: Livers of athymic and euthymic mice infected with virulent B . abortus were cultured and examined morphologically to determine the effects of T cell dysfunction on persistence of intracellular bacteria . Bacterial Ag was detected immunohistochemically and by ultrastructural immunogold techniques . RESULTS: Bacteria in livers of euthymic mice rose to high titers at postinoculation Day (PID) 6 but rapidly declined and were slowly cleared . In athymic mice, bacteria did not reach such high titers and persisted in all mice to PID 121 . Granulomas were similar in size, structure, and number in both groups of mice through PID 28 . Thereafter, euthymic mice developed many granulomas that disappeared by PID 121 . In contrast, athymic mice failed to maintain granuloma formation but had diffuse lymphohistiocytic pericholangiitis with brucella Ag in subepithelial stellate cells, intraepithelial monocytes, and luminal macrophages . Intact bacteria were identified in lysosomes of macrophages and neutrophils only in acute infection . CONCLUSIONS: Athymic mice have normal or enhanced resistance to B . abortus in the first 10 days of infection but fail to maintain granuloma formation, do not clear brucellae from the liver, and develop persistent infection of the biliary tract . Brucellar Ag persists in chronically infected livers in periductal inflammatory tissues.

J Bacteriol, 1995 Jul, 177(13), 3870 - 2
Pathway of butyrate catabolism by Desulfobacterium cetonicum; Janssen PH et al.; Desulfobacterium cetonicum 480 oxidized butyrate to 1 mol of acetate and 2 mol of CO2; this reaction was coupled to reduction of sulfate to sulfide . Butyrate was activated by coenzyme A (CoA) transfer from acetyl-CoA, and butyryl-CoA was oxidized to acetyl-CoA by a classical beta-oxidation pathway . Acetyl-CoA was oxidized through the acetyl-CoA/carbon monoxide dehydrogenase pathway . There was a rapid exchange of 14CO2 into the intermediate CoA esters and into acetate and butyrate, showing that all of the steps involved in the oxidation of butyrate to acetyl-CoA are reversible.

Mol Cell Endocrinol, 1995 Jul, 112(1), 83 - 93
Study of TTF-1 gene expression in dog thyrocytes in primary culture; Van Renterghem P et al.; TTF-1 is a homeodomain-containing transcription factor mainly expressed in the thyroid where it controls the tissue-specific expression of the thyroglobulin, thyroperoxidase and TSH receptor genes . It is therefore potentially implicated in the hormonal control exerted by thyrotropin via the second messenger cyclic AMP on the transcription of these genes in thyrocytes . In order to investigate whether there exists a relationship between the stimulation of the cAMP pathway and TTF-1 gene expression in these cells, we have compared the amounts of TTF-1 protein, its state of phosphorylation and its subcellular distribution in control and cAMP-stimulated dog thyrocytes in primary culture . Dog TTF-1 was expressed in bacteria as a fusion protein and antibodies were raised against the dog TTF-1 moiety . Stimulation of the thyrocytes by cyclic AMP agonist only marginally increased TTF-1 gene expression as shown for the mRNA by RNase protection assay and for the protein by immunoblotting and immunoprecipitation of extracts from 35S-methionine labelled cells . The phosphorylation state of TTF-1 was investigated by immunoprecipitation of extracts from 32P-labelled thyrocytes . Phosphorylation level appeared to be essentially unaffected by forskolin treatment of the cells . We also looked for differences in the use of phosphorylation sites by partial proteolytic digestion of immunoprecipitated 32P-labelled TTF-1 with Glu-C and Asp-N endoproteases . Comparison of radioactivity distribution amongst the generated fragments did not reveal any difference in the pattern of TTF-1 phosphorylation in control and forskolin conditions . Lastly, in situ detection of TTF-1 by immunofluorescence demonstrated that the protein was localized in the nucleus of the cells, irrespective of the culture conditions . No major change in TTF-1 gene expression upon stimulation of the thyrocyte with a cAMP agonist could thus be detected in this study . The absence of an obvious modification of the TTF-1 protein itself in response to cAMP stimulation may indicate that other transcription factor(s) or co-factor(s) are involved in the control exerted by cAMP on the expression of thyroid-specific genes.

Zhonghua Zhong Liu Za Zhi, 1995 Jul, 17(4), 258 - 62
{Cloning of anti-breast cancer monoclonal antibodies from phage antibody libraries}; Zhang G et al.; Rodent monoclonal antibodies (McAbs) with anti-tumor specificity can be isolated from hybridomas by screening reactivity against tumor cells . Here, as an approach to bypass hybridoma technology, we attempted to isolate anti-tumor rodent McAbs directly from phage antibody libraries . We first used RT-PCR reactions to amplify diverse repertoires of heavy (Fd part) and light chains from spleen lymphocytes of Balb/c mice immunized with human cancer cell line BCap37 and then, by recombination of the repertoires in bacteria, generated a repertoire (1.0 x 10(7)) of Fab fragments displayed on filamentous phage . Four rounds of selection against living cancer cells showed specific enrichment of phage antibodies . After the fourth round of selection 174 out of 182 clones exhibited cancer cell binding capacity . The specificity of those clones was verified by reactivity against 12 human tumor cell lines, human lymphocytes and fibroblasts . In the production of monoclonal antibodies toward human cancer, this strategy may provide an alternative approach, and even surpass the hybridoma technology.

Clin Infect Dis, 1995 Jul, 21(1), 97 - 101
Is Blastocystis hominis a cause of diarrhea in travelers? A prospective controlled study in Nepal; Shlim DR et al.; Although the pathogenicity of Blastocystis hominis has been extensively debated in the medical literature, controlled studies of the association between B . hominis and diarrhea are lacking . We conducted a case-control study among expatriates and tourists in Kathmandu, Nepal, in which we compared the prevalence of the organism among patients with diarrhea to that among a control group without diarrhea . B . hominis was detected in 56 (30%) of 189 patients with diarrhea, compared with 40 (36%) of 112 asymptomatic controls . Patients with diarrhea were significantly more likely to have > or = 10 B . hominis organisms per high-power (400x) field than were controls . However, among the 25 patients with this concentration of organisms, other enteric pathogens were detected in 17 (68%) . Only 8 (4%) of 189 patients with diarrhea had > or = 10 B . hominis organisms per high-power field detected in the absence of other pathogens, compared with 5 (5%) of 112 asymptomatic controls . Thus, B . hominis in higher concentrations was not associated with diarrhea . There were no specific symptoms associated with B . hominis infection, and the presence of higher concentrations of the organism in stool was not associated with more-severe symptoms . Despite the high prevalence of the organism among travelers and expatriates in Nepal, the results of this study suggest that B . hominis does not cause diarrhea in this population.

Am J Vet Res, 1995 Jul, 56(7), 941 - 9
Effects of three occlusive dressing materials on healing of full-thickness skin wounds in dogs; Ramsey DT et al.; The effects of 3 occlusive dressing materials and a standard, nonadherent dressing material on healing of full-thickness skin defects were evaluated in dogs . Two wounds measuring 2 x 2 cm were created bilaterally (4 wounds/dog) on the dorsolateral aspect of the trunk of 12 Beagles . Wound treatments were evenly distributed between 4 sites, using a Latin square design . Treatments evaluated were: equine amnion (group A), biosynthetic hydrogel dressing (group B), transparent polyethylene sheeting (group T), and a semi-occlusive rayon/polyethylene, nonadherent dressing (group C) . Rates of contraction and epithelialization of group-A wounds were significantly greater than those of wounds of groups C, B, and T . On days 14, 21, and 28, mean percentage of wound contraction and mean percentage of total wound healed in group A exceeded those wounds in groups C, B, and T . On day 28, wounds in group A were significantly smaller than wounds in groups B and T, but were not significantly smaller than wounds in group C . All wounds in group A achieved 100% healing during the 28-day study period . Mean time for complete healing of group-A wounds was 21 days . The percentages of wounds completely healed by day 28 for groups B, C, and T were 25, 67, and 25%, respectively . Results indicate that use of equine amnion as an occlusive biological dressing on full-thickness wounds in dogs increases rate of healing.

Z Gastroenterol, 1995 Jul, 33(7), 408 - 13
{Hydrogen metabolism in the large intestine--physiology and clinical implications}; Christl SU et al.; During the anaerobic metabolism of the colonic bacterial flora short chain fatty acids and the gases hydrogen (H2) and carbon dioxyde (CO2) are produced . In about 50% of a European and North-American population and in 90% of rural black Africans, methane is generated from H2 and CO2 . In methane-negative individuals, sulfate reducing bacteria utilize H2 to reduce sulfate to sulfide . Methanogenesis and sulfate reduction are usually mutually exclusive . A competition exists for the common substrate hydrogen which is potentially regulated by the availability of sulfate in the colonic lumen . Other bacteria can use H2 to reduce CO2 to acetate (homoacetogenesis) . Methane is an inert gas and has probably no direct effect in man . The metabolites of sulfate reduction (mercaptides, hydrogen sulfide) are toxic and hydrogen sulfide is supposed to play a role in the pathogenesis of ulcerative colitis . All H2-utilizing metabolisms reduce the gaseous volume in the colon and thus prevent flatulence . In patients with pneumatosis cystoides intestinalis methanogenesis is absent and sulfate reduction is insignificant . This deficiency provides an explanation for the massive H2-excretion in those patients and their symptoms.

Recenti Prog Med, 1995 Jul-Aug, 86(7-8), 299 - 303
Effects of lactitol {correction of lactilol} on hepatic encephalopathy and plasma amino-acid imbalance; Trovato GM et al.; Hepatic encephalopathy in liver cirrhosis is due to several factors, including amino acid imbalance and hyperammonemia . Lactitol {correction of lactilol}, a non adsorbable disaccharide, improves hepatic encephalopathy increasing bowel movements, modifying colonic bacteria and pH, and reducing blood ammonium . Ten patients with liver cirrhosis and longstanding stable hepatic encephalopathy were treated, after a period of drugs wash-out, with lactitol . A significant improvement of hepatic encephalopathy was observed, with a significant decrease of blood ammonium, related with the increase of stool frequency/day . Atrial natriuretic peptide decreased as well . Moreover, an increase of the ratio of plasma aliphatic amino acids (valine, leucine and isoleucine)/aromatic amino acid (tyrosine and phenylalanine) was observed . Lactitol is an effective drug in the treatment of chronic hepatic encephalopathy; its mechanism of action involves not only a decrease of blood ammonium but also modifications of the degree of plasma amino acid imbalance, and fluid and circulatory adjustments.

J Periodontol, 1995 Jul, 66(7), 653 - 7
Occurrence of Actinobacillus actinomycetemcomitans and anti-leukotoxin antibodies in some members of an extended family affected by Papillon-Lefèvre syndrome; Stabholz A et al.; Eighteen (18) members of an extended family in which numerous individuals have Papillon-Lefevre syndrome (PLS) were examined . In all, 6 affected members and 12 non-affected members were included . All patients underwent a clinical examination which, in the dentate persons, included plaque index, bleeding on probing, probing depth, and periodontal attachment loss and a set of full mouth periapical x-rays . Subgingival bacterial samples were also collected from 2 teeth in the dentate patients for cultures and identification of Actinobacillus actinomycetemcomitans . Serum samples were collected from all participants and assayed for antileukotoxin antibodies . The results indicate that there is a high prevalence of leukotoxic strains of A . actinomycetemcomitans in persons suffering from PLS, as well as in unaffected family members . The ubiquitous presence of A . actinomycetemcomitans in the family units suggests a close association between A . actinomycetemcomitans and the periodontal disease associated with the syndrome; it also suggests that A . actinomycetemcomitans by itself is not sufficient for the expression of periodontal disease and that other factors, some of which must be genetic, are necessary for lesion development.

J Nat Prod, 1995 Jul, 58(7), 992 - 1002
The structures of new lanostane triterpenes from the fruiting bodies of Hebeloma senescens; Garlaschelli L et al.; Three new lanostane triterpenes, hebelomic acids B{4}, E{5}, and F{6}, were isolated from the inedible mushroom Hebeloma senescens . The latter two compounds are acyl derivatives of the new triterpene senescensol (12-deoxycrustulinol) {12} . The structures of compounds 4-6, including the absolute configuration of the 3-hydroxy-3-methylglutaric acid moiety, were established on the basis of spectral and chemical evidence.

J Med Assoc Thai, 1995 Jul, 78 Suppl 2, S95 - 8
An outbreak of post-operative endophthalmitis in Lampang Hospital; Pitaksiripan S et al.; An outbreak of post-operative endophthalmitis involving 48 patients from October 1991 to October 1992 in Lampang Hospital was reported . There were 3 waves of clustered cases, i.e . from October 1991 to January 1992, April-June 1992, and August-October 1992 . Investigation revealed several risk factors: defects in sterilization of surgical instruments, poor operating room hygiene, contaminated tap water and the use of multiple-dose fluids and medication . Bacteria isolated from vitreous fluid showed different bacteria, indicating multiple sources of infection or failure of asepsis . Each episode of infection was brought under control by removing the risk factors and emphasis on aseptic techniques . The value of an effective survey programme for the detection of post-operative endophthatmitis was emphasized.

J Med Assoc Thai, 1995 Jul, 78 Suppl 2, S102 - 4
Pneumonia in mechanically ventilated patients in Nan Hospital intensive care unit; Thanamee N et al.; A study on ventilator-associated pneumonia was done in the I.C.U., Nan Hospital from April 1991 to March 1992 . Of the 536 patients, 40 had pneumonia (7.5%) . One half of the cases with pneumonia were on mechanical ventilation for 3-7 days . The incidence rate of pneumonia was 17.5, 6.5, 2.5 and 0 per cent in paediatric, medical, surgical and gynaecologic patients . Nine patients died of pneumonia . Low birth weight was the commonest predisposing factor of death along with prolonged ventilatory support and resistant bacteria . Breaching of aseptic and nursing care techniques was also observed.

Plant Cell Physiol, 1995 Jul, 36(5), 789 - 97
Isolation and characterization of a cDNA that encodes maize glutamate dehydrogenase; Sakakibara H et al.; A full-length cDNA clone, pMGDH1, encoding maize NADH-glutamate dehydrogenase (NADH-GDH) was isolated from a maize root cDNA library . The identity of the cDNA was established by the coincidence of the structure of the purified protein with that inferred from the nucleotide sequence of the cDNA . pMGDH1 had a cDNA insert of 1,638 bp and the open reading frame encoded 411 amino acid residues . The deduced amino acid sequence was similar to putative partial sequences of GDHs from higher plants and to the sequences of GDHs from organisms as diverse as mammals and bacteria . The NH2-terminal sequence deduced from the open reading frame had a typical structure that is associated with the import of proteins into the mitochondrial matrix . The cDNA hybridized to an RNA of about 1.6 kb . This transcript was more abundant in roots than in leaves and was localized in the bundle sheath cells in leaf tissues . Analysis of genomic DNA by Southern hybridization suggested the existence of gene(s) for another NADH-GDH subunit(s).

Curr Opin Rheumatol, 1995 Jul, 7(4), 270 - 8
Structural polymorphism and function of HLA-B27; Lopez de Castro JA; Over the past year, new subtypes have been described and significant advances have been made in understanding peptide binding to HLA-B27 . These developments help in interpreting how antigen presentation is modulated by HLA-B27 polymorphism, in determining the similarities and differences in T cell antigenicity among subtypes, and in finding the basis of HLA-B27 evolution . New insights into the pathogenetic link between enteric bacteria and HLA-B27 come from demonstration of a direct relationship between gut flora and joint inflammation in transgenic rats and from involvement of HLA-B27 in modulating bacterial invasion of mammalian cells . The association between HLA-B27 and spondyloarthropathy may be related to antigen-presenting specificity, but a different mechanism, presentation of a B27-derived peptide by class II antigens, may contribute to the pathogenesis of acute anterior uveitis, emphasizing the need for a comprehensive understanding of the structure, antigenicity, and function of HLA-B27.

Acta Otorrinolaringol Esp, 1995 Jul-Aug, 46(4), 317 - 9
{Meningitis and cerebrospinal liquid fistula as a complication from the surgery of endolymphatic sac}; Munoz Colado M et al.; A 28 years old woman diagnosed of Meniere's disease was surgically treated with a drainage from endolymphatic system and a valve placed . Six years after surgery, the patient presented a bacteria meningitis an a cerebrospinal fluid leak to mastoid was found . New surgery was needed . Complications of endolymphatic system surgery, its treatment and frequency are discussed . This case is a very rare.

Clin Exp Immunol, 1995 Jul, 101(1), 39 - 44
Human autoantibodies directed against the RNA recognition motif of La (SS-B) bind to a conformational epitope present on the intact La (SS-B)/Ro (SS-A) ribonucleoprotein particle; Rischmueller M et al.; In systemic autoimmunity, the human B cell response to the La (SS-B) autoantigen is polyclonal and directed to both conserved and human-specific epitopes . This study has further characterized the B cell epitope(s) present within the conserved central region of the La protein, LaC (amino acids 111-242) containing the RNA recognition motif (RRM, aa 111-187) . Ten overlapping and non-overlapping protein fragments spanning LaC were expressed in bacteria as NH2-terminal fusions with glutathione-S-transferase . The fusion proteins were tested by ELISA for reactivity with a panel of human anti-La sera in order to define the nature of the epitopes . Ninety-two percent of patient sera containing anti-La antibodies reacted with the region of La containing the RRM . Fine mapping of this reactivity using deletion mutants indicated that the deletion of 19 amino acids from either the NH2-terminal or COOH-terminal region of the RRM was associated with loss of antibody reactivity, suggesting that the immunodominant epitope expressed in this region is discontinuous . Autoantibodies affinity-purified from the La RRM fragment to remove other specificities immunoprecipitated newly synthesized native La (SS-B)/Ro (SS-A) complexes, providing additional evidence that autoantibodies were recognizing a conformational epitope . The findings indicate that the human autoantibody response to La involves recognition of a conformational determinant involving the conserved RRM region without necessarily interfering with the RNA-dependent association of the La/Ro ribonucleoprotein.

Z Rheumatol, 1995 Jul-Aug, 54(4), 241 - 9
Cartilage destruction in septic arthritis--electron microscopy and historical considerations; Gauger M et al.; Light and electron microscopic investigations of joint cartilage in 13 cases of septic arthritis revealed sites of cartilaginous matrix infiltrated with polymorphonuclear granulocytes . This finding confirms rather unknown morphological works done by investigators on this diseases in the late 19th and beginning of the 20th century . Around neutrophils ultrastructural signs of direct chondrolytic activity were observed . In three cases bacteria were discovered inside the cartilaginous matrix . It is assumed that the invasion of polymorphonuclear granulocytes into the cartilaginous matrix is an important process in septic arthritis leading to cartilage destruction.

Mol Microbiol, 1995 Jul, 17(2), 221 - 30
Deletion of DNA lying close to the glkA locus induces ectopic sporulation in Streptomyces coelicolor A3(2); Kelemen GH et al.; Streptomyces coelicolor A3(2) J1668 sporulated ectopically in the substrate hyphae (the Esp phenotype) with the same time course as sporulation in the aerial hyphae . Examination of related strains implied that the Esp phenotype was caused by the deletion of DNA that lies close to, but is distinct from, the glucose kinase gene (glkA) . Co-transduction of the Esp phenotype with the deletion present in J1668 confirmed this hypothesis . The size of the deletion was found to be 7.4 kb . Construction of a strain carrying both the J1668 deletion and a whiG mutation demonstrated that the Esp phenotype depends on at least one of the genes required for the differentiation of aerial hyphae into spores.

J Comp Pathol, 1995 Jul, 113(1), 75 - 80
Histopathology of C57BL/6 mice inoculated orally with Mycobacterium paratuberculosis; Veazey RS et al.; The susceptibility of C57BL/6 mice to oral inoculation with Myobacterium paratuberculosis was evaluated histopathologically . Granulomatous lesions containing acid-fast bacteria developed in the mesenteric lymph nodes in over 50% of the mice by 11 months after inoculation . The results suggest that C57BL/6 mice may be useful for studying infection, pathogenesis, and other aspects of paratuberculosis.

Mol Microbiol, 1995 Jul, 17(1), 183 - 96
A multiple site-specific DNA-inversion model for the control of Omp1 phase and antigenic variation in Dichelobacter nodosus; Moses EK et al.; The molecular cloning and sequence analysis of four structurally variant linked genes (omp1A,B,C,D) that encode the major outer membrane protein of Dichelobacter nodosus strain VCS1001 are described . The isolation of rearranged copies of omp1A and omp1B, and the identification in the 5' regions of all four genes of short cross-over-site sequences that were similar to the Din family of cross-over-site sequences, suggested that site-specific DNA inversion was involved in omp1 rearrangement . Evidence for site-specific inversion of the 497 bp DNA fragment, which was located between the divergently orientated omp1A and omp1B genes, and which contained the promoter and 5' coding sequence of Omp1, was obtained by polymerase chain reaction-mediated amplification of inverted forms of these genes . However, to account for all of the omp1 gene copies cloned in this study, a more widespread inversion phenomenon must be involved in the rearrangement of these genes and a model for multiple site-specific DNA inversions at the omp1 locus is described . In this model the four structurally variant omp1 genes can be assembled from one of four structurally variant C-terminal coding regions and a conserved N-terminal coding region and can be expressed from a single promoter . It is postulated that this genetic capability endows D . nodosus with the ability to switch the antigenic specificity of one of its major surface proteins.

J Biol Chem, 1995 Jun 30, 270(26), 15838 - 43
Two distinct cell attachment sites in entactin are revealed by amino acid substitutions and deletion of the RGD sequence in the cysteine-rich epidermal growth factor repeat 2; Dong LJ et al.; The basement membrane glycoprotein, entactin, has previously been shown to promote cell attachment and chemotaxis . We have constructed a panel of glutathione S-transferase fusion proteins that encompasses the four major structural domains of entactin, G1, G2, E, and G3 . These proteins have been synthesized in bacteria and purified by affinity chromatography . The connecting stalk of entactin, E, which contains four cysteine-rich EGF homology repeats and the integrin receptor RGD recognition sequence, has been modified by deletion of the RGD sequence and substituting glutamic acid for aspartic acid . Attachment assays reveal that the RGD sequence is one of the major cell attachment sites in entactin and that this sequence is recognized by the alpha v beta 3 integrin receptor . Analysis of cell attachment on mutant forms of full-length entactin expressed in the baculovirus expression system revealed a second attachment site that was independent of the RGD sequence . This second site was localized to a peptide of 39 amino acid residues in the second globular G2 domain of entactin . This peptide represents a cysteine-rich EGF repeat . Inhibition of cell attachment by anti-integrin receptor antibodies indicates that the second attachment site is recognized by a member of the beta 1 family of integrin receptors, possibly alpha 3 beta 1.

Biochemistry, 1995 Jun 27, 34(25), 8130 - 43
ENDOR studies of the primary donor cation radical in mutant reaction centers of Rhodobacter sphaeroides with altered hydrogen-bond interactions; Rautter J et al.; The electronic structure of the cation radical of the primary electron donor was investigated in genetically modified reaction centers of Rhodobacter sphaeroides . The site-directed mutations were designed to add or remove hydrogen bonds between the conjugated carbonyl groups of the primary donor, a bacteriochlorophyll dimer, and histidine residues of the protein and were introduced at the symmetry-related sites L168 His-->Phe, HF(L168), and M197 Phe-->His, FH(M197), near the 2-acetyl groups of the dimer and at sites M160 Leu-->His, LH(M160), and L131 Leu-->His, LH(L131), in the vicinity of the 9-keto carbonyls of the dimer . The single mutants and a complete set of double mutants were studied using EPR, ENDOR, and TRIPLE resonance spectroscopy . The changes in the hydrogen bond situation of the primary donor were accompanied by changes in the dimer oxidation midpoint potential, ranging from 410 to 710 mV in the investigated mutants {Lin, X., Murchison, H . A., Nagarajan, V., Parson, W . W., Williams, J . C . & Allen, J . P . (1994) Proc . Natl . Acad . Sci . U.S.A . 91, 10265-10269} . It was found that the addition or removal of a hydrogen bond causes large shifts of the spin density between the two halves of the dimer . Measurements on double mutants showed that the unpaired electron can be gradually shifted from a localization on the L-half of the dimer to a localization on the M-half, depending on the hydrogen bond situation . As a control, the effects of the different hydrogen bonds on P.+ in the mutant HL(M202), which contains a BChlL-BPheM heterodimer as the primary donor with localized spin on the BChl aL {Bylina, E . J., & Youvan, D . C . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 7226-7230; Schenck, C . C., Gaul, D., Steffen M., Boxer S . G., McDowell L., Kirmaier C., & Holten D . (1990) in Reaction Centers of Photosynthetic Bacteria (Michel-Beyerle M . E., Ed.) pp 229-238, Springer, Berlin} were studied . In this mutant only small local changes of the spin densities (< or = 10%) in the vicinity of the hydrogen bonds were observed . The effects of the introduced hydrogen bonds on the spin density distribution of the dimer in the mutants are discussed in terms of different orbital energies of the two BChl a moieties which are directly influenced by hydrogen bond formation . The observed changes of the spin density distribution for the double mutants are additive with respect to the single mutations.(ABSTRACT TRUNCATED AT 400 WORDS)

Orv Hetil, 1995 Jun 25, 136(26), 1387 - 91
{Epidemiology of Helicobacter pylori infection in Hungary (comparative sero-epidemiologic study)}; Tamassy K et al.; In the last decade pathogenetical role of Helicobacter pylori infection has been proved in development of gastroduodenal alterations . DNA-RNA hybridisation and protein profile studies proved that Helicobacter pylori is an organism distinct from other bacteria . Therefore serology became a useful method to study the epidemiology of Helicobacter pylori infection in various populations . In Hungary sera were collected from adults aged 20-60 in blood banks (Military Hospital, Tolna County Hospital) in 1993 . The samples were classified in 5 year increment groups and questionnaires were filled out . Anti-H . pylori IgG were tested using Cobas Core kit (Roche Diagnostic) . The overall rate of seropositivity was 63.3% . The prevalence according to age was the following: 20-24 yrs = 44%, 25-29 yrs = 40%, 30-34 yrs = 52%, 35-39 yrs = 64%, 40-44 yrs = 75%, 45-49 yrs = 73%, 50-54 yrs = 77%, 55-59 yrs = 83% . There were no statistical difference between gender, living in urban or rural areas at the time of collection or in childhood, between the level of education, type of labour or social status . However we found statistical correlation between anti-H . pylori seropositivity and epigastric symptoms . The same characteristics of Helicobacter pylori infection were found in Hungary as well as in other countries . In groups between 20 and 30 years has been proved lower prevalence in Hungary than in Poland, Bulgaria.

J Biol Chem, 1995 Jun 23, 270(25), 15299 - 306
Chloroplasts can accommodate inclusion bodies . Evidence from a mutant of Chlamydomonas reinhardtii defective in the assembly of the chloroplast ATP synthase; Ketchner SL et al.; We identified two neighboring missense mutations in the chloroplast atpA gene which are responsible for the defect of ATP synthase assembly in the FUD16 mutant from Chlamydomonas reinhardtii . The two corresponding amino acid substitutions, Ile184-->Asn and Asn186-->Tyr, occurred at strictly conserved sites among the alpha and beta subunits of (C)F1 complexes from bacteria, mitochondria, and chloroplasts . The altered region in the alpha polypeptide chain is located 7 amino acids downstream of the P-loop, which forms most of the conserved nucleotide binding site . Although the resulting chloroplast mutant fails to accumulate most of the ATP synthase subunits, it displays an increased intracellular content in both the alpha and beta subunits . We demonstrate that the two subunits do not bind to the thylakoid membranes but associate and overaccumulate in the chloroplast stroma as inclusion bodies . Increased rates of synthesis of the two subunits in the mutant point to an early interaction between the two subunits during their biogenesis.

Biochemistry, 1995 Jun 20, 34(24), 7854 - 60
FT-IR and near-infrared FT-Raman study of aggregation of bacteriochlorophyll c in solutions: evidence for involvement of the ester group in the aggregation; Sato H et al.; Ultraviolet-visible (UV-Vis) absorption, Fourier-transform infrared (FT-IR), and near-infrared (NIR)-excited FT-Raman spectra have been measured for bacteriochlorophyll c (BChl-c) in acetone, tetrahydrofuran (THF), pyridine-d5, carbon disulfide (CS2), and water-saturated carbon tetrachloride (CCl4) to investigate its aggregation in vitro . The UV-Vis absorption spectra can be classified into two groups . Group I (acetone, THF, and pyridine-d5 solutions) gives a spectrum with a Qy band around 665 nm while group II (CS2 and water-saturated CCl4 solutions) shows a spectrum typical of BChl-c aggregates with a broader red-shifted Qy band . All the NIR-FT-Raman spectra, which are preresonant with the Qy band, are very close to those of chlorophyll a (Chl-a) measured in the corresponding solutions . Bands due to a C = O stretching mode of free and strongly hydrogen-bonded 13(1)-keto carbonyl groups appear near 1685 and 1645 cm-1, respectively . In contrast to the FT-Raman spectra, FT-IR spectra of the pyridine-d5 solution and group II are largely different from those of Chl-a in the corresponding solutions, suggesting that BChl-c forms quite different types of aggregates . It is clear from the IR spectra that the ester carbonyl group plays an important role in the aggregation for the pyridine-d5 and group II solutions . Of particular note is that bands due to C = O stretching modes of the ester group are observed at 1733, 1719, and 1705 cm-1 in the spectrum of BChl-c in water-saturated CCl4.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1995 Jun 20, 92(13), 6067 - 71
The Ste locus, a component of the parasitic cry-Ste system of Drosophila melanogaster, encodes a protein that forms crystals in primary spermatocytes and mimics properties of the beta subunit of casein kinase 2; Bozzetti MP et al.; Males of Drosophila melanogaster lacking the Y chromosome-linked crystal locus show multiple meiotic alterations including chromosome disorganization and prominent crystal formation in primary spermatocytes . These alterations are due to the derepression of the X chromosome-linked Stellate sequences . To understand how the derepression of the Stellate elements gives rise to these abnormalities, we have expressed the protein encoded by the Stellate sequences in bacteria and produced an antibody against the fusion protein . Immunostaining of crystal- testes has clearly shown that the Stellate protein is a major component of the crystals . Moreover, in vitro experiments have shown that this protein can interact with the catalytic alpha subunit of casein kinase 2 enzyme, altering its activity.

J Biol Chem, 1995 Jun 16, 270(24), 14768 - 75
Mutagenesis of apobec-1, the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme, reveals distinct domains that mediate cytosine nucleoside deaminase, RNA binding, and RNA editing activity; MacGinnitie AJ et al.; Apolipoprotein (apo) B48 is synthesized by mammalian small intestine as a result of post-transcriptional RNA editing . This process is mediated by an enzyme complex containing a catalytic subunit, apobec-1, which is homologous to other cytidine deaminases, particularly in a domain (H/C)-(A/V)-E-(X)24-30-P-C-(X)2-C which coordinates zinc, apobec-1, expressed as a glutathione S-transferase fusion protein, demonstrates both apoB RNA editing and cytidine deaminase activity . His61, Cys93, and Cys96, the putative zinc-coordinating residues, were mutated to Arg, Ser, and Ser, respectively, with loss of RNA editing activity and either great reduction or abolition of cytidine deaminase activity . Mutation of the catalytically active Glu63 residue to Gln and Pro92 to Leu abolished both cytidine deaminase and RNA editing activity . The conservative His61-->Cys mutation, which should coordinate zinc, retained both editing and cytidine deaminase activity . Thus, zinc binding is required for both apoB RNA editing and cytidine deaminase activity . Mutation of the first four leucines within the heptad repeat of the leucine-rich region (LRR) of apobec-1 resulted in reduced RNA editing but preservation of wild-type cytidine deaminase activity . GST/APOBEC-1 was also demonstrated to cross-link to apoB RNA . Mutation of His61-->Arg abolished RNA binding, while the Glu63-->Gln and Cys96-->Ser mutant proteins showed wild-type levels of RNA binding . The remaining mutants had reduced levels of activity . Overexpression of wild-type apobec-1 in McA 7777 cells resulted in a 5-6-fold increase in editing of endogenous apoB . Transfection of the His61-->Cys, LRR, and Cys93-->Ser mutants increased endogenous editing 2-3-fold, while Glu63-->Gln and His61-->Arg mutants acted as dominant negatives, reducing endogenous editing . These data suggest that apobec-1 has distinct functional domains which modulate activity in the context of the apoB mRNA editing enzyme.

Biochem J, 1995 Jun 15, 308 ( Pt 3), 859 - 64
Probing the determinants of protein stability: comparison of class A beta-lactamases; Vanhove M et al.; Five class A beta-lactamases produced by various mesophilic bacterial species have been compared . Although closely related in primary and overall structures, these enzymes exhibit very different stabilities . In order to investigate the factors responsible for these differences, several features deduced from the amino acid composition and three-dimensional structures were studied for the five proteins . This analysis revealed that higher stability appeared to correlate with increased numbers of intramolecular hydrogen bonds and of salt bridges . By contrast, the global hydrophobicity of the protein seemed to play a relatively minor role . A strongly unfavourable balance between charged residues and the presence of a cis-peptide bond preceding a non-proline residue might also contribute to the particularly low stability of two of the enzymes.

Rev Prat, 1995 Jun 15, 45(12), 1514 - 9
{Future vaccines}; Tron F; Powerful genetic and immunological techniques allow the production of new vaccines . Recombinant proteins and synthetic peptides represent new categories of subunit vaccines, illustrated, respectively, by recombinant hepatitis B vaccines and a peptide-derived malaria vaccine . Virus and bacteria can be used as vectors of foreign genes encoding antigens of vaccinal interest, to build-up new forms of live vaccines . One may expect from these new strategies of vaccine production a better control of viral, bacterial and parasitic diseases and a dramatic change in vaccine recommendations for children and adults.

Eur J Biochem, 1995 Jun 15, 230(3), 920 - 5
Structure and expression of Hemolin, an insect member of the immunoglobulin gene superfamily; Lindstrom-Dinnetz I et al.; Hemolin is an insect protein which belongs to the immunoglobulin superfamily and is strongly induced upon bacterial infection . It has been isolated from two moths, Hyalophora cecropia and Manduca sexta . We have isolated and sequenced a genomic clone for hemolin in H . cecropia, in order to resolve its organization and as a basis for investigating hemolin gene regulation . According to Southern-blot analysis, hemolin is encoded by a single gene, Hemolin . It contains six exons ranging over 32-603 bp . The introns are positioned both within and between the immunoglobulin-like domains, a feature typical for cell-adhesion molecules belonging to the immunoglobulin superfamily . By an RNase protection assay, we show that the Hemolin transcript is strongly induced not only by bacteria, but also by lipopolysaccharide and phorbol 12-myristate 13-acetate . Analysis of the upstream region and introns revealed potential binding sites for the Cecropia immunoresponsive factor (CIF), which recognizes the kappa B-like consensus GGGRA YYYYY.

Eur J Biochem, 1995 Jun 15, 230(3), 899 - 905
Quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni . Purification, characterization, and reconstitution of the apoenzyme with pyrroloquinoline quinone analogues; de Jong GA et al.; Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydrogenase (QH-EDH) apoenzyme was isolated from ethanol-grown Comamonas testosteroni . The purified apoenzyme, showing a single band of 71 kDa on native gel electrophoresis, could be only partially converted into active holoenzyme by addition of PQQ in the presence of calcium ions . In addition to a band with a molecular mass of 71 kDa, additional bands of 51 kDa and 25 kDa were observed with SDS/PAGE . Analysis of the N-terminal sequences of the bands and comparison with the DNA sequence of the gene, suggested that the latter two originate from the former one, due to scission occurring at a specific site between two vicinal residues in the protein chain . The extent of scission appeared to increase during growth of the organism . After addition of PQQ to apoenzyme, holoenzyme and nicked, inactive enzyme could be separated . Holoenzyme prepared in this way was found to contain equimolar amounts of PQQ, Ca2+ and covalently bound haem . EPR spectra of fully oxidized apo-QH-EDH and holo-QH-EDH showed g values typical for low-spin haem c proteins . In partially oxidized holo-QH-EDH an organic radical signal attributed to the semiquinone form of PQQ was observed . Binding of PQQ leads to conformational changes, as reflected by changes of spectral and chromatographic properties . Reconstitution of apoenzyme with PQQ analogues resulted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols . Compared with PQQ, analogues with a large substituent had a lower affinity for the apoenzyme . Results with other analogues indicated that possession of the o-quinone/o-quinol moiety is not essential for binding but it is for activity.

Nucleic Acids Res, 1995 Jun 11, 23(11), 1841 - 4
Sequence identity of the n-1 product of a synthetic oligonucleotide; Temsamani J et al.; After synthesis and purification of an oligonucleotide, the final product usually contains a low level of n-1 congeneric species . We have sequenced the n-1 population of a 25mer phosphodiester oligonucleotide . The n-1 band was cut from the gel and eluted . Oligonucleotides were tailed with dA and annealed to a dT-tailed plasmid . The recombinant plasmid was ligated and used to transform competent bacteria . Our results show that the n-1 population was heterogeneous . The frequency of truncated nucleotides at the 3'-end was much higher than at the 5'-end of the oligomer . No truncated nucleotides were found in the last four nucleotides at the 5'-end . Our results also show that the chain of oligonucleotides can grow on unreacted sites of a controlled-pore glass support.

Genomics, 1995 Jun 10, 27(3), 425 - 34
Construction of a panel of transgenic mice containing a contiguous 2-Mb set of YAC/P1 clones from human chromosome 21q22.2; Smith DJ et al.; Libraries of the entire human genome, or regions of the genome, have been made in bacteria, yeast, and somatic cells . We have expanded this strategy using overlapping YACs and P1s from human 21q22.2 (the Down syndrome region) to create a panel of transgenic mice containing DNA that encompasses this region of the human genome . Together the members of the in vivo library, each with a unique transgene (four YACs and four P1s), contain approximately 2 Mb of contiguous DNA . The integrity, stable inheritance, and expression of a coding sequence for each member of the YAC panel are demonstrated, and the uses of the panel are described.

Science, 1995 Jun 9, 268(5216), 1448 - 54
Bent helix formation between RNA hairpins with complementary loops; Marino JP et al.; The initial interaction between the ColE1 plasmid specific transcripts RNA I and RNA II, which function as antisense regulators of plasmid replication, comprises a transient complex between complementary loops found within the RNA secondary structures . Multidimensional heteronuclear magnetic resonance spectroscopy was used to characterize complexes formed between model RNA hairpins having seven nucleotide complementary loops . Seven base pairs are formed in the loop-loop helix, with continuous helical stacking of the loop residues on the 3' side of their helical stems . A sharp bend in the loop-loop helix, documented by gel electrophoresis, narrows the major groove and allows bridging of the phosphodiester backbones across the major groove in order to close the hairpin loops at their 5'-ends . The bend is further enhanced by the binding of Rom, a ColE1 encoded protein that regulates replication.

Cell, 1995 Jun 2, 81(5), 687 - 93
Identification of a nuclear receptor that is activated by farnesol metabolites; Forman BM et al.; Nuclear hormone receptors comprise a superfamily of ligand-modulated transcription factors that mediate the transcriptional activities of steroids, retinoids, and thyroid hormones . A growing number of related proteins have been identified that possess the structural features of hormone receptors, but that lack known ligands . Known as orphan receptors, these proteins represent targets for novel signaling molecules . We have isolated a mammalian orphan receptor that forms a heterodimeric complex with the retinoid X receptor . A screen of candidate ligands identified farnesol and related metabolites as effective activators of this complex . Farnesol metabolites are generated intracellularly and are required for the synthesis of cholesterol, bile acids, steroids, retinoids, and farnesylated proteins . Intermediary metabolites have been recognized as transcriptional regulators in bacteria and yeast . Our results now suggest that metabolite-controlled intracellular signaling systems are utilized by higher organisms.

J Biol Chem, 1995 Jun 2, 270(22), 13399 - 405
Superoxide radical and iron modulate aconitase activity in mammalian cells; Gardner PR et al.; Aconitase is a member of a family of iron-sulfur-containing (de)hydratases whose activities are modulated in bacteria by superoxide radical (O2-.)-mediated inactivation and iron-dependent reactivation . The inactivation-reactivation of aconitase(s) in cultured mammalian cells was explored since these reactions may impact important and diverse aconitase functions in the cytoplasm and mitochondria . Conditions which increase O2- . production including exposure to the redox-cycling agent phenazine methosulfate (PMS), inhibitors of mitochondrial ubiquinol-cytochrome c oxidoreductase, or hyperoxia inactivated aconitase in mammalian cells . Overproduction of mitochondrial Mn-superoxide dismutase protected aconitase from inactivation by PMS or inhibitors of ubiquinol-cytochrome c oxidoreductase, but not from normobaric hyperoxia . Aconitase activity was reactivated (t1/2 of 12 +/- 3 min) upon removal of PMS . The iron chelator deferoxamine impaired reactivation and increased net inactivation of aconitase by O2-. . The ability of ubiquinol-cytochrome c oxidoreductase-generated O2- . to inactivate aconitase in several cell types correlated with the fraction of the aconitase activity localized in mitochondria . Extracellular O2- . generated with xanthine oxidase did not affect aconitase activity nor did exogenous superoxide dismutase decrease aconitase inactivation by PMS . The results demonstrate a dynamic and cyclical O2-.-mediated inactivation and iron-dependent reactivation of the mammalian {4Fe-4S} aconitases under normal and stress conditions and provide further evidence for the membrane compartmentalization of O2-..

J Bioenerg Biomembr, 1995 Jun, 27(3), 275 - 83
Relationship between the oxidation potential of the bacteriochlorophyll dimer and electron transfer in photosynthetic reaction centers; Allen JP et al.; The primary electron donor in the photosynthetic reaction center from purple bacteria is a bacteriochlorophyll dimer containing four conjugated carbonyl groups that may form hydrogen bonds with amino acid residues . Spectroscopic analyses of a set of mutant reaction centers confirm that hydrogen bonds can be formed between each of these carbonyl groups and histidine residues in the reaction center subunits . The addition of each hydrogen bond is correlated with an increase in the oxidation potential of the dimer, resulting in a 355-mV range in the midpoint potential . The resulting changes in the free-energy differences for several reactions involving the dimer are related to the electron transfer rates using the Marcus theory . These reactions include electron transfer from cytochrome c2 to the oxidized dimer, charge recombination from the primary electron acceptor quinone, and the initial forward electron transfer.

Scanning Microsc, 1995 Jun, 9(2), 419 - 25; discussion 425-7
High resolution electron microscopy of the interface between dental calculus and denture resin; Hayashi Y; Dental calculus may grow on the denture surface . In order to demonstrate the mechanism of deposition, the interface between calculus and denture resin was investigated using a high resolution electron microscope . Ultrathin sections were also used for electron diffraction of selected areas to reveal any mineral phase . The mineral layers without mineralized bacteria adjacent to the denture surface revealed a marked variation in thickness and crystal shape . Three types of crystal shape were observed at the junction: needle-like, rod-like and plate-like crystals . High resolution electron microscopy (HREM) showed that both rod-like and plate-like crystals were an aggregation of fine crystallites . The lattice fringes of the fine crystallites were observed among the near atomic structures of resin polymer at the interface in all three types of crystals . The electron diffraction patterns of selected areas revealed that needle-like and rod-like crystals were composed of hydroxyapatite (OH-AP), while plate-like crystals were composed of a mixture of OH-AP and whitlockite . These findings indicate that, after saliva penetrates through the acrylic resin, calcium and phosphate ions in the saliva are trapped in the molecular chains of the resin polymer, while the local ion concentration then increases to reach supersaturation, whereas a spontaneous precipitation would occur at the superficial layer of the denture resin . Furthermore, a thin intermediate layer of crystallites might be indispensable for the scaffolding process in the calculus formation on the denture surface.

Environ Health Perspect, 1995 Jun, 103 Suppl 5, 9 - 12
Molecular analysis of isophthalate and terephthalate degradation by Comamonas testosteroni YZW-D; Wang YZ et al.; Comamonas testosteroni YZW-D was isolated from Passaic River sediment for its ability to degrade isophthalate and terephthalate . Degradation of the two isomeric compounds proceeds via separately inducible catabolic pathways that converge at protocatechuate . Analysis of the catabolic pathways by which these two isomers are degraded demonstrated that a cis-dihydrodiol intermediate is involved in both pathways . The genes for the conversion of isophthalate and terephthalate to protocatechuate were cloned on a single fragment of genomic DNA from C . testosteroni YZW-D . The two operons were located by subcloning and mutant complementation experiments . The regions coding for the two degradative pathways were sequenced . Analysis of the nucleotide sequence for the isophthalate degradation operon located genes for a dioxygenase, a transport protein, a cis-dihydrodiol dehydrogenase, and a reductase . Analysis of the nucleotide sequence for the terephthalate degradation operon located genes for a regulatory protein, a transport protein, a dioxygenase large subunit, a dioxygenase small subunit, a cis-dihydrodiol dehydrogenase, and a reductase.

Environ Health Perspect, 1995 Jun, 103 Suppl 5, 113 - 5
Plasmid transfer for enhancing degradation capabilities; Rittmann BE et al.; The kinetics of plasmid conjugation for the TOL and RP4 plasmids depend strongly on the donor cells' specific growth rate and substrate concentration, both of which determine the cells' energy availability . Although transfer rates can be large when energy availability is high, normal biological processes have low energy availability . Therefore, we propose and evaluate preliminarily a simple scheme to create a small zone of high energy availability.

Acta Otorhinolaryngol Ital, 1995 Jun, 15(3 Suppl 48), 1 - 24
{The treatment of allergic vasomotor rhinitis: diagnostic problems and local immunotherapy}; Motta G et al.; Treatment of allergic vasomotor rhinitis is to be regarded with the factors that modify the symptomatology . In fact must be considered the morphological changes (septum deviation, adenoids, turbinates hypertrophy, polyps) infections (bacteria, chlamydiae, micetes) and specific allergens . Identification of allergens and sensitization threshold is to be studied; then specific hyposensitization will be assessed . In the present study, 68 subjects having nasal reactivity have been observed and underwent to different ways of treatment: 18 with permanent stenosis identified by rhinomanometry and not modified with vasoconstrictors were operated; 25 of the 50 patients with normal nasal cavities showed contemporary infections: after a specific antimicrobic or antimycotic treatment, a clear improvement was obtained documented by rhinomanometry before and after nasal stimulations . The 50 patients with normal morphology underwent a local hyposensitization against the allergens . As a matter of fact: a) in all cases a clear improvement was obtained in phase of increasing; b) after one year of maintainance just 13 over 50 (26%) returned to previous conditions . The Authors remark how local immunotherapy by the nasal way give good possibilities in a high percentage of cases in the following conditions: correct clinical evaluation, especially concerning the identification of factors determining vasomotor rhinitis; employment of precise techniques for diagnosis; observation of clinical data and results turning out from instrumental investigations, especially concerning the nasal provocation test evaluated by rhinomanometry.

Rinsho Shinkeigaku, 1995 Jun, 35(6), 643 - 7
{A case report of pyomyositis--early diagnosis and follow-up by MRI}; Ohira T et al.; We report a rare case of pyomyositis in a 28-year-old Japanese woman who was not immunocompromised . She was admitted because of high fever, sore throat, and severe tenderness and swelling of the right calf . Redness, swelling, and tenderness indicated presence of acute inflammation in the calf . CT of the lower extremities demonstrated low density areas in the right soleus muscle and surrounding fascia with marked swelling, which were of high signal on T2 weighted images of MRI . There was no finding of abscess formation . A tentative clinical diagnosis of acute pyomyositis was made, and antibiotics therapy with a combination of fosfomycin and sulbactam/cefoperazone was started although the arterial blood culture was negative for bacteria . Associated acute tonsilitis was the most probable focus of pyomyositis . Antibiotics relieved her symptoms, and the inflammation subsided in several weeks . No surgical procedure was necessary . MRI taken tree weeks after the onset demonstrated abscess formation between the soleus and gastrocnemius muscles . Slight high intensity indicating scar formation remained in the area of the former abscess six weeks after the onset . MRI was very useful not only in making the early diagnosis but also in the follow-up of pyomyositis.

J Allergy Clin Immunol, 1995 Jun, 95(6), 1221 - 8
IgE antibodies to recombinant forms of Fel d I: dichotomy between fluid-phase and solid-phase binding studies; Slunt JB et al.; BACKGROUND: The major cat allergen Fel d I consists of two polypeptide chains linked by disulfide bonds, each of which has been expressed in bacteria . To investigate the antigenic structure of Fel d I, antibody binding to the native molecule and to each recombinant chain were compared . METHODS: Polyclonal human IgE and IgG antibodies and monoclonal antibodies (mAbs) to Fel d I were compared for binding to Fel d I, chain 1, or chain 2 by fluid-phase inhibition radioimmunoassay, RAST, and immunoabsorption . RESULTS: In the fluid-phase assay, neither recombinant chain significantly inhibited the binding of antibody to native Fel d I at concentrations of up to 10 micrograms/ml . Partial inhibition was observed when chain 1 was used, which inhibited the binding of two mAbs by 40% and 75% . In contrast, when the solid-phase RAST assay was used, IgE antibodies bound both chains with high specificity, and there was a good quantitative correlation between IgE antibody binding to Fel d I and both chain 1 (r = 0.58, p < 0.01) and chain 2 (r = 0.47, p < 0.01) . Up to 70% of IgG or IgE anti-Fel d I antibodies could be absorbed by either chain 1 or chain 2, and both chains in combination produced similar absorption values in response to native Fel d I . Four mAbs were fully absorbed by chain 1, but not chain 2, and three mAbs were not absorbed by either chain . CONCLUSIONS: The results demonstrate a dichotomy between antibody binding to recombinant Fel d I chains, which may be explained by confirmational differences between the chains in the fluid phase or on solid supports . The results also suggest that chain 1 is an important site for mAb-defined B-cell epitopes on Fel d I.

Bull Tokyo Med Dent Univ, 1995 Jun, 42(2), 57 - 65
Properties of alkaline phosphatase in the gingival crevicular fluid; Kina JR et al.; The isoenzymic properties of the alkaline phosphatase (ALP) of the gingival crevicular fluid (GCF) were investigated and compared with those in other cells, such as human polymorphonuclear leukocytes (PMNs), and human periodontal ligament cells (PDLs), and with those of three species of periodontopathic bacteria: Porphyromonas gingivalis 381 (P . gingivalis), Prevotella intermedia ATCC25611 (P . intermedia), and Capnocytophaga sputigena ATCC33123 (C . sputigena) . The biochemical properties of the isoenzymes were analyzed by the following methods: enzyme assays, inhibition pattern using three chemical inhibitors, 4 to 20% gradient polyacrylamide gel electrophoresis, thermostability, immunological specificity, and phosphatidylinositol-specific phospholipase C (PI-PLC) treatment . The inhibition experiment showed that ALP of the PMNs and PDLs possessed almost the same enzymatic properties of tissue-nonspecific ALP (bone/liver/kidney; TNSALP), and the ALP of the three species of periodontopathic bacteria possessed specific properties that were different from those of TNSALP, intestinal, or placental ALP . The ALP of the GCF was only slightly susceptible to levamisole (1 mM), L-phenylalanine (20 mM), and SDS (1%) . An electrophoresis thermostability test demonstrated that the enzyme activity of the GCF was separated into one or two bands . The main heat-labile slow band contained the phosphatidylinositol (PI)-moiety-anchored ALP and possessed immunological specificity against anti-bone type ALP . The minor fast band was heat stable and showed mobility similar to that in P . gingivalis . These results indicated that the ALP of the GCF consisted of several ALP isoenzyme types whose possible origins are considered to be derived from phosphatidylinositol (PI) anchored ALP and periodontopathic bacterial ALP.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1995 Jun, 61(6), 2257 - 61
Identification of grass-associated and toluene-degrading diazotrophs, Azoarcus spp., by analyses of partial 16S ribosomal DNA sequences; Hurek T et al.; The genus Azoarcus includes nitrogen-fixing, grass-associated strains as well as denitrying toluene degraders . In order to identify and group members of the genus Azoarcus, phylogenetic analysis based on partial sequences of 16S rRNA genes (16S rDNAs) is proposed . 16S rRNA-targeted PCR using specific primers to exclude amplification in the majority of other members of the beta subclass of the class Proteobacteria was combined with direct sequencing of the PCR products . Tree inference from comparisons of 446-bp rDNA fragments yielded similar results for the three known Azoarcus spp . sequences and for analysis of the complete 16S rDNA sequence . These three species formed a phylogenetically coherent group with representatives of two other Azoarcus species which were subjected to 16S rRNA sequencing in this study . This group was related to Rhodocyclus purpureus and Thauera selenatis . New isolates and also sequences of so far uncultured bacteria from roots of Kallar grass were assigned to the genus Azoarcus as well . Also, strains degrading monoaromatic hydrocarbons anaerobically in the presence of nitrate clustered within this genus, albeit not with grass-associated isolates . All representative members of the five species harboring rhizospheric bacteria were able to form N2O from nitrate and showed anaerobic growth on malic acid with nitrate but not on toluene . In order to visualize different Azoarcus spp . by whole-cell in situ hybridizations, we generated 16S rRNA-targeted, fluorescent probes by in vitro transcription directly from PCR products which spanned the variable region V2 . Hybridization was species specific for Azoarcus communis and Azoarcus indigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1995 Jun, 61(6), 2099 - 107
Phenotypic and genetic diversity of chlorine-resistant Methylobacterium strains isolated from various environments; Hiraishi A et al.; Strains of pink-pigmented facultative methylotrophs which were isolated previously from various environments and assigned tentatively to the genus Methylobacterium were characterized in comparison with authentic strains of previously known species of this genus . Most of the isolates derived from chlorinated water supplies exhibited resistance to chlorine, whereas 29 to 40% of the isolates from air, natural aquatic environments, and clinical materials were chlorine resistant . None of the tested authentic strains of Methylobacterium species obtained from culture collections exhibited chlorine resistance . Numerical analysis of phenotypic profiles showed that the test organisms tested were separated from each other except M . organophilum and M . rhodesianum . The chlorine-resistant isolates were randomly distributed among all clusters . The 16S ribosomal DNA (rDNA) sequence-based phylogenetic analyses showed that representatives of the isolates together with known Methylobacterium species formed a line of descent distinct from that of members of related genera in the alpha-2 subclass of the Proteobacteria and were divided into three subclusters within the Methylobacterium group . These results demonstrate that there is phenotypic and genetic diversity among chlorine-resistant Methylobacterium strains within the genus.

J Leukoc Biol, 1995 Jun, 57(6), 883 - 90
Ultraviolet radiation reduces phagocytosis and intracellular killing of mycobacteria and inhibits nitric oxide production by macrophages in mice; Jeevan A et al.; Exposure of mice to a single or multiple low doses of ultraviolet radiation (UVR) decreases the induction of the delayed-type hypersensitivity (DTH) response to Mycobacterium bovis BCG and Mycobacterium lepraemurium (MLM) and impairs the clearance of bacteria from the lymphoid organs . This study is an attempt to address the mechanism by which UV radiation impairs the clearance of bacteria from the lymphoid organs by determining whether alterations in macrophage function such as ingestion and intracellular killing of mycobacteria or production of reactive nitrogen intermediates might be responsible for these effects . BALB/c or C3H/HeN mice were exposed to a single dose of UVB (280-320 nm) radiation ranging from 0.35 to 45 kJ/m2, and at regular intervals after irradiation, the peritoneal and splenic macrophages were collected, cultured, and infected with live BCG or MLM . Phagocytosis was assessed at 6 h by counting the number of acid-fast bacteria per macrophage after Ziehl-Neelsen staining . The rate of intracellular killing was assessed by lysing the macrophages at 6, 12, 24, and 48 h after BCG infection, plating the suspension on 7H11 agar, and counting the number of colony-forming units 21 days later . Similarly, the nitric oxide production, as measured by nitrite, by macrophages obtained from UVB-irradiated and nonirradiated mice in response to BCG was assessed . There was a significant reduction in the uptake of organisms by both peritoneal and splenic macrophages collected from UV-irradiated mice . The intracellular killing of organisms was also significantly reduced, as was the production of nitric oxide by peritoneal macrophages infected with BCG in vitro . These results indicate that UVR affects macrophage functions and are consistent with our hypothesis that impaired clearance of bacteria in vivo results from an alteration in macrophage function.

Chest, 1995 Jun, 107(6), 1532 - 7
Empyema thoracis . Therapeutic management and outcome; LeMense GP et al.; STUDY OBJECTIVE: We evaluated treatment and outcome of patients with thoracic empyema at a teaching institution . DESIGN AND SETTING: Retrospective chart review over a 44-month period at a university hospital . PATIENTS AND MEASUREMENTS: Charts of patients with a hospital discharge diagnosis of thoracic empyema were reviewed . Age, symptoms, alcohol use, empyema etiology, culture results, number of loculations, date and success of each procedure, length of hospital stay, and hospital discharge status were recorded for each patient . Success of procedure, recovery time, time between procedures, and total hospitalization time were compared between procedures and between subgroups . RESULTS: Charts from 43 patients were reviewed . Twenty-four of 43 (56%) cases were parapneumonic empyemas . Forty of 43 (93%) patients had symptoms attributable to their empyema, with fever being the most common (65%) . Seventy-nine procedures were needed to treat the 43 patients (1.84 procedures per patient) . Success rates ranged from 11% (3/27) for tube thoracostomy to 95% (21/22) for decortication (p = 0.0001) . Delay between procedures averaged 6.2 +/- 1.1 (mean +/- SEM) days between the first and second procedure (n = 27), and 10.4 +/- 5.1 days between the second and third procedure (n = 8) . Mean recovery after successful intervention ranged from 9 to 19.3 days depending on the procedure (p = NS) . Comparisons between multiloculated and uniloculated empyemas, parapneumonic and nonparapneumonic empyemas, and culture proven and biochemically proven empyemas showed no significant difference in procedure success rates or length of hospital stay . CONCLUSION: Multiple therapeutic options exist for the treatment of thoracic empyema . Optimal therapy requires selection of the most appropriate first procedure for each patient with early postprocedure imaging to avoid inordinate delays between interventions.

Biochem J, 1995 Jun 1, 308 ( Pt 2), 585 - 90
The complete amino acid sequence confirms the presence of pseudoazurin in Thiosphaera pantotropha; Chan C et al.; The complete amino acid sequence, obtained by direct protein sequencing, of the pseudoazurin from Thiosphaera pantotropha is reported . It shows sequence identities varying from 46 to 66% with previously sequenced pseudoazurins . Previously identified conserved residues with key functions in pseudoazurins are found in the protein from T . pantotropha with the exception of glycine-39, the carbonyl group of which has been considered as a ligand to the copper, which is replaced by a serine residue . Electrospray-ionization MS (ESI-MS) has shown that pseudoazurin from T . pantotropha often contains two polypeptide species differing in molecular mass by 16 Da, presumably owing to oxidation of a methionine residue to a sulphoxide derivative . These two species have different endoproteinase Arg-C digestion patterns . Conditions for ESI-MS were identified that permitted either the retention or the loss of the single copper ion associated with the pseudoazurin . The aberrant tendency of T . pantotropha pseudoazurin to form a disulphide-bridged dimer on SDS/PAGE under some conditions is described.

Biochem J, 1995 Jun 1, 308 ( Pt 2), 375 - 9
The structure of the quinoprotein alcohol dehydrogenase of Acetobacter aceti modelled on that of methanol dehydrogenase from Methylobacterium extorquens; Cozier GE et al.; The 1.94 A structure of methanol dehydrogenase has been used to provide a model structure for part of a membrane quinohaemoprotein alcohol dehydrogenase . The basic superbarrel structure and the active-site region are retained, indicating essentially similar mechanisms of action, but there are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site.

J Bacteriol, 1995 Jun, 177(11), 3071 - 9
Particulate methane monooxygenase genes in methanotrophs; Semrau JD et al.; A 45-kDa membrane polypeptide that is associated with activity of the particulate methane monooxygenase (pMMO) has been purified from three methanotrophic bacteria, and the N-terminal amino acid sequence was found to be identical in 17 of 20 positions for all three polypeptides and identical in 14 of 20 positions for the N terminus of AmoB, the 43-kDa subunit of ammonia monooxygenase . DNA from a variety of methanotrophs was screened with two probes, an oligonucleotide designed from the N-terminal sequence of the 45-kDa polypeptide from Methylococcus capsulatus Bath and an internal fragment of amoA, which encodes the 27-kDa subunit of ammonia monooxygenase . In most cases, two hybridizing fragments were identified with each probe . Three overlapping DNA fragments containing one of the copies of the gene encoding the 45-kDa pMMO polypeptide (pmoB) were cloned from Methylococcus capsulatus Bath . A 2.1-kb region was sequenced and found to contain both pmoB and a second gene, pmoA . The predicted amino acid sequences of these genes revealed high identity with those of the gene products of amoB and amoA, respectively . Further hybridization experiments with DNA from Methylococcus capsulatus Bath and Methylobacter albus BG8 confirmed the presence of two copies of pmoB in both strains . These results suggest that the 45- and 27-kDa pMMO-associated polypeptides of methanotrophs are subunits of the pMMO and are present in duplicate gene copies in methanotrophs.

J Bacteriol, 1995 Jun, 177(11), 3010 - 20
Comparative ultrastructural and functional studies of Helicobacter pylori and Helicobacter mustelae flagellin mutants: both flagellin subunits, FlaA and FlaB, are necessary for full motility in Helicobacter species; Josenhans C et al.; Helicobacter mustelae causes chronic gastritis and ulcer disease in ferrets . It is therefore considered an important animal model of human Helicobacter pylori infection . High motility even in a viscous environment is one of the common virulence determinants of Helicobacter species . Their sheathed flagella contain a complex filament that is composed of two distinctly different flagellin subunits, FlaA and FlaB, that are coexpressed in different amounts . Here, we report the cloning and sequence determination of the flaA gene of H . mustelae NCTC12032 from a PCR amplification product . The FlaA protein has a calculated molecular mass of 53 kDa and is 73% homologous to the H . pylori FlaA subunit . Isogenic flaA and flaB mutants of H . mustelae F1 were constructed by means of reverse genetics . A method was established to generate double mutants (flaA flaB) of H . mustelae F1 as well as H . pylori N6 . Genotypes, motility properties, and morphologies of the H . mustelae flagellin mutants were determined and compared with those of the H . pylori flaA and flaB mutants described previously . The flagellar organizations of the two Helicobacter species proved to be highly similar . When the flaB genes were disrupted, motility decreased by 30 to 40% . flaA mutants retained weak motility by comparison with strains that were devoid of both flagellin subunits . Weakly positive motility tests of the flaA mutants correlated with the existence of short truncated flagella . In H . mustelae, lateral as well as polar flagella were present in the truncated form . flaA flaB double mutants were completely nonmotile and lacked any form of flagella . These results show that the presence of both flagellin subunits is necessary for complete motility of Helicobacter species . The importance of this flagellar organization for the ability of the bacteria to colonize the gastric mucosa and to persist in the gastric mucus remains to be proven.

J Bacteriol, 1995 Jun, 177(11), 2990 - 7
Characterization of an aerobic repressor that coordinately regulates bacteriochlorophyll, carotenoid, and light harvesting-II expression in Rhodobacter capsulatus; Ponnampalam SN et al.; For most species of purple photosynthetic bacteria, the presence of molecular oxygen represses synthesis of carotenoids and bacteriochlorophyll . In this study we characterize a strain of Rhodobacter capsulatus, DB469, which contains a genomic disruption of an open reading frame in the photosynthesis gene cluster termed ORF469 . Characterization of the steady-state level of bacteriochlorophyll synthesis demonstrates that disruption of ORF469 results in a 2.5-fold increase in aerobic synthesis of bacteriochlorophyll over that observed with the parent strain . Utilizing reporter plasmids that contain transcriptional fusions of lacZ to various carotenoid and bacteriochlorophyll biosynthesis genes, we also demonstrate that disruption of ORF469 leads to an approximate twofold increase in bacteriochlorophyll and carotenoid gene expression under anaerobic growth conditions . Similar analysis with reporter plasmids that contain translational fusions of lacZ to the puf, puh, and puc operons demonstrates that disruption of ORF469 leads to elevated levels of aerobic transcription of light harvesting-II genes (puc), without affecting light harvesting-I or reaction center gene expression (puf and puh, respectively) . Gel mobility analysis demonstrates that DB469 cells lack a DNA-binding protein that interacts with a palindromic sequence in the bchC promoter region . The results of this study indicate that ORF469 codes for a DNA-binding protein that acts as an aerobic repressor of promoters for bacteriochlorophyll, carotenoid, and light harvesting-II gene expression.

Am J Respir Crit Care Med, 1995 Jun, 151(6), 1974 - 80
Mucosal T-lymphocytes in central airways of lung transplant recipients; Fournier M et al.; The immunohistochemical profile of mucosal lymphocytes was investigated in the central airways of lung transplant recipients . Bronchial and transbronchial biopsies (BB and TBB, respectively) and bronchoalveolar lavage for culture of bacteria and viruses were performed during a fibroscopic procedure in patients without evidence of chronic rejection, 3 to 10 mo after surgery . Analysis was restricted to samples without concurrent airway infection: 23 pairs of BB and TBB from 18 transplant recipients were analyzed . An immunohistochemical technique was used to identify and score mucosal cells that reacted with monoclonal antibodies against CD4, CD8, CD45-Ro (memory T-cells), and HLA-DR molecules . The same procedure was applied in nine nonsmoking control subjects (NS group) . Data from transplant recipients were allocated to R+ (n = 11) or R- groups (n = 12), depending on the presence or absence of histologic evidence of acute rejection on TBB . A statistically significant depletion of every immunoreactive cell subset was observed in the R+ and the R- groups, but not in the NS group . Conversely, no significant difference for either score of immunoreactive cells were found between R+ and R- groups . The immunosuppressive regimen is suspected to play to play a major role in this depletion of bronchial mucosal T-cells . The acute lung rejection process does not appear to affect concurrently the immunohistochemical profile of immunoreactive cells in the bronchial mucosa.

J Virol, 1995 Jun, 69(6), 3584 - 96
Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs; Ou SH et al.; Human immunodeficiency virus type 1 (HIV-1) gene expression is modulated by both viral and cellular factors . A regulatory element in the HIV-1 long terminal repeat known as TAR, which extends from nucleotides -18 to +80, is critical for the activation of gene expression by the transactivator protein, Tat . RNA transcribed from TAR forms a stable stem-loop structure which serves as the binding site for both Tat and cellular factors . Although TAR RNA is critical for Tat activation, the role that TAR DNA plays in regulating HIV-1 gene expression is not clear . Several studies have demonstrated that TAR DNA can bind cellular proteins, such as UBP-1/LBP-1, which repress HIV-1 gene expression and other factors which are involved in the generation of short, nonprocessive transcripts . In an attempt to characterize additional cellular factors that bind to TAR DNA, a lambda gt11 expression cloning strategy involving the use of a portion of TAR DNA extending from -18 to +28 to probe a HeLa cDNA library was used . We identified a cDNA, designated TAR DNA-binding protein (TDP-43), which encodes a cellular factor of 43 kDa that binds specifically to pyrimidine-rich motifs in TAR . Antibody to TDP-43 was used in gel retardation assays to demonstrate that endogenous TDP-43, present in HeLa nuclear extract, also bound to TAR DNA . Although TDP-43 bound strongly to double-stranded TAR DNA via its ribonucleoprotein protein-binding motifs, it did not bind to TAR RNA extending from +1 to +80 . To determine the function of TDP-43 in regulating HIV-1 gene expression, in vitro transcription analysis was performed . TDP-43 repressed in vitro transcription from the HIV-1 long terminal repeat in both the presence and absence of Tat, but it did not repress transcription from other promoters such as the adenovirus major late promoter . In addition, transfection of a vector which expressed TDP-43 resulted in the repression of gene expression from an HIV-1 provirus . These results indicate that TDP-43 is capable of modulating both in vitro and in vivo HIV-1 gene expression by either altering or blocking the assembly of transcription complexes that are capable of responding to Tat.

J Hosp Infect, 1995 Jun, 30(2), 85 - 93
Wound disinfection with ultraviolet radiation; Taylor GJ et al.; Bacteria were counted concurrently in the air and wounds during the first 20 min of total joint arthroplasty procedures in two theatres: a conventional plenum ventilated theatre with ultraviolet C (UVC) tubes installed and a filtered vertical laminar flow theatre . Four theatre environments were tested: conventional theatre and clothing; conventional theatre with UVC protective clothing, with UVC set to produce 100 or 300 microW cm-2 s-1 irradiation; and filtered vertical laminar flow air with staff wearing cuffed cotton/polyester clothing . When used, the UVC was activated 10 min after starting an operation to assess the effect of UVC clothing alone, and of UVC radiation on bacteria already present in the wound . Compared with conventional theatres, UVC clothing reduced air counts by 38%, UVC at 100 microW cm-2 s-1 by 81%, at 300 microW cm-2 s-1 by 91%, and laminar flow by 92% . Wounds counts fell correspondingly by 66% with UVC clothing, 87% with UVC at 100 microW cm-2 s-1 and 92% both with UVC at 300 microW cm-2 s-1 and laminar flow . In conventional and laminar flow theatres air and wound counts correlated closely but in UVC theatres wound counts were lower than levels expected from prevailing air counts suggesting that UVC kills bacteria in wounds as well as in air.

J Cell Sci, 1995 Jun, 108 ( Pt 6), 2241 - 51
Expression of the beta 6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling; Breuss JM et al.; The alpha v beta 6 integrin was identified in cultured epithelial cells and functions as a fibronectin receptor . We have now used monoclonal antibodies to determine in vivo expression patterns of the beta 6 subunit in normal and pathological human or primate tissues, and during experimental wound healing or induced lung injury . The results indicate that beta 6 expression is restricted to epithelia and is up-regulated in parallel with morphogenetic events, tumorigenesis, and epithelial repair . During development of the kidney, lung, and skin, we found that beta 6 is expressed by specific types of epithelial cells, whereas it is mostly undetectable in normal adult kidney, lung and skin . In contrast, we detected high-level expression in several types of carcinoma . For example, beta 6 is almost invariably neo-expressed in squamous cell carcinomas derived from the oral mucosa, often focally localized at the infiltrating edges of tumor islands . Expression of beta 6 is also upregulated in migrating keratinocytes at the wound edge during experimental epidermal wound healing . Similarly, beta 6 expression is induced in type II alveolar epithelial cells during lung injury caused by injection of live bacteria . We also observed beta 6 expression in adult lungs and kidneys at focal sites of subclinical inflammation, as well as in a variety of clinical specimens from patients with chronic or acute inflammation of the lungs or kidneys . From these findings and earlier results, we hypothesize that alpha v beta 6 affects cell spreading, migration and growth during reorganization of epithelia in development, tissue repair, and neoplasia.

Clin Lab Med, 1995 Jun, 15(2), 209 - 34
The laboratory diagnosis of pneumonia . The role of the community hospital pathologist; Yungbluth M; Pneumonia is one of the most serious infections seen in community hospital practice, with virulent bacteria and viruses producing infections in the healthy host and a variety of opportunistic organisms capable of causing disease in the immunocompromised patient . Accurate laboratory diagnosis is extremely important for correct clinical management of pneumonia, and the community hospital pathologist can take an active role in daily review of respiratory tract specimens to optimize and coordinate this important laboratory testing . This article discusses strategies for improving sputum Gram stain interpretation and for the use of both routine and supplementary cultures in community-acquired pneumonias and outlines a comprehensive consultative approach for rapid and reliable pneumonia diagnosis in the compromised patient.

Farmaco, 1995 Jun, 50(6), 431 - 8
Study of the Mannich reaction: beta-amino-methylation of N-aryl and N-azaheteroaryl-substituted 2,5-dimethylpyrroles, compounds with potential biological activity; Biava M et al.; Owing to the increasing need of drugs for the treatment of a variety of fungal and bacterial opportunistic infections, a study has been started with the aim of synthesizing structures amenable to a number of easily-to-perform structural modifications in order to meet the requirement of bypassing resistance phenomena . This paper reports on the synthesis of several N-(alpha-azaheteroaryl)-substituted 2,5-dimethyl-pyrroles bearing in one beta-position (or in both beta-positions) aminomethyl groups, introduced via a Mannich reaction . Electronic and steric effects by the N-(azaheteroaryl) substituents and the 2- and 5-methyl groups on the course of the Mannich reaction are discussed along with the results of in vitro tests against many Candida species, some bacteria, and several pathogenic plant fungi.

Protein Expr Purif, 1995 Jun, 6(3), 319 - 28
Pyrophosphate-dependent phosphofructokinase from Giardia lamblia: purification and characterization; Li Z et al.; The pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) from Giardia lamblia has been purified to homogeneity using two methods . The purified enzyme is shown to be homogenous by its migration as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a single peak on reverse-phase HPLC . The purified enzyme is a monomeric protein of Mr 64 kDa as determined by SDS-PAGE and native PAGE . The enzyme fractionated as a 67-kDa protein by HPLC gel filtration . The pH optima in either the glycolytic or the gluconeogenic direction is near neutral, which is unlike any of the protozoan or bacterial enzymes . The enzyme displayed typical Michaelis-Menten kinetics . The Km values for pyrophosphate and fructose 6-phosphate were 0.039 +/- 0.005 and 0.25 +/- 0.0196 mM, respectively . The initial velocity for the reverse reaction was also found to be hyperbolic . Different nucleoside triphosphates, including ATP, could not substitute for pyrophosphate as the phosphoryl donor . Similar to the pyrophosphate-dependent enzyme from other anaerobic protozoans and bacteria, the Giardial enzyme was not activated by fructose 2,6-bisphosphate, although this metabolite is a competitive inhibitor with respect to fructose 1,6-bisphosphate in the reverse reaction . The pH optima and the molecular weight properties of the Giardial enzyme are different from those of the two classes of the pyrophosphate-dependent phosphofructokinases.

Burns, 1995 Jun, 21(4), 297 - 300
Treatment of partial thickness burns of the hand with the preshaped, semipermeable Procel Burn Cover: results of a multicentre study in the burn centres of Berlin, Duisburg and Munich; Jostkleigrewe F et al.; The results of a prospective clinical study conducted in three German burn centres are reported . The subject of the evaluation was to show the effectiveness of a new, preshaped, semipermeable burn dressing that is resistant to fluids and bacteria but highly permeable to vapour . The dressing was used in conjunction with 1 per cent silver sulphadiazine cream in treating partial thickness burns of the hand . In 49 patients, 72 partial thickness burned hands were treated . The application proved to be very easy . The time for a dressing change was short (5-10 min) . The duration of treatment was 13 days on average . Complications due to infections did not occur . Because of the semipermeable properties of the dressing material, skin macerations occurred in only a few instances (13 per cent) as a result of inappropriate cream application or extremely high exudation rates, and these did not adversely affect the healing process . Patients achieved the ability to perform activities of daily living early with positive results for the patients, the physician and the nursing team.

Burns, 1995 Jun, 21(4), 267 - 72
Prophylactic anti-lipopolysaccharide freeze-dried plasma in major burns: a double blind controlled trial; Jones EB; A double-blind controlled trial to test the efficacy of freeze-dried plasma containing a high titre of anti-lipopolysaccharide (anti-LPS) IgG in preventing or reducing sepsis in burns was carried out on 60 consecutive consenting adult burn victims with burns between 20 and 55 per cent of body surface area . Statistics failed to demonstrate a reduction in the mortality (P = 0.12) but did demonstrate a reduction in the incidence of burn wound infection (P = 0.0443), a fall in endotoxin levels in the first week (P = 0.041) and a rise in the anti-endotoxin levels after 5 units of anti-LPS plasma (P = 0.027) . The logistics of carrying out such a trial are difficult but a larger patient population and modification of the original protocol might demonstrate a significant fall in mortality and morbidity . It was felt that some of the positive benefits of the high titre anti-LPS plasma might be due to factors other than the anti-LPS IgG.

Burns, 1995 Jun, 21(4), 255 - 8
The association of circulating endotoxaemia with the development of multiple organ failure in burned patients; Yao YM et al.; In this study, we examined the relationship of plasma endotoxin levels to the development of multiple organ failure (MOF), and the outcome in patients with thermal injury . A prospective cohort study of 17 patients admitted with burns covering more than 70 per cent of body surface area was undertaken . Circulating endotoxin concentrations were measured by modified limulus amoebocyte lysate assay in serial samples of plasma . Seven out of 17 burned patients developed MOF according to multiple criteria . The plasma endotoxin concentrations of patients with MOF were 0.512-1.127 EU/ml, which were significantly higher than that of 10 patients without MOF (0.216-0.553 EU/ml), on 3, 14, 21 and 28 days postburn (p < 0.05-0.01) . A significantly higher incidence of positive endotoxin tests (> or = 0.120 EU/ml) was found in patients who developed MOF as compared to those patients who did not develop MOF during the observation period (p < 0.05) . As the mean endotoxin levels increased, the incidence of MOF and death rate also increased, and persistent endotoxemia carried a poor prognosis . The present investigation provides further evidence that endotoxemia in severely burned patients commonly occurs . Circulating endotoxin has also been found to be strongly associated with the development of MOF and mortality following major burn injury.

Aust Dent J, 1995 Jun, 40(3), 201 - 6
Infection emanating from an 'innocent' facial puncture wound . Case reports; Csillag GD; All accidental wounds are contaminated by bacteria . The factors involved in the conversion of a wound from contaminated to infected are identical, irrespective of the site and classification of the wound, although puncture wounds are particularly suited for the growth of anaerobic organisms . A case is presented of facial infection emanating from an 'innocent' puncture wound . The management of orofacial puncture wounds is discussed and illustrated with a second case.

Fundam Appl Toxicol, 1995 Jun, 26(1), 20 - 31
A collaborative evaluation of seven alternatives to the Draize eye irritation test using pharmaceutical intermediates; Sina JF et al.; Much of the data which have been generated on in vitro alternatives to the Draize eye irritation test have dealt with compounds within a specific chemical class or product category . However, in the pharmaceutical industry, it is often necessary to evaluate materials which are not related in structure or properties . It was thus decided to evaluate a diverse series of chemicals in seven in vitro methods for estimating ocular irritation . Thirty-seven test materials were chosen to represent a broad range of pH, solubility, and in vivo irritation potential . Assays were chosen to include as many different types of end points as practical . The group of assays was composed of TOPKAT (assessing structure-activity relationships), bovine corneal opacity-permeability (BCO-P; corneal opacity/toxicity), Eytex (protein coagulation), neutral red uptake (cytotoxicity), MTT in living dermal equivalent (cytotoxicity), Microtox (cytotoxicity in bacteria), and CAMVA (inflammation/toxicity) . The results of the study indicated that, in general, the cytotoxicity end points did not correlate well with the in vivo data . The BCO-P, CAMVA, and Eytex assays had the best overall concordance (88.9, 75.8, and 75.0%, respectively) with this set of compounds . Estimation of irritation potential based on structure-activity (TOPKAT) was possible for only approximately 50% of the compounds; however, the assay showed 100% sensitivity (i.e., no false negatives), but low specificity (i.e., negatives correctly identified only 54.5% of the time) . These data suggest that for screening of chemicals of diverse structure and properties, the more mechanism-based assays, as opposed to general cytotoxicity assays, hold more promise and should be further evaluated.

Hum Mol Genet, 1995 Jun, 4(6), 1063 - 72
Characterization of myotonic dystrophy kinase (DMK) protein in human and rodent muscle and central nervous tissue; Whiting EJ et al.; Myotonic dystrophy (DM) is the most common form of inherited neuromuscular disease in adults and is characterized by progressive muscle wasting and myotonia . The mutation responsible for DM has been identified as the amplification of a polymorphic (CTG)n repeat in the 3' untranslated region of a gene encoding a serine/threonine kinase (DMK) . We have produced a polyclonal rabbit antibody preparation against a fusion protein encoding the C-terminal amino acids 471-629 of the human DMK gene . This antibody specifically detects products of both full length and truncated human DMK genes expressed in bacteria and in insect cells . On immunoblots, we observed protein species of approximately 74 and 82 kDa in cardiac muscle, skeletal muscle, ependyma and choroid plexus . By immunofluorescence, DMK was found to localize post-synaptically at the neuromuscular junction of skeletal muscle, at intercalated discs of cardiac tissue and at the apical membrane of the ependyma and choroid plexus . We have also detected two to three species (approximately 45-50 kDa) in other regions of the brain . Synaptic localization of DMK in the cerebellum, hippocampus, midbrain and medulla was noted . These results suggest that DMK plays a specialized role in intercellular communication.

Ophthalmologe, 1995 Jun, 92(3), 289 - 92
{The aqueous humor-vitreous body barrier and the blood-aqueous humor barrier after YAG laser capsulotomy in capsular sac vs ciliary sulcus fixation of the intraocular lens}; Schalnus RW et al.; An intact posterior capsule between aqueous and vitreous may act as a barrier to substances of low and high molecular weight, e.g., prostaglandins, hyaluronic acid, or the angiogenic factor . After phacoemulsification followed by posterior YAG capsulotomy, an increased diffusion rate of such molecules into the vitreous and increased permeability of blood aqueous barrier (BAB) may occur . These barriers were quantified in eyes that underwent YAG capsulotomy after sulcus or intracapsular IOL implantation in order to determine the safest surgical procedure with respect of maintenance of these barriers . PATIENTS AND METHODS: Between 2 to 6 h after topical fluorescein application, the time-dependent decrease in dye concentration ratio between aqueous and anterior vitreous leads to the diffusion rate D(av) {10(-3)min-1} between aqueous and vitreous; D(av) was evaluated fluorophotometrically before and 3 weeks after capsulotomy (3 to 5 mm) in human eyes of each group . In order to quantify BAB function, aqueous laser flare was measured in eyes with sulcus and capsular fixation of IOL before, 3 h, and 3 weeks after YAG capsulotomy . RESULTS: After YAG surgery D(av) increased 2.7-fold (P < 0.001) in eyes with a sulcus implant compared to the values obtained in the group that had an intracapsular PCL . Aqueous laser flare was increased to 140% (P < 0.001) in eyes with sulcus fixation and to 95% (P < 0.001) in eyes with capsular fixation of PCL . Laser flare values became normal 3 weeks after laser treatment (P > 0.05) . CONCLUSION: Intracapsular PCL implantation more effectively maintains the protective aqueous vitreous barrier and BAB after posterior capsulotomy than sulcus implantation . This possibly reduces the incidence of cystoid macular edema (diffusion of prostaglandins), retinal detachment (loss of hyaluronic acid of the vitreous), endophthalmitis (spread of bacteria) or rubeosis iridis (angiogenic factor) after YAG capsulotomy.

Biokhimiia, 1995 Jun, 60(6), 827 - 36
{Structure of an acidic polysaccharide found in a bacteriolytic complex of lysoamidase}; Likhosherstov LM et al.; A structural study of an acidic polysaccharide, a component of the lysoamidase bacteriolytic complex has been carried out . Analysis of the monosaccharide composition of the original polysaccharide, of the product of carboxyl groups reduction and of the 13C-NMR and 1H-NMR spectra of the original and the O-deacetylated polysaccharides using two-dimensional NMR spectroscopy has made it possible to establish the structure of the repeating trisaccharide unit of the polysaccharide as follows: {formula: see text} where ManNAcA and GalNAcA are 2-acetamido-2-deoxymannuronic acid and 2-acetamido-2-deoxygalacturonic acid, respectively . Also small amount of a neutral polysaccharide containing of rhamnose is present in the lysoamidase preparation.

J Neurosci Res, 1995 Jun 1, 41(2), 270 - 8
Beta-amyloid precursor protein is modified with O-linked N-acetylglucosamine; Griffith LS et al.; The beta-amyloid precursor protein (APP) has been implicated in the etiology of Alzheimer's disease (Kang et al.: Nature 325:733-736, 1987; Selkoe: Science 248:1058-1060, 1990; Selkoe: In Cowan et al . (eds): "Annual Review of Neuroscience." Palo Alto, CA: Annual Reviews, Inc., pp 489-519, 1994) and numerous studies have shown that beta-amyloid is involved in amyloid plaque formation (Rumble et al.: N Engl J Med 320:1446-1452, 1989; Sisodia et al.: Science 248: 492-495, 1990) . Evidence is presented that APP is modified with N-acetylglucosamine linked to cytoplasmic serine or threonine residues (O-GlcNAc) . This is the first report of a plasma membrane protein modified with this carbohydrate . It has been postulated that this modification, which is ubiquitous in all organisms studied to date except bacteria (Haltiwanger et al.: Biochem Soc Trans 20:264-269, 1992; Dong et al.: J Biol Chem 268:16679-16687, 1993; Elliot et al.: J Neurosci 13:2424-2429, 1993; Kelly et al.: J Biol Chem 268:10416-10424, 1993), may function as an alternative to phosphorylation (Dong et al., 1993) and is involved in the multimerization of proteins (Haltiwanger et al., 1992; Dong et al., 1993) . O-GlcNAc occurs at "PEST" sequences (Rogers et al.: Science 234:364-368, 1986) and it has been suggested that this modification within such a sequence leads to increased proteolytic stability of the molecule (Dong et al., 1993).(ABSTRACT TRUNCATED AT 250 WORDS)

Hautarzt, 1995 Jun, 46(6), 417 - 20
{Draining sinus in acne and rosacea . A clinical, histopathologic and experimental study}; Jansen T et al.; The draining sinus is an unpleasant complication of acne conglobata, acne fulminans, acne inversa, rosacea conglobata and rosacea fulminans (pyoderma faciale) . It is most common on the face, especially in the nasolabial folds, and on the neck below the mandibular line . Clinically, it is an elongated (2-5 cm long), elevated, periodically inflamed lesion, which sporadically discharges pus . The lesion persists with no tendency to spontaneous regression . Histopathologically, it consists of elaborate, epithelialized galleries connected to the skin surface at multiple points . It contains corneocytes, hairs, bacteria, serum, inflammatory cells and epitheloid granulomas . A surgical thread placed into the skin provides a model in which the generation of sinus tracts can be studied . Therapy is difficult . Intralesional corticosteroid injection, cryosurgery and isotretinoin are not always very effective . Sometimes complete excision of the lesion is necessary . The draining sinus is a special form of scar analogous to the pilonidal cyst.

J Pharmacol Toxicol Methods, 1995 Jun, 33(3), 121 - 8
Diadenosine polyphosphates: their biological and pharmacological significance; Baxi MD et al.; Diadenosine polyphosphates are members of a group of dinucleoside polyphosphates that are ubiquitous in bacteria to mammals . In recent years, the diadenosine polyphosphates have received considerable attention in view of their multiple biological activities and potential pharmacological activities . Diadenosine polyphosphates have been identified as modulators of cardiovascular and neurotransmitter-like activities in recent years, besides their previously described role in cell proliferation and as signal molecules when cells are undergoing stress . Diadenosine polyphosphates and their synthetic analogues are being evaluated for their potential as pharmacological agents . This article discusses the various biological functions and physiological significance of the diadenosine polyphosphates.

Inflammation, 1995 Jun, 19(3), 313 - 31
Identification and characterization of rhesus macaque interleukin-8; Minnerly JC et al.; To establish a direct link between IL-8 and inflammation in vivo, we first isolated the gene encoding rhesus macaque IL-8 . The open reading frame directs the translation of a 101 amino acid (aa) precursor, which is 94% identical to human IL-8 . Rhesus IL-8 was expressed in bacteria and purified to homogeneity with ion-exchange chromatography . Pure rhesus IL-8 was biologically active as measured by its ability to bind specifically to either rhesus (Kd = 0.5 nM) or human (Kd = 2 nM) IL-8 receptors and to promote in vitro chemotaxis of rhesus (EC50 = 2 nM) or human neutrophils (EC50 = 4 nM) . Moreover, a mouse monoclonal antibody, DM/C7, which neutralizes human IL-8 activity, also recognized and neutralized (IC50 = 0.5-3.0 microgram/ml) rhesus IL-8 in vitro . Systemic administration of DM/C7 completely inhibited the dermal inflammation of rhesus ears induced by the external application of phorbol myristoyl acetate . These observations reveal that rhesus IL-8 is structurally and functionally similar to human IL-8 and suggests that IL-8 plays a prominent role in a primate model of inflammation.

Kansenshogaku Zasshi, 1995 Jun, 69(6), 723 - 8
{Detection of Mycoplasma pneumoniae from throat swab by polymerase chain reaction}; Okazaki N et al.; Polymerase chain reaction (PCR) with primers directed against the 16S-rRNA gene of Mycoplasma pneumoniae was used to diagnose M . pneumoniae infections, and the results were compared with those of culture and serology methods . Eighty (22%) of 363 throat swab samples from patients with acute respiratory complaints gave positive results by using the PCR method . Sixty-seven (18.5%) of the samples were positive in culture method . Of 35 samples which were unreliable culture results due to contamination with other bacteria, 13 gave positive results in the PCR method . Of the 97 cases obtained throat swabs and paired sera, 28 (28.9%) showed positive results by the PCR assay, and 29 (29.9%) by serology method (particle agglutination test) . The positive rate was increased to 36% by using both the PCR and the serology methods . From these results it was concluded that the PCR method is useful for laboratory diagnosis of M . pneumoniae infections.

Rinsho Byori, 1995 Jun, 43(6), 557 - 61
{Virulence and pathogenesis of Helicobacter pylori infection}; Fujioka T; Recent studies on the relationship between Helicobacter pylori and gastritis or gastroduodenal ulcers have indicated that this organism can induce chronic active gastritis with neutrophil infiltration . However the mechanisms by which bacteria colonized and damage the host are often complex and multiple . The ammonia produced by H . pylori, which has strong urease activity, has been reported to have a cytotoxic effect on the gastric mucosa . With H . pylori the major areas of interest regarding pathogenesis are also related to other factors such as oxygen radicals, cytotoxins and cytokines . There may also be virulence differences between H . pylori strains which may determine the severity of the gastric mucosal damage . Clearly this is an important area for future research.

Immunol Rev, 1995 Jun, 145, 211 - 28
Idiotypic DNA vaccines against B-cell lymphoma; Stevenson FK et al.; Idiotypic antigens are clearly defined tumor-associated protein antigens, which can induce protective immunity against lymphoma . Because each patient requires an individual vaccine, idiotypic antigens also provide ideal candidates for exploring the feasibility of replacing protein antigens by DNA vaccines . Component idiotypic variable region genes can be identified in patients' tumor biopsies and rapidly assembled as scFv sequences . These can be used to produce recombinant scFv protein in bacteria, or as direct naked DNA vaccines . A preliminary small trial of DNA vaccines for chemotherapy-resistant patients with lymphoma has begun . Intramuscular idiotypic DNA vaccination in a mouse model induces low levels of anti-idiotypic antibody in serum . Levels can be increased dramatically by coinjection of DNA plasmids encoding either IL-2 or GM-CSF, and specific proliferative anti-idiotypic T cells are induced . However protective immunity remains to be demonstrated, and a possible reason for this may lie in the continued secretion of idiotypic scFv antigen which blocks antibody activity by formation of immune complexes . Methods for regulating secretion of antigen are required before this category of tumor antigen can be fully exploited as a vaccine . The power of DNA technology should allow analysis and manipulation of pathways of antigen presentation to induce maximal therapeutic attack on neoplastic B cells . In addition, lymphoma presents a model for application of DNA technology to the wide range of human tumors known to harbor potential tumor antigens.

FEMS Immunol Med Microbiol, 1995 Jun, 11(3), 247 - 55
Immunochemical characterisation and epitope mapping of a novel fimbrial protein (Pg-II fimbria) of Porphyromonas gingivalis; Ogawa T et al.; Mouse monoclonal antibody (mAb) Pgf-II specific for a 72-kDa major cell-surface protein (72K-CSP) derived from Porphyromonas gingivalis OMZ 409 was prepared . Immunoblotting analysis revealed that mAb Pgf-II reacted with 72K-CSP but not with 41-kDa fimbrial subunit protein (41K-fimbrilin) derived from P . gingivalis 381 . Electron microscopic observation revealed that P . gingivalis OMZ 409 possessed peritrichous, thin fimbriae on their surface . Immunogold electron microscopy also demonstrated that mAb Pgf-II bound to the 72K-CSP examined with the gold particles arranged along the fibril array originating from the cell surface of the bacteria . These findings suggested that P . gingivalis 72K-CSP was identifiable as another fimbriae (termed Pg-II fimbriae) different from the fimbriae (termed Pg-I fimbriae) composed of a 41K-fimbrilin . Using multipin peptide synthesis technology, 102 sequential overlapping peptides covering the entire 514 amino-acid stretch of Pg-II fimbriae were synthesised . Seven immunodominant regions within Pg-II fimbrial protein molecule, which definitely reacted with the serum of patients with periodontal diseases, were detected.

Arch Microbiol, 1995 Jun, 163(6), 391 - 9
Isolation and characterization of sulfur globule proteins from Chromatium vinosum and Thiocapsa roseopersicina; Brune DC; Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope . The proteins associated with sulfur globules of Chromatium vinosum and Thiocapsa roseopersicina were isolated by extraction into 50% aqueous acetonitrile containing 1% trifluoroacetic acid and 10 mM dithiothreitol . The extracted proteins were separated by reversed-phase HPLC, revealing three major proteins from C . vinosum and two from T . roseopersicina . All of these proteins have similar, rather unusual amino acid compositions, being rich in glycine and aromatic amino acids, particularly tyrosine . The molecular masses of the C . vinosum proteins were determined to be 10,498, 10,651, and 8,479 Da, while those from T . roseopersicina were found to be 10,661 and 8,759 Da by laser desorption time-of-flight mass spectrometry . The larger T . roseopersicina protein is N-terminally blocked, probably by acetylation, but small amounts of the unblocked form (mass = 10,619) were also isolated by HPLC . Protein sequencing showed that the two larger C . vinosum proteins are homologous to each other and to the large T . roseopersicina protein . The 8,479 Da C . vinosum and 8,759 Da T . roseopersicina proteins are also homologous, indicating that sulfur globule proteins are conserved between different species of purple sulfur bacteria.

Antimicrob Agents Chemother, 1995 Jun, 39(6), 1369 - 71
Protection of mice from Mycobacterium avium infection by recombinant interleukin-12; Kobayashi K et al.; Treatment with interleukin-12 (IL-12) significantly reduced the number of viable bacteria in mice infected with Mycobacterium avium . IL-12 itself, however, could not inhibit directly mycobacterial growth in vitro . IL-12 exerts antimycobacterial activity in vivo with a low level of toxicity, possibly by enhancing the host defense against the infection.

Photochem Photobiol, 1995 Jun, 61(6), 607 - 14
Genetic and molecular analyses of UV radiation-induced mutations in the fem-3 gene of Caenorhabditis elegans; Hartman PS et al.; The utility of a new target gene (fem-3) is described for investigating the molecular nature of mutagenesis in the nematode Caenorhabditis elegans . As a principal attribute, this system allows for the selection, maintenance and molecular analysis of any type of mutation that disrupts the gene, including deletions . In this study, 86 mutant strains were isolated, of which 79 proved to have mutations in fem-3 . Twenty of these originally tested as homozygous inviable . Homozygous inviability was expected, as Stewart and coworkers had previously observed that, unlike in other organisms, most UV radiation-induced mutations in C . elegans are chromosomal rearrangements of deficiencies (Mutat . Res . 249, 37-54, 1991) . However, additional data, including Southern blot analyses on 48 of the strains, indicated that most of the UV radiation-induced fem-3 mutations were not deficiencies, as originally inferred from their homozygous inviability . Instead, the lethals were most likely "coincident mutations" in linked, essential genes that were concomitantly induced . As such, they were lost owing to genetic recombination during stock maintenance . As in mammalian cells, yeast and bacteria, the frequency of coincident mutations was much higher than would be predicted by chance.

J Hosp Infect, 1995 Jun, 30 Suppl, 543 - 51
Endoscope decontamination: where do we go from here?
Babb JR, Bradley CR.
Thorough cleaning and disinfection or sterilization of endoscopes and associated equipment will reduce the likelihood of misdiagnosis and post-procedural infection . It will also prevent instrument deterioration and malfunction . With a rapid escalation in demand for endoscopy, particularly that associated with minimally invasive surgery, it is important that we have the processing technology to match the diagnostic and therapeutic value of these instruments without exposing staff and patients to unnecessary risk . Wherever possible staff should purchase heat tolerant endoscopic equipment that is readily accessible for cleaning . Automated processors, e.g . washer disinfectors and ultrasonic cleaners, improve the quality of the decontamination process but machines must have a self-disinfect function to prevent instrument recontamination during processing . Sterile, or filtered bacteria-free, water is essential for bronchoscopes and invasive instruments . Glutaraldehyde is still the most widely used disinfectant, particularly for the heat sensitive flexible endoscopes, but it is irritant and sensitizing and a safer alternative is sought . Peracetic acid is more rapidly efficacious and probably less irritant and, provided it does not damage endoscopes and processing equipment, may prove a suitable alternative . Unfortunately there are no nationally agreed test methods for assessing this and other new endoscope disinfectants and therefore no register of suitable or approved products . There is also no proven safe alternative to ethylene oxide for sterilizing invasive heat labile flexible endoscopes . It is important that, if toxic disinfectants and sterilants are used, staff and patients are suitably protected from exposure . Update training is essential for all processing staff if infection risks are to be minimized and sensitization problems avoided.

J Hosp Infect, 1995 Jun, 30 Suppl, 514 - 20
The disposal of clinical wastes; Blenkharn JI; The disposal of clinical wastes is often poorly conducted and inadequately supervised despite the publication of clear and definitive working guidelines and the introduction of increasingly stringent legislative control . The move away from landfill disposal of clinical wastes, and the further development of high temperature incinerators able to meet increasingly tight emission limits, is to be applauded but has inevitably increased the cost of waste disposal . Moreover, such developments fail to address the continuing 'shop floor' problems whereby wastes enter an inappropriate waste stream or colour coded wastes containers are used for inappropriate purposes thus undermining the value of a nationally approved hazard warning policy . The development of newer waste treatments, including microwave exposure of macerated wastes, may reduce costs and aid in the control of environmental pollution . However, stringent control of this and existing technologies remains essential . Additionally, increasing resources must be directed to improvements in primary waste disposal practices whereby all health care staff have a clear responsibility to ensure correct disposal of wastes without risk to themselves, their colleagues and others, or to the environment.

J Hosp Infect, 1995 Jun, 30 Suppl, 397 - 408
Disinfectant testing on surfaces; van Klingeren B; During the last decade a consensus view has evolved in Europe on the quantitative testing of disinfectant efficacy in suspension tests, as well as in surface tests . Harmonization of the different national test methods is being pursued within the framework of the European Committee for Standardisation (CEN/TC216) . An expert subgroup of the Committee has drafted a test method to measure the microbicidal activity of disinfectants on bacteria attached to surfaces . The outcome of an initial collaborative study from seven laboratories is that this test system yields reproducible results in different laboratories; thus fulfilling the requirements for a basic surface disinfectant test.

J Clin Periodontol, 1995 Jun, 22(6), 442 - 8
The effect of triclosan on developing gingivitis; Ramberg P et al.; The aim of the study was to examine whether triclosan has an effect on developing gingival inflammation . 10 volunteers, with clinically healthy gingivae were enrolled . The study was performed as a 2-week, double-blind, cross-over, experimental gingivitis trial . Between each plaque accumulation period, there was a wash-out phase of 4 weeks . A baseline examination was performed which included assessment of plaque and gingivitis . The volunteers were asked to refrain from mechanical oral hygiene measures for 2 weeks . During this period, they rinsed 2x daily with one of the randomly assigned mouthrinse preparations . Solution A (period A): 0.06% triclosan+ 2%tween 80 . Solution B (period B): 0.06% triclosan+ 0.25% sodium lauryl sulphate . Re-examinations were performed on days 4, 7, 11 and 14 . The mean plaque score increased during period A to 2.2 (day 4), 2.8 (day 7), 3.1 (day 11) and 3.1 (day 14) . The corresponding scores for period B were significantly lower; 1.2 (day 4), 1.8 (day 7), 2.0 (day 11) and 2.2 (day 14) . The mean gingivitis scores at baseline were 0.17 (periods A and B) . The mean gingivitis scores increased to 0.45 (day 4), 0.69 (day 7), 0.83 (day 11) and 0.96 (day 14) when the subjects rinsed with solution A and 0.42 (day 4), 0.64 (day 7), 0.78 (day 11) and 0.92 (day 14) in period B . There were no statistically significant differences between periods A and B with respect to gingivitis . Thus, although significantly more plaque formed during period A than period B, no differences could be found between the gingivitis scores in the 2 periods.

Baillieres Clin Gastroenterol, 1995 Jun, 9(2), 351 - 69
The major complications of coeliac disease; Wright DH; Neoplasms constitute the major complication of coeliac disease, and high-grade T-cell lymphoma of the small intestine (enteropathy-associated T-cell lymphoma) is the most common neoplasm in this category . HLA genotyping indicates that in patients with enteropathy-associated T-cell lymphoma have the coeliac disease associated DQA1*0501, DQB1*0201 phenotype, although additional HLA-DR/DQ alleles may represent risk factors for lymphoma development . Molecular biological and immunohistochemical studies have shown that the intestinal mucosa distant from the tumour contains clonal populations of small T cells, often of the same clone as the high-grade T-cell lymphoma . These findings suggest that enteropathy-associated T-cell lymphoma arises in the setting of coeliac disease and evolves from reactive intraepithelial lymphocytes through a low-grade lymphocytic neoplasm to a high-grade tumour, which is usually the cause of the presenting symptoms . Most cases of chronic ulcerative enteropathy (ulcerative jejunitis) are probably part of the same disease process . If the ulceration occurs at a time when the neoplastic T-cells are of a low grade, morphological recognition of tumour cells in the ulcers may be impossible . Carcinoma of the pharynx and oesophagus, and adenocarcinoma of the small intestine, are increased in frequency in patients with coeliac disease . The increased risk of carcinoma of the oesophagus may be related to vitamin A deficiency . A number of reports have indicated an increased prevalence of various types of chronic hepatitis in patients with coeliac disease, but no coherent view of the cause of this association has emerged . Similarly, patients with coeliac disease have been reported to have various forms of fibrosing lung disease of uncertain causation . In recent years, there have been several reports, mainly from Italy, of a syndrome of epilepsy and bilateral brain calcification occurring in coeliac patients . The pathogenesis of this condition is not known and its prevalence in other communities is uncertain . Splenic atrophy occurs frequently in patients with coeliac disease and is related to the severity of the disease and degree of dietary control . Splenic atrophy predisposes to infection with capsulated bacteria, although mortality studies indicate that infection with these organisms is not a major cause of death in patients with coeliac disease.

Tuber Lung Dis, 1995 Jun, 76(3), 234 - 9
Preliminary evaluation of a Mycobacterium tuberculosis lipooligosaccharide (LOS) antigen in the serological diagnosis of tuberculosis in HIV seropositive and seronegative patients; Daleine G et al.; OBJECTIVE: To assess the value of detection of specific IgG or IgM antibodies against three polar glycolipids of Mycobacterium tuberculosis in HIV seropositive and seronegative patients with documented active tuberculosis . DESIGN: Using an ELISA test, concentrations of IgM and IgG antibodies against phenolic glycolipid Tb1 (PGL Tb1), 2.3 diacyl trehalose (DAT) and polar lipooligosaccharide (LOS) were measured in the serum of 36 patients with active tuberculosis: 16 tuberculosis patients were coinfected with HIV (TB HIV+), and 20 were HIV seronegative (TB HIV-) . 104 control sera were tested: they were collected from 50 healthy blood donors and from 54 asymptomatic HIV seropositive patients . Specificity, sensitivity and predictive positive and negative values were calculated . RESULTS: For the LOS antigen tested, sensitivity, specificity and predictive positive value of specific IgG antibodies were 75%, 95% and 84% respectively among the 36 tuberculosis patients . No significant results were obtained either with the IgM against the LOS antigen or with IgG against the DAT and PGL Tb1 antigens . Significant sensitivity and positive predictive values were demonstrated only with the ELISA test measuring specific IgG against the LOS antigen . The sensitivity was greater in the TB HIV+ subgroup (94%) as compared with the TB HIV- (60%) subgroup . These results were not related to different potential factors such as the site of disease (pulmonary or extrapulmonary) or by the acid-fast bacteria load as detected by the smear microscopic (Ziehl-Neelsen) examination . CONCLUSIONS: The results suggest that of the 3 antigens tested, only the LOS antigen is a potential marker for detecting the development of tuberculosis in HIV patients.

J Med Microbiol, 1995 Jun, 42(6), 386 - 93
Modulatory action of Helicobacter pylori on histamine release from mast cells and basophils in vitro; Lutton DA et al.; Helicobacter pylori is important in the aetiology of peptic ulceration . Despite inducing an inflammatory response in the mucosa, the organism persists, suggesting that it has efficient protective mechanisms . Some bacterial and viral products modulate histamine secretion from inflammatory cells . Therefore, this study examined the modulatory effects of H . pylori preparations on histamine release from rat peritoneal mast cells and human basophils . Eleven clinical isolates of H . pylori were prepared in different ways: as whole washed bacteria, washed sonicated bacteria, and formalin-killed bacteria, and as outermembrane and lipopolysaccharide (LPS) extracts . Histamine release from mast cells or basophils was not elicited by any of these bacterial preparations alone . However, when mixed with various secretory stimulants, the bacterial preparations caused inhibition of histamine release from rat mast cells (calcium ionophore A23187, compound 48/80, concanavalin A, anti-rat IgE) and human basophils (A23187, N-formyl Met-Leu-Phe) . The degree of inhibition ranged from 48% to 97% . These results indicate that H . pylori exerts an inhibitory effect on cells of the immune system that contributes to its persistence within the gastric mucosa.

J Commun Dis, 1995 Jun, 27(2), 84 - 91
Epidemiology of diarrhoea in two major cities in Saudi Arabia; Milaat WA et al.; The epidemiological pattern of diarrhoeal diseases, causative agents and risk factors of their occurrence in two referral hospitals of Saudi Arabia is presented in this study . Stool specimens from 1726 admitted diarrhoeal cases were examined for parasites, yeast, enteropathogenic bacteria and rotavirus using the ELISA test . 41.3% of cases were due to rotavirus (RVGE) while 53.1% of cases showed no causative pathogens . Mean age of all cases was 20.2 months and RVGE cases showed a steady rise from the neonatal period onward, reaching a peak between 6-14 months . Males accounted for higher percentage of all diarrhoeal cases . Mothers of diarrhoea cases were mostly housewives with low educational level . Bottle fed children showed higher proportion (53.1%) of diarrhoea than other types of feeding suggesting the faeco-oral route of infection and the effect of poor sanitation . A pattern of higher RVGE cases was seen in warmer months in Al-taif and in cooler months in Jeddah . Findings demonstrated the interaction between host, pathogen and environmental factors in the epidemiology of infectious diarrhoeas in developing countries and the areas of possible preventionPIP: The epidemiological pattern of diarrheal diseases, causative agents, and risk factors of their occurrence in two referral hospitals of Saudi Arabia is presented in this study . Stool specimens from 1726 admitted diarrheal cases were examined for parasites, yeast, enteropathogenic bacteria, and rotavirus using the ELISA test . 41.3% of cases were due to rotavirus, while 53.1% of cases showed no causative pathogens . Mean age of all cases was 20.2 months and rotavirus gastroenteritis (RVGE) cases showed a steady rise from the neonatal period onward, reaching a peak between 6 and 14 months . Males accounted for a higher percentage of all diarrheal cases . Mothers of diarrheal cases were mostly housewives with low educational status . Bottle-fed children showed higher proportions (53.1%) of diarrhea than children fed otherwise, suggesting the feco-oral route of infection and the effect of poor sanitation . A pattern of higher RVGE cases was seen in warmer months in Al-taif and in cooler months in Jeddah . Findings demonstrated the interaction between host, pathogen, and environmental factors in the epidemiology of infectious diarrheas in developing countries and the areas of possible prevention . author's modified

Electrophoresis, 1995 Jun, 16(6), 881 - 7
Theory of the mobility-shift assay of nonspecific protein-DNA complexes governed by conditional probabilities: the HU:DNA complex; Cann JR et al.; Complexes of an 88 bp DNA and the HU protein were studied by both experimental and theoretical electrophoretic mobility-shift analyses . Experimental analysis defined the stoichiometry of binding and estimated an apparent intrinsic dissociation constant (Kd = 1 to 3 x 10(-7) M) for the HU:DNA complexes . The theory of conditional probabilities was applied to the binding of HU to DNA in order to fix the initial equilibrium composition of mixtures to be assayed theoretically by the mobility-shift procedure . Electrophoretic mobility-shift patterns were obtained by numerical solution of a set of simultaneous transport-reaction equations, in which the chemical kinetic term is formulated in terms of dissociation of the different DNA:HU complexes in gel cages . The computed patterns simulated the experimental patterns describing the titration of a fixed concentration of an 88 bp DNA fragment with dimeric HU . These insightful results provide guidelines for interpretation of the electrophoretic behavior of systems in which a ligand binds nonspecifically to DNA . In particular, the narrow unresolved zone observed both experimentally and theoretically beyond 50-60% saturation is a reaction zone characteristic of noncooperative ligand-binding governed by conditional probabilities . The discrepancy between the theoretically assigned and experimental values of the intrinsic binding constant is attributed to an HU-induced change in the conformation of DNA.

Minerva Stomatol, 1995 Jun, 44(6), 319 - 23
{Odontogenic sinusitis with cutaneous fistulization . A case report}; Garbarino R et al.; A 70-year-old male patient presenting with a cutaneous node of the genal region that was first diagnosed as basal cell carcinoma is described . Owing to the following observation, the authors were prompted to a diagnosis of cutaneous odontogenic sinusitis: a) examination of the lesion which emitted pus upon gentle squeezing; b) presence in the vestibular fornix of a large fibrous cord; c) the serious conditions of the mouth and the radiologic results of homolateral sinusitis . The importance of the sinusitis picture, and the necessity to completely remove all bacteria foci prior to surgery on the abdominal aorta the patient was scheduled to undergo, prompted the authors to avulse the cutaneous lesion with tooth extraction, after a "Caldwell-Luc" operation of the homolateral paranasal sinus . Full restitutio ad integrum was achieved.

Mol Microbiol, 1995 Jun, 16(5), 825 - 34
The PTR family: a new group of peptide transporters; Steiner HY et al.; The transport of peptides into cells is a well-documented biological phenomenon which is accomplished by specific, energy-dependent transporters found in a number of organisms as diverse as bacteria and humans . Until recently, the majority of peptide transporters cloned and characterized were found to be proteins of the ATP-binding cassette (ABC) family . We report the identification of a new family of peptide transporters, which we call the PTR family . This group of proteins, distinct from the ABC-type peptide transporters, was uncovered by sequence analyses of a number of recently discovered peptide transport proteins . Alignment of these proteins demonstrated a high number of identical and similar residues and identified conserved glycosylation and phosphorylation sites, as well as a structural motif unique to this group of proteins . Cluster analysis among the proteins indicated these sequences were indeed related and could be further divided into two subfamilies . A phylogenetic analysis of these new peptide transport sequences, compared to over 50 other peptide and membrane-bound transporters, showed that these proteins comprise a distinct, separate group of proteins.

Biochemistry, 1995 May 30, 34(21), 7154 - 60
Non-sialate inhibitor of influenza A/WSN/33 neuraminidase; Wu JC et al.; An N1 strain of influenza A virus neuraminidase (A/WSN/33 NA) was purified and used to screen for inhibitors . As a result, a well-known tuberculostatic, 4'-formylacetanilide thiosemicarbazone (or thiacetazone), was identified . Thiacetazone is a non-sialate compound and inhibits the enzyme in a noncompetitive manner with respect to the substrate sialic acid . Mechanistic studies indicate that the inhibition was due to the competition of thiacetazone with Ca2+, which maintains N1 neuraminidase in an active conformation . The Ki for the inhibition was estimated to be about 4 microM . Equilibrium exchange experiments revealed that when purified A/WSN/33 NA was incubated with 5 microM 45CaCl2, 2 mol of 45Ca2+ ion was exchanged into each mole of NA tetramer and subsequently displaced from the enzyme upon the introduction of the inhibitor . Inhibition of plaque formation by thiacetazone in an MDCK cell culture that had been infected with the influenza A/WSN/33 virus was demonstrated . Thiacetazone was highly specific for A/WSN/33 neuraminidase, since little effect was noted when it was tested against NAs from the other strains of influenza virus or from bacteria . This compound might represent a group of non-sialate inhibitors of influenza NA that bind to a noncatalytic or an allosteric site on the enzyme.

Gene, 1995 May 26, 158(1), 97 - 100
Complementation of a Streptomyces lividans Leu- mutant by the Actinoplanes teichomyceticus leuC gene; Castelli P et al.; A leucine auxotroph of Streptomyces lividans (Sl), designated PC196, was unable to convert alpha-isopropylmalate into the beta-isomer . A DNA fragment from Actinoplanes teichomyceticus (At) cloned into the Streptomyces vector pIJ702 complemented PC196 . Sequence analysis of the 3.0-kb insert revealed one complete ORF with high similarity to other leuC genes encoding the large subunit of isopropylmalate isomerase (IPMI), and the 5' end of a second ORF corresponding to leuD, which encodes the smaller subunit of IPMI . Further subcloning established that Sl strain PC196 is defective in the large subunit of IPMI.

Gene, 1995 May 26, 158(1), 151 - 2
Cloning and sequencing of the Bordetella pertussis cpn10/cpn60 (groESL) homolog; Fernandez RC et al.; The nucleotide sequence downstream from the Bordetella resistance to killing (brk) locus in Bordetella pertussis was determined . Analysis of the sequence revealed an operon consisting of two highly predicted open reading frames (ORFs) . The deduced amino-acid sequence of each ORF has strong homologies to the heat-shock proteins/chaperonins Cpn60 and Cpn10 . The promoter contains consensus sequences for both sigma 32 and sigma 70 binding, and it possesses the CIRCE regulatory inverted repeat . A potential Rho-independent terminator was identified and appears to be shared with the brkA gene.

J Biol Chem, 1995 May 26, 270(21), 12877 - 84
Candidate genes for the phycoerythrocyanin alpha subunit lyase . Biochemical analysis of pecE and pecF interposon mutants; Jung LJ et al.; The rod substructures of the Anabaena sp . PCC 7120 phycobilisome contain the light harvesting proteins C-phycocyanin and phycoerythrocyanin (PEC) . Even at low light intensities, PEC represents no more than 5% of the phycobilisome protein . The beta subunits of both proteins carry thioether-linked phycocyanobilin (PCB) at beta-Cys-82 and beta-Cys-155; however, C-phycocyanin has PCB at alpha-Cys-84 whereas PEC alpha subunit carries phycobiliviolin at this position . The Anabaena sp . PCC 7120 pec operon is made up of five genes . PecB and pecA encode the beta and alpha subunits of PEC, pecC encodes a linker polypeptide associated with PEC in the rod substructure, and pecE and pecF are genes of unknown function that show a high degree of homology to cpcE and cpcF, that encode a C-phycocyanin alpha subunit PCB lyase (Fairchild, C . D., Zhao, J., Zhou, J., Colson, S . E., Bryant, D . A., and Glazer, A . N . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 7017-7021) . Insertional mutants in pecE and pecF, and an interposon mutant in which a portion of both pecE and pecF was deleted, were constructed . All three types of mutants grew 1.3 times slower than wild-type under limiting light conditions and showed a 20% reduction in the PCB content of whole cells relative to chlorophyll alpha . Holo-PEC was missing from the phycobilisomes of all three types of mutants and the level of the PEC linker polypeptide was reduced relative to the wild-type . However, approximately 30% of the wild-type level of the PEC beta subunit was present in all of these phycobilisomes . In contrast, the PEC alpha subunit was barely detectable in the pecE and pecF mutants, but was present in the pecEF deletion mutant as a PCB-adduct in a 1:1 ratio with the PEC beta subunit . The identity of this "unnatural" adduct was confirmed by isolation of the subunit and amino-terminal sequencing . These biochemical results support the inference that pecE and pecF encode a PEC alpha subunit phycobiliviolin lyase, and, in conjunction with earlier findings, demonstrate that phycobiliprotein bilin lyases show high selectivity (rather than absolute specificity) for both the bilin and the polypeptide substrate.

Nucleic Acids Res, 1995 May 25, 23(10), 1775 - 81
Cloning and functional analysis of the TATA binding protein from Sulfolobus shibatae; Qureshi SA et al.; Archaea (formerly archaebacteria) comprise a domain of life that is phylogenetically distinct from both Eucarya and Bacteria . Here we report the cloning of a gene from the Archaeon Sulfolobus shibatae that encodes a protein with strong homology to the TATA binding protein (TBP) of eukaryotes . Sulfolobus shibatae TBP is, however, almost as diverged from other archaeal TBPs that have been cloned as it is from eukaryotic TBPs . DNA binding studies indicate that S.shibatae TBP recognizes TATA-like A-box sequences that are present upstream of most archaeal genes . By quantitatively immunodepleting S.shibatae TBP from an in vitro transcription system, we demonstrate that Sulfolobus RNA polymerase is capable of transcribing the 16S/23S rRNA promoter weakly in the absence of TBP . Most significantly, we show that addition of recombinant S.shibatae TBP to this immunodepleted system leads to transcriptional stimulation and that this stimulation is dependent on the A-box sequence of the promoter . Taken together, these findings reveal fundamental similarities between the transcription machineries of Archaea and eukaryotes.

Biochim Biophys Acta, 1995 May 24, 1271(1), 123 - 6
Mitochondria: beyond oxidative phosphorylation; Schatz G; Mitochondria are essential eukaryotic organelles that perform many functions in addition to oxidative phosphorylation . Some of these functions are still poorly understood or as yet undiscovered, but may resemble already known functions in bacteria . The rapidly growing sequence information on bacterial genes, the polymerase chain reaction, and gene disruption in yeast thus offer a powerful approach for discovering new mitochondrial functions.

Gene, 1995 May 19, 157(1-2), 87 - 92
MmeI, a class-IIS restriction endonuclease: purification and characterization; Tucholski J et al.; Two restriction endonucleases, MmeI and MmeII, from Methylophilus methylotrophus were purified to homogeneity . Both enzymes belong to the class-II restriction endonucleases (ENases) but exhibit very different enzymatic and physical properties . MmeII is a typical member of class-II ENases . It is a polymeric protein composed of 50-kDa subunits . In contrast to MmeII, MmeI is a monomeric protein of 101 kDa, cleaving a DNA molecule 20/18 nucleotides away from the asymmetric recognition sequence (5'-TCCRAC-3'); therefore, it is classified as a member of subclass-IIS . MmeI has an pI of 7.85 and is active in the pH range 6.5 to 10 with the optimum at 7 to 8 . Increasing salt concentration creates an inhibitory effect on MmeI: 40 mM KCl decreases activity by 50%, 100 mM completely inhibits DNA cleavage . Tris.HCl (pH 7.5) at a concentration exceeding 20 mM inhibits MmeI activity . Mg2+ stimulates MmeI in the range of 0.2 to 35 mM, with the optimum between 0.5 and 10 mM.

Gene, 1995 May 19, 157(1-2), 61 - 3
Cloning and characterization of the gene encoding the DsaV methyltransferase; Gopal J et al.; A gene encoding the M.DsaV methyltransferase was cloned and characterized . The enzyme methylates the internal cytosines in the 5'-CCTGG recognition sequence, as determined by a novel rapid method employing 3H label and exonuclease III.

Biochemistry, 1995 May 16, 34(19), 6278 - 87
1.4 A structure of photoactive yellow protein, a cytosolic photoreceptor: unusual fold, active site, and chromophore; Borgstahl GE et al.; A photosensing protein directs light energy captured by its chromophore into a photocycle . The protein's structure must accommodate the photocycle and promote the resulting chemical or conformational changes that lead to signal transduction . The 1.4 A crystallographic structure of photoactive yellow protein, determined by multiple isomorphous replacement methods, provides the first view at atomic resolution of a protein with a photocycle . The alpha/beta fold, which differs from the original chain tracing, shows striking similarity to distinct parts of the signal transduction proteins profilin and the SH2 domain . In the dark state structure of photoactive yellow protein, the novel 4-hydroxycinnamyl chromophore, covalently attached to Cys69, is buried within the major hydrophobic core of the protein and is tethered at both ends by hydrogen bonds . In the active site, the yellow anionic form of the chromophore is stabilized by hydrogen bonds from the side chains of Tyr42 and buried Glu46 to the phenolic oxygen atom and by electrostatic complementarity with the positively charged guanidinium group of Arg52 . Thr50 further interlocks Tyr42, Glu46, and Arg52 through a network of active site hydrogen bonds . Arg52, located in a concavity of the protein surface adjacent to the dominant patch of negative electrostatic potential, shields the chromophore from solvent and is positioned to form a gateway for the phototactic signal . Overall, the high-resolution structure of photoactive yellow protein supports a mechanism whereby electrostatic interactions create an active site poised for photon-induced rearrangements and efficient protein-mediated signal transduction.

J Immunol, 1995 May 15, 154(10), 5331 - 7
Increased opsonization of a prtH-defective mutant of Porphyromonas gingivalis W83 is caused by reduced degradation of complement-derived opsonins; Schenkein HA et al.; Periodontitis is a disease of the supporting structures of the teeth that is caused by bacteria whose common ecologic niche is the gingival crevice or the periodontal pocket . Tissue destruction occurs in spite of both local and systemic immune responses against such bacteria . Porphyromonas gingivalis is considered to be an important pathogen in some forms of human periodontitis and is particularly interesting because of its multiplicity of virulence factors . We have previously observed that phagocytosis-resistant invasive strains of P . gingivalis proteolytically degrade C3 and IgG and accumulate less C3-derived opsonins during complement activation . We recently have cloned the prtH gene from P . gingivalis W83 that encodes a 97-kDa active protease, which has the capacity to degrade purified C3 protein . By using this cloned gene we created an allelic exchange mutant of P . gingivalis W83, designated V2296, in which the prtH gene was inactivated . This mutant was previously shown to be less virulent than its parent strain W83 in a mouse model of bacterial invasiveness . In the present study we have assessed the relative capacity of V2296 and W83 to be opsonized by complement and to be taken up by PMNs . The data demonstrate that V2296, in comparison with its parent strain W83, is less able to degrade C3 and that it accumulates significantly greater numbers of molecules of C3-derived opsonins on the bacterial surface in the form of C3b and iC3b during complement activation . Furthermore, opsonized V2296 is taken up in much higher numbers by human PMNs than W83, suggesting that the prtH gene product may be important in evasion of host defense mechanisms.

J Mol Biol, 1995 May 12, 248(4), 835 - 44
Modifying filamentous phage capsid: limits in the size of the major capsid protein; Iannolo G et al.; Ff filamentous phages are long thin cylindrical structures that infect bacteria displaying the F pilus and replicate without lysing the host . These structures are exploited to display peptides by fusing them to the amino terminus of either the bacterial receptor protein (pIII) or the major coat protein (pVIII) . We have analysed a vast collection of phage mutants containing substitutions and insertions in the amino terminus of pVIII to ask whether any chemical group of this solvent exposed region of the phage capsid has any key function in the phage life cycle . Any of the five amino-terminal residues can be substituted by most amino acids without affecting phage assembly suggesting that this region does not play any essential role in morphogenesis . However, a deletion of three residues delta (Gly3Asp4Asp5) results in a phage clone with an decreased ability to produce infective particles . By engineering phages designed to display peptides by fusion to the amino terminus of the major coat protein we have found that phage viability is affected by peptide length while peptide sequence plays a minor "tuning" role . Most peptides of six residues are tolerated irrespective of their sequence while only 40% of the phages carrying an amino-terminal extension of eight residues can form infective particles . This fraction drops to 20% and 1% when we attempt to insert peptides 10 and 16 amino acids long . We have used this information to build phage libraries where each phage displays approximately 2700 copies of a different octapeptide all over the phage surface.

Proc Natl Acad Sci U S A, 1995 May 9, 92(10), 4631 - 5
Mutational analysis of the conserved region 2 site of adenovirus E1A and its effect on binding to the retinoblastoma gene product: use of the "double-tagging" assay; Wang ZX et al.; We have explored the feasibility of using a "double-tagging" assay for assessing which amino acids of a protein are responsible for its binding to another protein . We have chosen the adenovirus E1A-retinoblastoma gene product (pRB) proteins for a model system, and we focused on the high-affinity conserved region 2 of adenovirus E1A (CR2) . We used site-specific mutagenesis to generate a mutant E1A gene with a lysine instead of an aspartic acid at position 121 within the CR2 site . We demonstrated that this mutant exhibited little binding to pRB by the double-tagging assay . We also have shown that this lack of binding is not due to any significant decrease in the level of expression of the beta-galactosidase-E1A fusion protein . We then created a "library" of phage expressing beta-galactosidase-E1A fusion proteins with a variety of different mutations within CR2 . This library of E1A mutations was used in a double-tagging screening to identify mutant clones that bound to pRB . Three classes of phage were identified: the vast majority of clones were negative and exhibited no binding to pRB . Approximately 1 in 10,000 bound to pRB but not to E1A ("true positives") . A variable number of clones appeared to bind equally well to both pRB and E1A ("false positives") . The DNA sequence of 10 true positive clones yielded the following consensus sequence: DLTCXEX, where X = any amino acid . The recovery of positive clones with only one of several allowed amino acids at each position suggests that most, if not all, of the conserved residues play an important role in binding to pRB . On the other hand, the DNA sequence of the negative clones appeared random . These results are consistent with those obtained from other sources . These data suggest that a double-tagging assay can be employed for determining which amino acids of a protein are important for specifying its interaction with another protein if the complex forms within bacteria . This assay is rapid and up to 1 x 10(6) mutations can be screened at one time.

J Biol Chem, 1995 May 5, 270(18), 10405 - 11
Histidine 289 is essential for hydrolysis of the alkyl-enzyme intermediate of haloalkane dehalogenase; Pries F et al.; Haloalkane dehalogenase (DhlA) from Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic cleavage of carbon-halogen bonds in a broad range of halogenated aliphatic compounds . Previous work has shown that Asp124, which is located close to the internal substrate-binding cavity, carries out a nucleophilic attack on the C-alpha of the alkylhalide, displacing the halogen . The resulting alkyl-enzyme intermediate is subsequently hydrolyzed . In order to study the role of His289 in the hydrolysis of the intermediate, a His289-->Gln mutant was constructed by site-directed mutagenesis . The purified mutant enzyme was not catalytically active with haloalkanes, but a halide burst stoichiometric to the amount of enzyme was observed with 1,2-dibromoethane . Using ion spray mass spectrometry, accumulation of the covalent alkyl-enzyme and binding of the alkyl moiety of the substrate to an Asp124-containing tryptic peptide were shown . Fluorescence-quenching experiments indicated that halide ions are strongly bound by the alkyl-enzyme but not by the substrate-free enzyme . The results show that His289 is the base catalyst for the dealkylation of the covalent intermediate, but that it is not essential for the initial nucleophilic attack of Asp124 on the C-1 atom of the haloalkane . Furthermore, the halide ion that is released in the first step probably leaves the active site only after hydrolysis of the alkyl-enzyme.

Cell, 1995 May 5, 81(3), 369 - 77
The anatomy of a bifunctional enzyme: structural basis for reduction of oxygen to water and synthesis of nitric oxide by cytochrome cd1; Fulop V et al.; Cytochrome cd1-nitrite reductase is a bifunctional enzyme that catalyzes the one-electron reduction of nitrite to nitric oxide and the four-electron reduction of oxygen to water . The 1.55 A crystal structure of the dimeric enzyme from Thiosphaera pantotropha is reported here . The protein was sequenced from the X-ray structure . Each subunit contains a covalent c heme with two axial His ligands (His-17, His-69) and a unique noncovalent d1 heme ligated by Tyr-25 and His-200 . The d1 heme is the mononuclear iron center where both oxygen and nitrite reduction take place . The two types of heme are located in separate domains whose arrangement suggests a mechanism requiring domain movement during catalysis.

Int Endod J, 1995 May, 28(3), 141 - 8
The smear layer: a phenomenon in root canal therapy; Sen BH et al.; When the root canals are instrumented during endodontic therapy, a layer of material composed of dentine, remnants of pulp tissue and odontoblastic processes, and sometimes bacteria, is always formed on the canal walls . This layer has been called the smear layer . It has an amorphous, irregular and granular appearance under the scanning electron microscope . The advantages and disadvantages of the presence of smear layer, and whether it should be removed or not from the instrumented root canals, are still controversial . It has been shown that this layer is not a complete barrier to bacteria and it delays but does not abolish the action of endodontic disinfectants . Endodontic smear layer also acts as a physical barrier interfering with adhesion and penetration of sealers into dentinal tubules . In turn, it may affect the sealing efficiency of root canal obturation . When it is not removed, the durability of the apical and coronal seal should be evaluated over a long period . If smear layer is to be removed, EDTA and NaOCl solutions have been shown to be effective, among various irrigation solutions and techniques, including ultrasonics, that have been tested . Once this layer is removed, it should be borne in mind that there is a risk of reinfecting dentinal tubules if the seal fails . Further studies are needed to establish the clinical importance of the absence or presence of smear layer.

Dev Comp Immunol, 1995 May-Jun, 19(3), 195 - 204
In vitro generation of reactive oxygen species by free coelomic cells of the annelid Eisenia fetida andrei: an analysis by chemiluminescence and nitro blue tetrazolium reduction; Valembois P et al.; Coelomocytes of the earthworm Eisenia fetida andrei were activated in vitro with various stimulants in order to investigate their capacity to produce reactive oxygen species . Analysis by luminol-enhanced chemiluminescence and nitro blue tetrazolium reduction suggests the production in vitro of reactive oxygen species by both categories of free coelomocytes, leucocytes and chloragocytes, while affecting different modalities: a respiratory burst-like reaction is exhibited by leucocytes in the presence of zymosan but not bacteria; a moderate production of reactive oxygen species spontaneously occurs when chloragocytes aggregate together . A substantial reduction of reactive oxygen species production, rapidly occurring after addition of exogenous superoxide dismutase, was interpreted as indicating a prominent role of the superoxide anion, O2.- . The factors modulating the coelomocyte generation of reactive oxygen species, as well as the presence of lipofuscin in brown bodies, support the opinion that production of reactive oxygen species may be an immune defense process occurring under in vivo conditions.

Gut, 1995 May, 36(5), 670 - 4
Detection of the intragastric sites at which Helicobacter pylori evades treatment with amoxycillin and cimetidine; Atherton JC et al.; Treatment of Helicobacter pylori infection with amoxycillin is known to reduce the bacterial load to undetectable levels, while not eradicating the infection . It seems, therefore, that bacteria escape treatment at a 'sanctuary site' . This study examined whether such a site existed in the gastric antrum, body, or fundus . Twenty two patients with H pylori infection and duodenal ulcer disease were treated for one week with amoxycillin (500 mg three times a day) and cimetidine (800 mg at night) . Before treatment, H pylori was detected throughout all stomachs, and 13C-urea breath testing at least 28 days after treatment confirmed that eradication of H pylori had occurred in no patients . While under treatment, H pylori was sought by conventional methods and by polymerase chain reaction assay and was found in the gastric fundus in 13 of 22 subjects, in the body in 10 of 22, and the antrum in three of 22: the difference between fundus and antrum was significant (p < 0.01) . The continued antral infection in three subjects may have resulted from generalised treatment failure as two of three had H pylori detected throughout the stomach, and these two had compiled relatively poorly with treatment . This study suggests that amoxycillin and cimetidine are relatively effective at clearing H pylori from the gastric antrum, but that escape from treatment may occur in the gastric body, and especially the fundus.

J La State Med Soc, 1995 May, 147(5), 181 - 4
Deep neck space infections; Beasley DJ et al.; The incidence of deep neck space infections has dramatically decreased since the advent of antibiotics, but with delayed treatment they carry the potential for significant morbidity and mortality . Odontogenic infections with involvement of the submandibular space are the most common source of deep neck space infections in adults, whereas in the pediatric population the most common cause is acute tonsillitis with involvement of the peritonsillar space . The newest group of patients at risk for deep neck space infections are intravenous drug abusers who inject the major vessels of the neck . Knowledge of neck spaces and fascial relationships is important in understanding the presentation, treatment, and complications of deep neck space infections . The spaces, which are created by various fasciae of the head and neck, are only potential spaces in that under normal conditions they cannot be examined clinically or radiographically . As the spaces are invaded by bacteria, a cellulitis or abscess occurs, and this infection may travel through paths of least resistance from one space to another.

Eur J Immunol, 1995 May, 25(5), 1461 - 4
A 17-kDa CD4-binding glycoprotein present in human seminal plasma and in breast tumor cells; Autiero M et al.; We previously isolated gp17, a human seminal plasma glycoprotein, which specifically interacts with the D1-D2 region of CD4, a T cell surface molecule involved in antigen recognition mediated by helper T cells also acting as a receptor for the human immunodeficiency virus . In this study we report that monoclonal antibodies (mAb) reacting with gp17 are able to inhibit the binding of gp17 to immobilized soluble CD4 . An immunohistochemical analysis shows that gp17 is also expressed in mammary tumor cells upon hormone treatment and in biopsies from breast cancer patients . A structural characterization of gp17, including amino acid sequencing, indicates that the protein has an extensive structural similarity with a glycoprotein designated as seminal actin-binding protein (SABP), also secreted by male sexual glands . SABP is in turn identical to gross cystic disease fluid protein-15 (GCDFP-15) or prolactin-inducible protein (PIP), a factor known as a highly specific and sensitive marker of primary and metastatic apocrine breast cancer . To establish further the correspondence of gp17 and GCDFP-15/PIP/SABP, the latter was expressed in bacteria from a cloned cDNA and purified by affinity chromatography to either anti-gp17 mAb-Sepharose or CD4-Sepharose . The purified recombinant protein is shown to inhibit the binding of labeled, pure g17 to immobilized soluble CD4 . The finding that breast cancer cells express a protein able to interact with the CD4 domains involved in the recognition of class II major histocompatibility antigens suggests a possible mechanism by which a tumor may affect the activity of tumor-infiltrated CD4+ T cells.

J Infect Dis, 1995 May, 171(5), 1258 - 65
Binding of human plasminogen and urokinase-type plasminogen activator to the Lyme disease spirochete, Borrelia burgdorferi; Klempner MS et al.; Many bacteria that spread in the skin produce enzymes that digest extracellular matrix components . Borrelia burgdorferi spreads from a skin inoculation site to form the characteristic erythema migrans skin lesion . It was determined that B . burgdorferi does not produce collagenase, elastase, hyaluronidase, or other enzymes that digest extracellular matrix components . However, B . burgdorferi bound human plasmin, plasminogen (Pgn), and urokinase-type plasminogen activator (uPA) . When spirochetes were sequentially incubated with Pgn and uPA, bioactive plasmin was generated on the surface of B . burgdorferi . B . burgdorferi did not produce an endogenous Pgn activator . Fluorochrome-conjugated uPA and Pgn colocalized to the terminus of the spirochete . In a mouse model, uPA-treated B . burgdorferi were more infectious than control spirochetes . Binding of host uPA and Pgn to form a bioactive extracellular matrix protease on B . burgdorferi represents a mechanism that could facilitate dissemination and localization of spirochetes to sites of vascular injury.

J Bacteriol, 1995 May, 177(10), 2938 - 41
Conversion of 2-chloro-cis,cis-muconate and its metabolites 2-chloro- and 5-chloromuconolactone by chloromuconate cycloisomerases of pJP4 and pAC27; Vollmer MD et al.; 2-Chloro-cis,cis-muconate, the product of ortho-cleavage of 3-chlorocatechol, was converted by purified preparations of the pJP4- and pAC27-encoded chloromuconate cycloisomerases (EC 5.5.1.7) to trans-dienelactone (trans-4-carboxymethylenebut-2-en-4-olide) . The same compound was also formed when (+)-2-chloro- and (+)-5-chloromuconolactone were substrates of these enzyme preparations . Thus, the pJP4- and pAC27-encoded chloromuconate cycloisomerases are able to catalyze chloride elimination from (+)-5-chloromuconolactone . The ability to convert (+)-2-chloromuconolactone differentiates these enzymes from other groups of cycloisomerases.

J Bacteriol, 1995 May, 177(10), 2727 - 36
Identification of a locus required for the regulation of bvg-repressed genes in Bordetella pertussis; Merkel TJ et al.; In Bordetella pertussis, the coordinate regulation of virulence factor expression is controlled by the products of the bvgAS locus . In the presence of modulating signals such as MgSO4, nicotinic acid, or reduced temperature, the expression of bvg-activated genes is reduced while the expression of bvg-repressed genes is induced . One model for the regulation of bvg-repressed genes predicts the existence of a repressor protein encoded by a bvg-activated gene . Once activated, the product of this bvg-activated gene would bind to and repress transcription from the bvg-repressed genes . We isolated five genetically independent transposon insertion mutants of B . pertussis that have a phenotype consistent with the knockout of a putative bvg-regulated repressor . These mutants constitutively expressed a vrg6-phoA transcriptional fusion but demonstrated normal bvgAS function . Genomic mapping and DNA sequence analysis of the sites of transposon insertion demonstrated that these mutants define a locus downstream of bvgAS . Introduction of an in-frame, 12-bp insertion within this locus also conferred the mutant phenotype, confirming that the phenotype seen in the transposon mutants is the result of disruption of a distinct gene, which we have designated bvgR, and is not a consequence of polar effects on bvgAS.

FEMS Microbiol Lett, 1995 May 1, 128(2), 119 - 25
Application of random amplified polymorphic DNA (RAPD) assays in identifying conserved regions of actinomycete genomes; Mehling A et al.; Various arbitrary primers as well as pUC18/19 'reverse' sequencing primers were used for random amplified polymorphic DNA assays . Use of a modified reverse primer led to amplification of one major approx . 1100-bp band from the chromosomal DNA of all actinomycetes tested; however, the band was not found when DNAs from other bacteria were used in comparable experiments . Hybridization experiments showed that these bands all contained similar genomic regions . Subsequent sequencing of four of these fragments showed they each contained the sequence of the 3' end of the 23S rRNA gene, the intergenic region and the start of the 5S rRNA gene.

Ann Allergy Asthma Immunol, 1995 May, 74(5), 406 - 10
Histamine levels and nasal cytology in children with chronic otitis media and rhinitis; Meltzer EO et al.; OBJECTIVE: Nasal and middle ear diseases are frequent health problems for young children . In some of these patients, allergic reactions may be contributing factors . The objective of this study was to determine whether the histamine level in nasal mucosal scrapings may be used as a marker for this subset of children . METHODS: A total of 50 children, aged 2 through 7 years, was categorized into five groups of ten subjects as: normal, allergic rhinitis, nonallergic rhinitis, allergic with otitis media and nonallergic with otitis media by history, physical examination, allergy skin testing, nasal cytology, and tympanometry . Nasal mucosal scrapings were obtained using the Rhino-probe technique . Eosinophils, basophilic cells, neutrophils, and bacteria in nasal cytograms were quantified . Histamine levels were measured by radioimmunoassay, the values normalized to the total protein content assayed by enzyme-linked immunoassay, and expressed in pcg/micrograms of total protein . RESULTS: The mean histamine level for each group was: normal = 0.20, allergic rhinitis = 10.14, nonallergic rhinitis = 0.13, allergic with otitis media = 5.34, nonallergic with otitis media = 0.24 pcg/micrograms of total protein . Mean levels of histamine were statistically significantly higher in the allergic groups than in the nonallergic and normal groups (P < .05) . Allergic groups had significantly more eosinophils and basophilic cells in the nasal cytograms than the nonallergic groups . By contrast, the cytograms of children with nonallergic rhinitis and nonallergic otitis had significantly more neutrophils than the normal and allergic groups . CONCLUSION: We conclude that measuring histamine in nasal mucosal scrapings could be useful in the evaluation of young children with rhinitis and otitis and in determining which patients may have allergic disease.

Comp Biochem Physiol B Biochem Mol Biol, 1995 May, 111(1), 17 - 25
Characterization of mammalian thioredoxin reductase, thioredoxin and glutaredoxin by immunochemical methods; Martinez-Galisteo E et al.; Specific polyclonal antibodies towards the oxidized form of bovine thioredoxin reductase (TR) have been obtained in rabbits, and purified . The antigenicity was lost upon reduction of TR by NADPH indicating a large conformational change upon reduction of the redox-active disulfide in the enzyme . The antibodies did not cross-react with other bovine NADPH-dependent dehydrogenases . No reactivity was observed with TR from bacteria, yeast or rat and only a slight reaction was obtained with TR from horse . Immunoaffinity purified anti-thioredoxin and anti-glutaredoxin antibodies were used to develop competitive indirect ELISA assays that were validated giving very good linearity, reproducibility, sensitivity and parallelism . The glutaredoxin (Grx) immunoassay is the first quantitative method described to measure the protein . When applied to a battery of calf tissues the contents of Grx varied from 7 to 120 micrograms per gram of fresh tissue . Skeletal and heart muscles gave the lowest values and spleen and salivary glands the highest . However, skeletal muscle showed the highest gluthathione-hydroxyethyl disulfide oxidoreductase specific activity.

Cell Immunol, 1995 May, 162(2), 326 - 32
Severe and prolonged inflammatory response to localized cowpox virus infection in footpads of C5-deficient mice: investigation of the role of host complement in poxvirus pathogenesis; Miller CG et al.; Poxviruses are a large, complex group of highly successful pathogens that cause disease in humans and other animals . They encode several proteins postulated to be involved in the evasion of host immunity and therefore serve as excellent models for understanding virus-host interaction during the early stages of viral infection . Vaccinia virus, the best characterized member of the poxviridae family, encodes a 35-kDa major secretory polypeptide termed vaccinia virus complement control protein (VCP), which is structurally related to the family of human and mouse complement control proteins . Members of the family of complement control proteins have been shown to inhibit complement-mediated opsonization of bacteria and induction of inflammatory and phagocytic responses in vitro . Insertional inactivation of the VCP gene results in attenuation of viral virulence in vivo . The role of host complement in the inflammatory response to poxvirus infection has not been systematically investigated . Prior to determining the role of VCP on inflammatory responses in vivo, we decided to investigate the role of host complement in the progression of viral infection . We have compared the effects of injection of cowpox virus, primarily a rodent virus, into footpads of congenic mice strains B10.D2/nSnJ (C5-sufficient) and B10.D2/oSnJ (C5-deficient) . The effects of the injection were monitored macroscopically by measuring the specific swelling response immediately following primary injection and subsequently after reinfection and by histological examination of the stained sections of the footpads . Our results indicate that there is a significant variation in the primary response in the two different mouse strains to cowpox virus infection . The specific swelling response observed in measurements from the footpads of the B10.D2/oSnJ mice was significantly greater, persisted for a longer duration, and was accompanied by severe ulceration, edema, induration, and hemorrhaging . Reinjection of the footpads after a 3-month period, during which time the swelling had subsided and the footpad had fully recovered to its original size and appearance, showed no significant differences between the two strains . This strongly suggests that the host complement plays a significant role during the initial response to poxvirus infection.

Biochem J, 1995 May 1, 307 ( Pt 3), 735 - 41
The role of the novel disulphide ring in the active site of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens; Avezoux A et al.; All cysteines in methanol dehydrogenase (MDH) from Methylobacterium extorquens are involved in intra-subunit disulphide bridge formation . One of these is between adjacent cysteine residues which form a novel ring structure in the active site . It is readily reduced, the reduced enzyme being inactive in electron transfer to cytochrome cL . The inactivation is not a result of major structural change or to modification of the prosthetic group pyrrolo-quinoline quinone (PQQ) . The reduced enzyme appears to remain active with the artificial electron acceptor phenazine ethosulphate but this is because the dye re-oxidizes the adjacent thiols back to the original disulphide bridge . No free thiols were detected during the reaction cycle with cytochrome cL . Carboxymethylation of the thiols produced by reduction of the novel disulphide ring led to formation of active enzyme . Reconstitution of inactive Ca(2+)-free MDH with Ca2+ led to active enzyme containing the oxidized bridge and reduced quinol, PQQH2, consistent with the conclusion that no hydrogen transfer occurs between these groups in the active site . It is concluded that the disulphide ring in the active site of MDH does not function as a redox component of the reaction . The disulphide ring has no special function in the process of Ca2+ incorporation into the active site . It is suggested that this novel structure might function in the stabilization or protection of the free radical semiquinone form of the prosthetic group (PQQH.) from solvent at the entrance to the active site.

Am J Hematol, 1995 May, 49(1), 76 - 82
Decreased production of TNF and IL-6 in whole blood of CLL patients; Dahlke E et al.; Monocyte derived cytokines, tumor necrosis factor (TNF) and interleukin-6 (IL-6), were determined in cell free plasma after stimulation of heparinized whole blood from chronic lymphocytic leukemia (CLL) patients with lipopolysaccharide (LPS) at 1 microgram/ml for 6 hr . Compared to control donors (390 U/ml), CLL patients in average had eight-fold lower levels of TNF bioactivity (50 U/ml) . The depressed levels were observed over a wide range of LPS concentrations (0.01 to 10 micrograms/ml) . Furthermore, after stimulation with S . aureus bacteria, CLL samples gave three-fold lower levels, as well . TNF levels were not decreased because of defective bioactivity of TNF, since strongly reduced levels of TNF protein were also detected in an immunoassay . Finally, interleukin-6 levels after LPS stimulation were decreased threefold . Flow cytometry analysis with CD14 antibodies demonstrated comparable numbers of monocytes for control donors and CLL patients (698 +/- 802 and 427 +/- 267, respectively) . This suggests that deficient cytokine production was not due to a reduction in monocyte number, but rather to a functional impairment . The deficiency in cytokine production observed after ex vivo stimulation of whole blood from CLL patients suggests that in vivo during bacterial infection, CLL patients will exhibit an inappropriate response as well.

J Bacteriol, 1995 May, 177(9), 2543 - 9
Comparative analyses reveal a highly conserved endoglucanase in the cellulolytic genus Fibrobacter; Lin C et al.; An RNA probe complementary to the endoglucanase 3 gene (cel-3) of Fibrobacter succinogenes S85 hybridized to chromosomal DNAs from isolates representing the genetic diversity of the genus . The probe was subsequently used to identify putative cel-3-containing clones from genomic libraries of representative Fibrobacter isolates . Comparative sequence analyses of the cloned cel-3 genes confirmed that cel-3 is conserved among Fibrobacter isolates and that the ancestral cel-3 gene appears to have coevolved with the genus, since the same genealogy was inferred from sequence comparisons of 16S rRNAs and cel-3 genes . Hybridization comparisons using a xylanase gene probe suggested similar conservation of this gene . Together the data indicate that the cellulolytic apparatus is conserved among Fibrobacter isolates and that comparative analyses of homologous elements of the apparatus from different members, in relationship to the now established phylogeny of the genus, could serve to better define the enzymatic basis of fiber digestion in this genus.

Infect Immun, 1995 May, 63(5), 1960 - 8
Lipoarabinomannans derived from different strains of Mycobacterium tuberculosis differentially stimulate the activation of NF-kappa B and KBF1 in murine macrophages; Brown MC et al.; The inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) is rapidly induced in macrophages after exposure to Mycobacterium tuberculosis . Recently it was shown that lipoarabinomannan (LAM) derived from an attenuated (H37Ra) strain of M . tuberculosis (AraLAM) was capable of macrophage activation and induction of TNF-alpha production, whereas LAM derived from the virulent Erdman strain (ManLAM) was considerably reduced in this activity . A critical component in the regulation of many genes central to immune function is the transcription factor NF-kappa B . Lipopolysaccharide (LPS)-mediated induction of TNF-alpha expression in murine macrophages has been demonstrated to be regulated in part by NF-kappa B . In this study, we demonstrate that AraLAM is capable of rapid activation of NF-kappa B- and KBF1-binding activities in C3H/HeN bone marrow-derived macrophages and the J774.A and RAW264.7 murine macrophagelike cell lines, whereas ManLAM is considerably less potent at stimulating NF-kappa B . Treatment of RAW264.7 cells with AraLAM or LPS results in the stimulation of DNA binding of both forms within 7.5 min, which peaks within 30 min and 1 h, respectively . Interestingly, treatment of RAW264.7 macrophage-like cells with AraLAM, LPS, or ManLAM for greater than 2 h resulted in significant accumulation of KBF1 . Inhibition of protein synthesis blocked the transient nature of NF-kappa B activation as well as the accumulation of KBF1 . Using Western immunodetection of the NF kappa B1 p50 subunit, we also show that AraLAM and LPS stimulate the loss of the NF kappa B1 p105 precursor . These results demonstrate that NF-kappa B and KBF1 are rapidly induced in response to AraLAM and may play a role in avirulent M . tuberculosis activation of TNF-alpha expression in macrophages . The differential temporal regulation of kappa B element DNA-binding activities and the transient stimulation of NF kappa B followed by the sustained accumulation of KBF1 may serve as a feedback switch ensuring transient induction of TNF-alpha transcription.

Gastroenterology, 1995 May, 108(5), 1560 - 5
Pneumatosis cystoides intestinalis and high breath H2 excretion: insights into the role of H2 in this condition; Levitt MD et al.; Patients with pneumatosis cystoides intestinalis have been reported to excrete excessive H2 because of a lack of H2-consuming intestinal bacteria . This study describes a patient with bacterial overgrowth and pneumatosis of the small intestine whose colonic flora avidly consumed H2 but whose small bowel flora produced but did not consume H2 . There is no commonly accepted mechanism whereby excessive luminal H2 causes intramural gas . An explanation is proposed in which an initial, transitory source of intramural gas is distinguished from the mechanism that results in the persistence of the gas . Independent of the initial source of gas, rapid diffusion of H2 from the lumen into an intramural gas bubble would cause N2, O2, and CO2 to diffuse from the blood into the bubble . As a result, the bubble would expand and then persist indefinitely as long as H2 continued to diffuse from the lumen to the intramural gas collection.

Gastroenterology, 1995 May, 108(5), 1386 - 95
The importance of local acid production in the distribution of Helicobacter felis in the mouse stomach; Danon SJ et al.; BACKGROUND/AIMS: Helicobacter felis colonizes the gastric mucosa of rodents . Preliminary studies showed differences in the distribution of the organism in different parts of the stomach that seemed related to the secretory capacity of the mucosa . The aim of this study was to determine the localization of H . felis in the mouse stomach and to investigate the influence of acid-suppressive agents . METHODS: Specific-pathogen-free BALB/c mice were infected with H . felis . Colonization was assessed in longitudinal sections of gastric tissue from animals untreated or treated with omeprazole or ranitidine . RESULTS: In untreated H . felis-infected animals, the preferred ecological niche was the antrum and cardia equivalent . The density of colonization correlated with the number of parietal cells per gland . Partial acid suppression with ranitidine produced a slight increase in the colonization of the body but was restricted to the upper portions of the gastric gland . Omeprazole treatment produced a greater colonization of the body with bacteria traversing the entire gland . Some reduction in antral colonization occurred . CONCLUSIONS: These results are consistent with the hypothesis that local acid output is a crucial determinant in the distribution of Helicobacter species in the stomach . Differences in local acid output may explain the different patterns of Helicobacter pylori-induced gastric pathology.

Scand J Immunol, 1995 May, 41(5), 457 - 61
S . typhi vaccine strain Ty21a can cause a generalized infection in whole body-irradiated but not in hydrocortisone-treated mice; Van Dissel JT et al.; Various mutations including galE- in the S.typhi vaccine strain Ty21a are thought to prevent proliferation of these micro-organisms in the host, and elimination of Ty21a would occur independent of the immune system of the host . To investigate this issue, we determined whether Ty21a can proliferate in immunosuppressed mice, and assessed the role of phagocytes in the eradication of Ty21a from tissues . Mice were rendered lymphocytopenic and monocytopenic by hydrocortisone s.c., or were made leucocytopenic by whole body irradiation . Bacteria were injected into a tail vene to evaluate eradication from the blood, liver and spleen, and into thigh muscle, i.e . a tissue that lacks resident macrophages . Ty21a were grown overnight in glucose {glu}, or galactose and glucose {gal.glu}; only the Ty21a {gal.glu} expressed somatic O-antigens . After i.v . injection of 10(4) to 10(6) micro-organisms, Ty21a were rapidly eliminated from the liver and spleen of normal and immunosuppressed mice, i.e . within 1 day a 95% reduction of bacterial counts was observed . After i.m . injection of 10(4) to 10(6) bacteria, the number of viable Ty21a decreased in normal and hydrocortisone-treated mice, but in irradiated mice the micro-organisms proliferated and caused generalized infection . In all cases, Ty21a {glu} was eliminated more rapidly than Ty21a {gal.glu}, confirming reports that killing of bacteria that lack O-antigens is more rapid than that of smooth bacteria of the same species . These results indicate that elimination of the vaccine strain against typhoid fever, Ty21a, from host tissues is not due to an intrinsic property of the micro-organisms that prevents proliferation but instead depends on the action of resident macrophages and exudate monocytes and granulocytes.

Haematologica, 1995 May-Jun, 80(3), 252 - 67
Lactoferrin: a general review; Levay PF et al.; Lactoferrin is a 703-amino acid glycoprotein originally isolated from milk . Plasma lactoferrin is predominantly neutrophil derived but indications are that it may also be produced by other cells . Lactoferrin in body fluids is found in the iron-free form, the monoferric form and in the diferric form . Three isoforms of lactoferrin have been isolated, ie two with RNase activity (lactoferrin-beta and lactoferrin-gamma) and one without RNase activity (lactoferrin-alpha) . Receptors for lactoferrin can be found on intestinal tissue, monocytes/macrophages, neutrophils, lymphocytes, platelets, and on certain bacteria . A wide spectrum of functions are ascribed to lactoferrin . These range from a role in the control of iron availability to immune modulation . More research is necessary however to obtain clarity with regard to the exact mechanism of action of lactoferrin.

J Anim Sci, 1995 May, 73(5), 1487 - 92
Leucaena toxicosis and its control in ruminants; Hammond AC; Leucaena (Leucaena spp., especially L . leucocephala) is an arboreal, tropical legume that ranges into the cool subtropics and equatorial elevations up to 1,000 m . One of its uses includes forage for livestock, but introduction of leucaena outside its indigenous range often has led to acute and chronic toxicosis . The major toxic constituents of leucaena are the nonprotein free amino acid mimosine and its ruminal degradation product, 3-hydroxy-4(1H)-pyridone (3,4-dihydroxypyridine; 3,4-DHP) . Leucaena also contains appreciable quantities of condensed tannins . In ruminants, mimosine is a depilatory agent and 3,4-DHP is a potent goitrogen . In the 1980s, Australian workers demonstrated that the geographical limits of leucaena toxicosis were due to the absence of ruminal bacteria capable of degrading 3,4-DHP, and successfully introduced 3,4-DHP degrading ruminal bacteria from a Hawaiian goat into goats and cattle in Australia . Simple in vitro screening methods have been developed for detection of 3,4-DHP degraders in ruminal samples and feces . Also, several strains of 3,4-DHP degrading ruminal bacteria have been characterized and have been given the genus and species designation, Synergistes jonesii . Ruminal inoculation with ruminal contents from adapted animals, enriched cultures of 3,4-DHP-degrading ruminal bacteria, and pure cultures of S . jonesii have all been used successfully to establish ruminal populations that are capable of degrading 3,4-DHP and preventing leucaena toxicosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Sci, 1995 May, 4(5), 1001 - 6
Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis; Ferreira GC et al.; 5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria . Lysine-313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965) . Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis . Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme . Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently . However, addition of glycine to the mutant enzymes led to the formation of external aldimines . The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction . In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity . In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive . This suggests that lysine-313 has a functional role in catalysis.

Biol Trace Elem Res, 1995 May, 48(2), 141 - 60
Subcellular distribution of selenium during uptake and its influence on mitochondrial oxidations in germinating Vigna radiata L; Easwari K et al.; The metabolic significance of Se in plants is not well documented, though the presence of many selenoenzymes in bacteria and the essentiality of Se in higher animals is established . Since germination is an active process in plant growth and metabolism, the effect of Se was investigated in germinating Vigna radiata L, a nonaccumulating Se-deficient legume . Growth and protein were enhanced in seedlings supplemented with selenium (Se) as sodium selenite in the medium up to 1 microgram/mL . The pattern of uptake of 75Se in the differentiating tissues and the subcellular distribution were investigated . The percentage of incorporation of 75Se was greater in the mitochondria at the lowest level (0.5 micrograms/mL) of Se supplementation compared to higher levels of Se exposure . Proteins precipitated from the postmitochondrial supernatant fractions, when separated by means of polyacrylamide gel electrophoresis (PAGE), indicated a major selenoprotein in the seedlings germinated at 2.0 micrograms/mL Se . In seedlings grown with supplemented Se, enhanced respiratory control ratio and succinate dehydrogenase activity were observed in the mitochondria of tissues, indicative of a role for Se in mitochondrial membrane functions.

Mol Plant Microbe Interact, 1995 May-Jun, 8(3), 424 - 34
Characterization and localization of new antifungal cysteine-rich proteins from Beta vulgaris; Kragh KM et al.; Two novel antifungal proteins, AX1 and AX2, were isolated from leaves of sugar beet infected with Cercospora beticola . AX1 (MW = 5078 +/- 3D) and AX2 (MW = 5193 +/- 3D) were N-terminally sequenced and identified as monomeric, basic proteins consisting of 46 amino acid residues, of which eight are cysteines . Both AX proteins strongly inhibit growth of C . beticola and other filamentous fungi, but have little or no effect against bacteria . Based on primary sequence homology (24 to 46% identity), they are related to the superfamily of gamma-thionins, which have been isolated recently from seeds of monocotyledons and Brassicaceae . Specific antibodies were raised against the AX proteins after conjugation to diphtheria toxoid . Using immunoblotting and immunohistology, we detected high concentrations of AX proteins extracellularly in cell walls and in globular bodies around necrotic lesions in sugar beet leaves infected with C . beticola, suggesting that AX proteins are involved in antifungal defense . Furthermore, AX proteins or serologically related proteins were detected in xylem, stomata, and stomatal cells as well as in sugar beet styles.

Virus Res, 1995 May, 36(2-3), 293 - 7
A novel human papillomavirus sequence based on L1 general primers; Togawa K et al.; Over 65 human papillomaviruses (HPVs) have been identified . The majority have been isolated on the basis of cloning systems in bacteria . Recently, general consensus or degenerate primers from the L1 region have been used in PCR to identify novel genotypes . In this fashion, we employed general primers in the L1 region followed by DNA sequencing to identify a novel HPV sequence in association with esophageal squamous cell carcinoma.

Shock, 1995 May, 3(5), 362 - 8
Comparison of plasma cytokine levels in rats subjected to superior mesenteric artery occlusion or hemorrhagic shock; Grotz MR et al.; The overall goal of this study was to compare the effects of systemic hypotension (hemorrhagic shock) versus local gut ischemia (superior mesenteric artery (SMA) occlusion) on cytokine production and bacteria/endotoxin translocation . Sham or actual SMA occlusion led to an increase in tumor necrosis factor (TNF), which was greatest at the end of the occlusion period, while the IL-6 response peaked 3 h post-SMA occlusion . The TNF and IL-6 response after hemorrhagic shock differed from that observed after SMA occlusion in that the peak response occurred later and was of lower magnitude (p < .05) . Although the animals subjected to SMA occlusion had a significantly increased incidence of bacterial translocation to both the mesenteric lymph nodes and systemic organs compared to rats subjected to hemorrhagic shock, in neither group was the blood level of endotoxin elevated and there was no association between bacterial translocation and cytokine levels . These results suggest that different models of intestinal ischemia have different cytokine profiles and that the early TNF response associated with SMA occlusion model is primarily due to the laparotomy.

Appl Environ Microbiol, 1995 May, 61(5), 1805 - 9
Precision and accuracy of recovery of Legionella pneumophila from seeded tap water by filtration and centrifugation; Boulanger CA et al.; Determination of the concentration of Legionella pneumophila in environmental water sites may be useful for the prediction of the risk of a particular site's causing Legionnaires' disease as well as for experimental studies of environmental growth or remediation . The precision and accuracy of recovery of two different L . pneumophila strains from seeded tap water samples were studied, with either filtration or centrifugation used to concentrate the bacteria . L . pneumophila grown on BCYE alpha agar or in Acanthamoeba castellanii was used to seed sterile tap water . Water samples were then either filtered (0.2-microns pore size) or centrifuged . An average of 53% (95% confidence interval {CI}, 47 to 58%; n = 45) of the seeded L . pneumophila organisms were recovered by filtration with flat polycarbonate membranes . This recovery was significantly higher (P < 0.01) than that obtained by filtration with cast membranes (mean, 13%; 95% CI, 11 to 38%; n = 4) or by centrifugation at 3,800 x g for 30 min (mean, 14%; 95% CI, 2 to 25%; n = 9) or at 8,150 x g for 15 min (mean, 32%; 95% CI, 28 to 36%; n = 19) . Recovery of L . pneumophila was not significantly different whether the bacteria were grown on plates or in amoebae . Use of a selective medium did not decrease the recovery efficiency, but preplating acid treatment of specimens caused an approximately 30% bacterial loss.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1995 May, 61(5), 1688 - 90
Randomly amplified polymorphic DNA analysis of Xylella fastidiosa Pierce's disease and oak leaf scorch pathotypes; Chen J et al.; Randomly amplified polymorphic DNA analysis was conducted with 14 primers to 17 strains of Xylella fastidiosa . There was a high degree of similarity among the seven Pierce's disease (PD) strains (Sxy > 0.93) and the seven oak leaf scorch (OLS) strains (Sxy > 0.96) . However, the two groups were different, with a similarity index of 0.67, confirming the presence of a PD DNA cluster and suggesting the presence of a new OLS cluster . The control plum leaf scald strains (two strains) together with the periwinkle wilt strain had a much smaller similarity index (0.44) compared with the PD and OLS clusters.

Neurol Clin, 1995 May, 13(2), 405 - 12
The sick building syndrome and mass hysteria; Rothman AL et al.; Significant overlaps of symptoms in SBS and MH exist including central nervous system manifestations, mucous membrane irritation, skin abnormalities, and eye symptomatology . Both occur with greater frequency in women with lower job rank and in patients with psychological and physical stresses . No specific cause has been identified in over 75% of the cases of SBS . Remotely, less than 25% have been alleged to be secondary to an environmental toxin but even removal of the inciting irritant does not improve the symptoms . Not surprisingly, litigation is lurking in the background with the chronic complainer . There has been no evidence of exposure to toxins or VOCs that exceed the NIOSH safety standards in any SBS cases . The importance of a thorough work up is to distinguish those cases that are secondary to bacteria or toxic contaminants and infection where significant morbidity and death have occurred in the building related illness . Although this is a separate entity it indicates that the building environment can be a significant cause of morbidity and mortality . Other variables such as temperature and humidity do influence the frequency of SBS symptomatology . It may be that we are not yet sophisticated enough to find the causes that are present within our current paradigms . Current levels of toxic data testing may not be sensitive enough to realize that they might be a cause of toxicity . It might be that at low levels with multiple toxins and VOCs present, the additive effects may cause toxic symptomatology whereas individually they do not . In summary, SBS is an emerging phenomena within our litigious society.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Cell Biol, 1995 May, 67(1), 32 - 41
Lipopolysaccharide-stimulated exocytosis of nonself recognition protein from insect hemocytes depend on protein tyrosine phosphorylation; Charalambidis ND et al.; Insect hemocytes (blood cells) synthesize the major nonself recognition protein (47 kDa) during 3rd instar larvae (V.J . Marmaras, S . Tsakas, Dev . Biol . 129, 294-303 (1988)) . In this study we show the presence of the 47 kDa protein in plasmatocytes (main hemocyte type) and prohemocytes . In plasmatocytes this protein appears to be localized both in vesicles and in the cell surface . The cell surface-associated 47 kDa protein was released from membrane fraction by 1 M NaCl, indicating that it is not tightly bound . Bacterial lipopolysaccharide (LPS) can function on isolated hemocytes from Ceratitis capitata larvae, inducing their spreading and degranulation . During degranulation (exocytosis) the plasmatocytes release the 47 kDa protein, among others . This protein could not be normally traced in serum, nor is it released by basal secretion . The secretion of the 47 kDa protein was found to be LPS-dependent, whereas its presence on plasmatocyte surface is LPS independent . LPS-stimulated exocytosis of the 47 kDa protein appears to be dependent on protein tyrosine phosphorylation . We have now demonstrated that LPS increases tyrosine phosphorylation of 19 and 22 kDa polypeptides in C . capitata hemocytes . Inhibition of the LPS-induced tyrosine phosphorylation mediated by tyrosine kinase inhibitor, genistein, was accompanied by the inhibition of the secretion of the 47 kDa protein . These results support the hypothesis that tyrosine protein phosphorylation is a signal reaction in hemocytes after LPS exposure . These LPS responses of insect plasmatocytes show strong similarities to mammalian macrophages (S . Weinstein et al., J . Immunol . 151, 3829-3838 (1993)) . In a model we propose that the LPS-independent cell surface-associated 47 kDa protein is responsible for the phagocytosis and for the formation of nodules and capsules, whereas the LPS-dependent secreting counterpart is responsible for the extracellular killing of bacteria.

Semin Pediatr Surg, 1995 May, 4(2), 77 - 82
The immune response to trauma; Harris BH et al.; The response to trauma begins in the immune system at the moment of injury . The loci are the wound, with activation of macrophages and production of proinflammatory mediators, and the microcirculation with activation of endothelial cells, blood elements, and a capillary leak . These processes are potentiated by ischemia and impaired oxygen delivery and by the presence of necrotic tissue, each exacerbating the inflammatory response . Hemorrhage alone may be a sufficient stimulus . Inflammation once was considered to be a host reaction to bacteria or other irritants . This concept was expanded by the discovery of autoimmune diseases, and we are now aware that some illnesses are the result of the body's response to an invader rather than the direct effect of the invader itself . The discoveries about the response to trauma described here add another dimension, showing inflammation to be a fundamental life process that begins at the molecular level at the moment of injury and that, depending on the severity of the stimulus and the effectiveness of initial treatment, may spread to include every cell, tissue, and organ in the body, for good or ill . An important part of these expanding concepts is the notion that all noxious stimuli activate the cytokine system as a final common pathway . Sepsis, hemorrhage, ischemia, ischemia-reperfusion, and soft tissue trauma all share an ability to activate macrophages and produce proinflammatory cytokines that may initiate the SIRS . Second-message compounds and effector molecules mediate the observed clinical phenomena.(ABSTRACT TRUNCATED AT 250 WORDS)

Biol Reprod, 1995 May, 52(5), 1167 - 78
Immunogenicity enhancement of recombinant rabbit 55-kilodalton zona pellucida protein expressed using the baculovirus expression system; Prasad SV et al.; In the present study we have used a molecular approach to evaluate the immunogenicity and antigenicity of glycosylated and non-glycosylated recombinant rabbit 55-kDa zona pellucida (ZP) protein . The 55-kDa cDNA was expressed in insect (Sf9) cells through use of a baculovirus expression system to obtain nonfusion glycosylated recombinant ZP protein (BV-55) . SDS-PAGE and immunoblot analysis demonstrated that the recombinant protein is expressed as two forms having relative molecular masses of 70 kDa and 80 kDa . Because cells treated with tunicamycin produce predominantly the 70-kDa form, this heterogeneity is presumed to be due to differential glycosylation . Further studies using lectin blot and immunoblot analyses showed that the BV-55 protein has both N-linked and O-linked oligosaccharides . However, this glycosylation is distinct from that of the native 55-kDa ZP protein, since it was not recognized by a monoclonal antibody associated with lactosaminoglycan-type carbohydrate epitopes in native ZP proteins . Immunogenicity studies demonstrated that antibodies against the BV-55 protein are developed by female rabbits and guinea pigs and that these antibodies recognize epitopes associated with native, enzyme-deglycosylated as well as nonglycosylated recombinant forms of the rabbit 55-kDa ZP protein . In contrast, recombinant protein expressed in bacteria did not elicit antibodies in either rabbits or guinea pigs . These results demonstrate that expression of the 55-kDa recombinant protein in the baculovirus expression system enhances its immunogenicity.

J Nat Prod, 1995 May, 58(5), 653 - 71
Chemical and biological investigation of the polar constituents of the starfish Luidia clathrata, collected in the Gulf of Mexico; Iorizzi M et al.; Ten new inverted question mark1-10 inverted question mark and three known inverted question mark11-13 inverted question mark polyhydroxysteroids were isolated, along with four known asterosaponins inverted question mark14-17 inverted question mark, from the starfish Luidia clathrata, collected from the offshore waters of the northern Gulf of Mexico . The EtOH extracts of this starfish showed feeding-deterrent properties against marine fish, and inhibited the settlement of larvae of barnacles and bryozoans, as well as the growth of several bacteria . The structures of the new compounds were determined by interpretation of their nmr spectral data and by comparison with the spectral data of known compounds . The assignment of the configurations of the side-chain stereogenic centers of compounds 1 and 3-10 were based on the comparison of their nmr data with those of the stereoisomeric model compounds after derivatization with the chiral auxiliary MTPA reagent . Larval settlement assays conducted on ten isolated compounds revealed they are all potent inhibitors of settlement . Two of these isolated compounds inhibited the growth of several bacteria.

Clin Infect Dis, 1995 May, 20(5), 1102 - 10
Ehrlichial diseases of humans: emerging tick-borne infections; Dumler JS et al.; The ehrlichioses are emerging zoonotic infections that are caused by obligate intracellular bacteria in the genus Ehrlichia . Two human ehrlichioses occur in the United States: monocytic ehrlichiosis (HME), which is caused by Ehrlichia chaffeensis that infects mononuclear phagocytes in blood and tissues, and granulocytic ehrlichiosis (HGE), an infection of granulocytes that is due to a phylogenetically distinct organism . Both infections cause undifferentiated fever with leukopenia, thrombocytopenia, and elevations in serum aminotransferase levels . Rash is an infrequent sign, and vasculitis is exceedingly rare . Severe or fatal ehrlichiosis is associated with secondary or opportunistic infections and delayed therapy . Ticks are the likely vectors, and deer are the likely reservoirs . HGE is associated with Ixodes species ticks and Lyme disease, a finding suggesting concurrent infection . In cases of HME, ehrlichial inclusions (morulae) are rarely detected; however, they are often seen in neutrophils of patients with HGE . A clinical diagnosis is confirmed with use of the polymerase chain reaction during the infection or by serology during convalescence . Therapy with doxycycline is highly efficacious.

J Am Board Fam Pract, 1995 May-Jun, 8(3), 217 - 20
Septic olecranon bursitis: recognition and treatment; Shell D et al.; BACKGROUND: The superficial location of the olecranon bursa places it at high risk for injury, possibly leading to the entry of bacteria into the bursal sac . Early differentiation between septic and nonseptic olecranon bursitis is paramount to direct therapy, to hasten recovery, and to prevent chronic inflammation . METHODS: A literature review was performed using MEDLINE files from 1967 to the present . Additional references from the bibliographies of these were also utilized . RESULTS AND CONCLUSIONS: Olecranon bursitis is a common condition that requires the treating physician to be aware of the predisposing factors, clinical signs and symptoms, and laboratory findings of both septic and nonseptic olecranon bursitis . With early recognition, prompt therapy, and preventive measures, the morbidity of septic olecranon bursitis can be considerably reduced, but surgical incision and drainage or excision could be required if conservative therapy fails.

Vet Immunol Immunopathol, 1995 May, 46(1-2), 21 - 33
Panleukopenia-like syndrome of FeLV caused by co-infection with FeLV and feline panleukopenia virus; Lutz H et al.; To study the effect of interferon on feline leukemia virus (FeLV) infection, 30 specific pathogen free (SPF) cats were infected with the apathogenic FeLV A Glasgow . Unexpectedly, between 5 and 8 weeks after FeLV infection, all 19 cats with persistent FeLV infection but not the FeLV-negative cats died from a panleukopenia-like syndrome . No feline panleukopenia virus (FPLV) antigen was found in feces by latex agglutination, enzyme-linked immunosorbent assay (ELISA) or immunoelectron microscopy . No enteropathogenic bacteria were found . Histopathology revealed changes resembling those of FPLV infection such as destruction of crypts and pancytopenia of bone marrow . Neither clinical signs nor seroconversion to FPLV could be induced by transmitting intestinal extracts to two SPF cats . However, FPLV antigen was demonstrated by immunofluorescence assay in intestinal cryostat sections of diseased animals . FPLV could also be demonstrated in intestinal extracts by immunoelectron microscopy, by latex agglutination and ELISA after anti-FPLV antibodies were removed from immune-complexed FPLV by ultracentrifugation over a CsCl gradient at pH 2.0 . From these experiments it was concluded that the panleukopenia-like syndrome of FeLV may not be caused by FeLV alone but at least in some cases by co-infection with FeLV and FPLV . In addition, some form of 'cooperation' between FeLV and FPLV must be postulated because neither virus alone induced symptoms.

Brain Res Mol Brain Res, 1995 May, 30(1), 37 - 47
Differential expression of heme oxygenase-1 in cultured cortical neurons and astrocytes determined by the aid of a new heme oxygenase antibody . Response to oxidative stress; Dwyer BE et al.; Heme oxygenase exists as two isoenzymes designated heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2) . HO-2 is made constitutively in many cell types whereas HO-1 is a stress protein inducible by heat, heavy metals, ultraviolet irradiation, and oxidative stress . Recombinant rat HO-1 was expressed in bacteria and antiserum designated HO-1713 was raised against the purified protein . HO-1713 detected recombinant rat HO-1 and recombinant rat HO-2 . In rat tissues it detected HO-1 and a second, unidentified band designated HO-L (heme oxygenase-like immunoreactivity) which was not HO-2 . Cultured rat cortical neurons and forebrain astrocytes were exposed to hydrogen peroxide (0.14-0.7 micromolar for 30 or 60 min) . Neurons which contained little detectable HO-1 and which were sensitive to hydrogen peroxide at the high end of the dose curve failed to induce HO-1 by Western blot analysis . In contrast, cultured rat forebrain astrocytes which contained HO-1 under normal culture conditions and which were resistant to injury by hydrogen peroxide, increased their content of immunoreactive HO-1 by 7-fold within 3 h after exposure . Our results support a protective role for HO-1 in oxidative injury and suggest that the relative inability of neurons to increase HO-1 after oxidative stress may contribute to their selective vulnerability vis-a-vis astrocytes . They also suggest that differential expression of heme oxygenase in studies utilizing CNS cultures may alter normal cell physiology and cell survival.

Laryngorhinootologie, 1995 May, 74(5), 274 - 81
{Etiology and differential diagnosis of sialadenitis}; Seifert G; The salivary glands are integrated into the body in such a complex manner that the analysis of the saliva allows conclusions about pathological function of other organ systems . Such interactions between salivary glands and the body as a whole play an important role in the etiology and pathogenesis of the different types of sialadenitis . An etiological classification is a proven method for arriving at a clinical diagnosis . Etiological factors include traumas, bacteria, viruses, ductal obstructions, disturbances of secretion, radiogenic damage, and immunological reactions (immunocomplex, tuberculin, or autoimmune types) . Frequently the etiology is influenced by several factors . After a brief discussion of the clinical diagnostic methods, we present the clinical and morphological findings concerning the various types of sialadenitis and their differential diagnosis.

Ann Acad Med Singapore, 1995 May, 24(3), 411 - 4
Incidental cholecystectomy--an old problem reconsidered; Wee SB et al.; This paper reexamines the issues and indications for incidental cholecystectomy when gallstones are present during laparotomy for an unrelated condition . Seventy-nine such patients were studied between 1988 and 1993--66 had incidental cholecystectomy (both elective and emergency) while asymptomatic gallstones were left alone in 13 patients for various reasons . There was little morbidity and no mortality arising directly from biliary surgery . Interestingly, four bile cultures from the asymptomatic gallbladders grew bacteria and this could explain why patients may develop severe disease postoperatively if their gallbladders were left alone . The added cholecystectomy resulted in an acceptable extension of 22 to 23 minutes in the operative time without compromising the patients' safety.

J Clin Immunol, 1995 May, 15(3), 121 - 9
Biology of human TH1 and TH2 cells; Romagnani S; Evidence is accumulating to suggest the existence of polarized human T-cell responses, reminiscent of TH1 and TH2 subsets described for mouse T cells . Human TH1 cells preferentially develop during infections by intracellular bacteria and trigger phagocyte-mediated host defense, whereas TH2 cells, which predominate during helminthic infestations and in response to common environmental allergens, are responsible for phagocyte-independent host response . Human TH1 and TH2 cells exhibit not only different functional properties but probably also distinct surface markers; TH2, but not TH1, clones express membrane CD30 and release the soluble form of CD30, a member of the TNF receptor superfamily . The cytokine profile of "natural immunity" evoked by different offending agents in the context of different host genetic backgrounds appears to be the most critical factor in determining the phenotype of the subsequent specific response . IL-12 and IFN-alpha and gamma produced by macrophages and NK cells favor the development of TH1 cells, whereas the early production of IL-4 by a still-unidentified cell type favors the development of TH2 cells . Clearly, polarized human TH1 and TH2 responses not only play different roles in protection, they can also promote different immunopathological reactions . Strong and persistent TH1 responses seen to be involved in organ-specific autoimmunity, contact dermatitis, and some chronic inflammatory disorders of unknown etiology . In contrast, polarized TH2 responses favor a reduced protection against the majority of infectious agents (including HIV) and, in genetically predisposed hosts, are responsible for triggering of allergic atopic disorders.

Curr Genet, 1995 May, 27(6), 559 - 64
The gene for ribosomal protein S10 is present in mitochondria of pea and potato but absent from those of Arabidopsis and Oenothera; Knoop V et al.; A novel group II intron has been identified in the pea (Pisum sativum) mitochondrial genome . The gene harbouring this intron is identified as rps10 (encoding protein S10 of the small ribosomal subunit) by similarity to its known homologues in bacteria and in the mitochondrion of the liverwort Marchantia polymorpha . The rps10 gene is transcribed in pea, the intron is removed, and RNA editing in the rps10 reading frame increases similarity to its homologue in the M . polymorpha mitochondrion . Contrary to the situation in bacteria and Marchantia, rps10 is not part of a ribosomal-protein gene cluster in pea . It is flanked upstream by the genes trnF and trnP, encoding phenylalanine- and proline-accepting tRNAs, and downstream by cox1, encoding subunit 1 of the cytochrome-c-oxidase . Southern hybridization shows that sequences homologous to rps10 exist in potato mitochondria but not in mitochondria of Oenothera berteriana and Arabidopsis thaliana . The pea rps10 intron is homologous to introns in rrn26 and cox3 in the Marchantia mitochondrial genome, while the Marchantia rps10 gene lacks an intron.

Clin Exp Allergy, 1995 May, 25(5), 423 - 31
Asthmatic symptoms and indoor levels of micro-organisms and house dust mites; Bjornsson E et al.; As a part of a worldwide investigation on the prevalence of respiratory symptoms, we have performed a study on the relationship between the indoor environment and asthma-like symptoms in the population of a central Swedish municipality . The study comprised 88 individuals, aged 20-45 years who underwent a structured interview, spirometry, a methacholine provocation test, skin-prick tests and blood samples for measurements of serum concentrations of eosinophil cationic protein (S-ECP), blood eosinophil count and total immunoglobulin E (S-IgE) . In the homes, the room temperature, air humidity, respirable dust, house dust mites (HDM) and airborne micro-organisms were measured . The relative humidity in all the homes was found to be above 33% . HDM were found in 13% of homes . In the homes of the 47 subjects with asthma related symptoms, significantly higher total levels of bacteria and mould (P < 0.05) and a higher proportion of detected HDM (OR = 5.3) was found than in subjects with no asthma related symptoms, after adjustment for age, sex, smoking, indoor temperature and air humidity . HDM were found to be an independent risk factor for asthma related symptoms (OR = 7.9) and nocturnal breathlessness (OR = 6.2) (P < 0.05), while the total level of bacteria was a risk factor for asthma related symptoms and wheezing (P < 0.05) . We conclude that although HDM is relatively infrequently found in the homes of central-Sweden, the presence of HDM is related to asthmatic symptoms . A relation between levels of airborne bacteria and asthma related symptoms was also found.

Mol Chem Neuropathol, 1995 May, 25(1), 1 - 17
Stimulation of group II phospholipase A2 mRNA expression and release in an immortalized astrocyte cell line (DITNC) by LPS, TNF alpha, and IL-1 beta . Interactive effects; Tong W et al.; Astrocytes are immunoactive cells in brain and have been implicated in the defense mechanism in response to external injury . Previous studies using cultured glial cells indicated the ability of astrocytes to respond to bacteria endotoxin and cytokines, resulting in the release of phospholipase A2 . In this study, we examined the interactive effects of lipopolysaccharides (LPS), interleukin 1 beta (IL-1 beta) and tumor necrosis factor (TNF alpha) to stimulate phospholipase A2 (PLA2) in an immortalized astrocyte cell line (DITNC) with many properties of type I astrocytes . Northern blot analysis using oligonucleotide probes derived from the cDNA encoding the rat spleen group II PLA2 indicated the ability of DITNC cells to respond to all three factors in the induction of gene expression and the release of PLA2 . After an initial lag time of 2 h, PLA2 release was proportional to time, reaching a plateau by 12 h . This event occurred at a time period preceding any signs of cell death . Cycloheximide at 1.25 microM completely inhibited cytokine-induced PLA2 release . When suboptimal amounts of TNF alpha were added to the DITNC culture together with IL-1 beta or LPS, a synergistic increase in the induction of PLA2 release could be observed . On the other hand, combination of IL-1 beta and LPS resulted only in an additive increase in PLA2 release . Antibodies to IL-1 beta and TNF alpha completely neutralized the effects of these two agents on PLA2 release . However, neither antibody was able to inhibit the PLA2 release induced by LPS, suggesting that the effect of LPS was not complicated by the release of IL-1 beta or TNF alpha . Taken together, results show that the immortalized astrocyte cell line (DITNC) can be used for studies to elucidate the molecular mechanism underlying the cytokine signaling cascade and subsequent induction of PLA2 synthesis.

Trends Microbiol, 1995 May, 3(5), 178 - 85
Biosynthesis of lipopolysaccharide O antigens; Whitfield C; Lipopolysaccharide O antigens are important virulence determinants for many bacteria . O-antigen synthesis is an interesting problem in cell-surface assembly . There are two known assembly pathways, which differ in the cellular location of their polymerization steps and in the direction of chain polymerization . Some reactions are shared with those for other surface polymers, such as capsular polysaccharides, and may be potential targets for therapeutic intervention.

J Clin Microbiol, 1995 May, 33(5), 1344 - 7
Diagnostic assay for Helicobacter hepaticus based on nucleotide sequence of its 16S rRNA gene; Battles JK et al.; Conserved primers were used to PCR amplify 95% of the Helicobacter hepaticus 16S rRNA gene . Its sequence was determined and aligned to those of related bacteria, enabling the selection of primers to highly diverged regions of the 16S rRNA gene and an oligonucleotide probe for the development of a PCR-liquid hybridization assay . This assay was shown to be both sensitive and specific for H . hepaticus 16S rRNA gene sequences.

Invest Ophthalmol Vis Sci, 1995 May, 36(6), 1076 - 83
Effect of tecogalan sodium on angiogenesis in vitro by choroidal endothelial cells; Sakamoto T et al.; PURPOSE . To examine the possible inhibitory effect of tecogalan sodium, derived from bacteria, on three important components of in vitro angiogenesis (endothelial proliferation, migration, and tube formation in a collagen gel) using bovine choroidal endothelial cells (CECs) . METHODS . The effects of tecogalan sodium (1, 5, 25, 125, and 250 micrograms/ml) on cultured CECs were examined when basic fibroblast growth factor (bFGF, 10 ng/ml), vascular endothelial growth factor (VEGF, 50 ng/ml), a combination of bFGF (10 ng/ml) and VEGF (50 ng/ml) (bFGF/VEGF) and 10% fetal calf serum (FCS) were used as angiogenic stimulants . For the proliferation assay, CECs were cultured and the cell numbers counted on days 1, 3, and 5 . For migration assay, CECs were seeded in the upper half of a Boyden chamber while an angiogenic growth factor was loaded in the lower half . After 6 hours of incubation, cell migration was evaluated by counting the numbers of migrated cells per microscopic field on the lower side of the filter . For the tube-forming assay, CECs were seeded in a type I collagen gel, and the length of the tube-like structures (an indicator of angiogenesis) formed by CECs per microscopic field was quantified by image analysis . The effect of neutralizing antibody for bFGF also was tested in these three assays . RESULTS . All tested angiogenic stimulants induced CEC proliferation . The stimulatory effect of bFGF and bFGF/VEGF was reduced by tecogalan sodium (IC50 for bFGF effect, 26.1 micrograms/ml) . However, the effect of VEGF and of 10% FCS was not altered by low doses of tecogalan sodium (< 25 micrograms/ml) . Chemotaxis of CECs was stimulated by bFGF alone and by bFGF/VEGF, and this effect was inhibited by tecogalan sodium (IC50 for bFGF, 3.2 micrograms/ml) . Stimulation of chemotaxis by VEGF alone and by 10% FCS was not affected by tecogalan sodium in low doses but was inhibited by high doses . Tube formation was stimulated by administration of each of the factors . Stimulation of tube formation by bFGF and by bFGF/VEGF was inhibited by tecogalan sodium (IC50 for bFGF, 18.2 micrograms/ml) . High doses of tecogalan sodium (125 and 250 micrograms/ml) also inhibited 10% FCS-induced proliferation, migration, and tube formation . CONCLUSION . bFGF, VEGF, and a combination of bFGF and VEGF stimulated proliferation, migration, and tube formation by CECs in vitro . These stimulatory effects, but especially those of bFGF, were inhibited by tecogalan sodium . If tecogalan sodium can be shown to have a similar effect in vivo, it might have the potential for pharmacologic control of subretinal neovascularization.

Cancer, 1995 May 1, 75(9), 2203 - 8
Detection of Helicobacter pylori infection in early stage gastric cancer . A comparison between intestinal- and diffuse-type gastric adenocarcinomas; Endo S et al.; BACKGROUND . Helicobacter pylori (H pylori) infection has been suggested to be a risk factor for gastric carcinogenesis . However, those previous studies have been concerned with advanced cancer cases . To the authors' knowledge, no detailed investigation on the prevalence of H pylori in early stage gastric cancer tissue has been performed . The relationship between early stage gastric cancer and the prevalence of H pylori was studied by a immunohistochemical staining analysis . METHODS . Sixty-eight patients who were endoscopically and surgically diagnosed as having early stage gastric cancer were enrolled in this study . All tissue specimens were obtained from patients by endoscopic biopsy, and were classified histopathologically as the intestinal-type of early stage gastric cancer in 34 patients (male-to-female ratio, 28:6; age, 64 +/- 11 years) and the diffuse-type of early stage gastric cancer (male-to-female ratio, 23:11; age, 57 +/- 14 years) in the other 34 patients . The amount of H pylori in tissue samples was graded from 0 (no characteristic bacteria) to 3 (numerous bacteria) using the fluorescent microscopic and an immunohistochemical technique . RESULTS . Twenty-nine of the 34 cases of the intestinal-type of gastric cancer had H pylori infection, as compared with 11 of the 34 cases of diffuse-type early stage gastric cancer . A significantly higher incidence (85%; P < 0.001) of H pylori infection and, thus, higher grading scores of the number of H pylori were found in the intestinal-type early stage gastric cancer . CONCLUSIONS . These findings suggest that the infection of H pylori may have a crucial relationship to the early stages of carcinogenesis of intestinal-type gastric cancer.

J Periodontal Res, 1995 May, 30(3), 172 - 80
Detection of antigenic proteins in Porphyromonas gingivalis using two-dimensional electrophoresis and western blots; Tani M et al.; Using two-dimensional (2D) electrophoresis and western blot assay, we analyzed antigenic proteins in Porphyromonas gingivalis uniquely recognized by antibodies in sera of periodontitis subjects . Proteins in the total membrane fraction of P . gingivalis 381 were resolved into at least 70 protein spots by 2D electrophoresis . In the gel stained with silver, the substance around 47 kDa protein on the acidic side (at an isoelectric point of about 4.5) was stained as a smear . Antigenic substances were characterized using purified IgGs from sera of 16 adult periodontitis (AP), 19 rapidly progressive periodontitis (RPP) and 14 periodontally healthy volunteers . Western blots demonstrated that 75 kDa protein reacted with IgGs from 75% of AP patients (p < 0.001), the antigenic substance around acidic 47 kDa protein reacted with IgGs from 81.3% of AP (p < 0.01) and 68.4% of RPP patients (p < 0.01) and the acidic 47 kDa protein reacted with 87.5% of AP (p < 0.01) and 78.9% of RPP patients (p < 0.01) . The reaction frequency was significantly different from that of the healthy volunteers . Also 51 kDa and 41 kDa proteins reacted with 47 and 43 of 49 IgG samples, respectively . The substance around acidic 47 kDa protein reacted with mouse antiserum to P . gingivalis-LPS . After treatment with pronase or heat, the antigenic reactions disappeared not only from the proteins, but also from the area around the acidic 47 kDa protein . When the fraction was digested with lipase, the antigenic reaction of the area decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Science, 1995 Apr 28, 268(5210), 563 - 6
Expression cloning of a protective Leishmania antigen; Mougneau E et al.; Parasite-specific CD4+ T cells have been shown to transfer protection against Leishmania major in susceptible BALB/c mice . An epitope-tagged expression library was used to identify the antigen recognized by a protective CD4+ T cell clone . The expression library allowed recombinant proteins made in bacteria to be captured by macrophages for presentation to T cells restricted to major histocompatibility complex class II . A conserved 36-kilodalton member of the tryptophan-aspartic acid repeat family of proteins was identified that was expressed in both stages of the parasite life cycle . A 24-kilodalton portion of this antigen protected susceptible mice when administered as a vaccine with interleukin-12 before infection.

Nature, 1995 Apr 27, 374(6525), 820 - 2
Connecting a promoter-bound protein to TBP bypasses the need for a transcriptional activation domain; Chatterjee S et al.; Biochemical analyses have suggested potential targets for transcriptional activation domains, which include several components of the RNA polymerase II machinery, as well as the chromatin template . Here we examine the mechanism of transcriptional activation in yeast cells by connecting a heterologous DNA-binding domain (LexA) to the TATA-binding protein (TBP) . LexA-TBP efficiently activates transcription from a promoter containing a LexA operator upstream of a TATA element . Activation is promoter-specific and is sensitive to mutations on the DNA-binding surface of TBP; hence it is not due to a fortuitous activation domain on TBP . Thus a promoter-bound protein lacking an activation domain can stimulate transcription if it is directly connected to TBP . This suggests that recruitment of TBP to the promoter can be a rate-limiting step for transcription in vivo, and that interactions between activation domains and factors that function after TBP recruitment can be bypassed for activation.

Nucleic Acids Res, 1995 Apr 25, 23(8), 1434 - 40
Multi-alphabet consensus algorithm for identification of low specificity protein-DNA interactions; Ulyanov AV et al.; A method for the identification and characterization of protein-DNA interactions is presented . We have developed an approach for finding unknown multiple patterns that occur imperfectly in a set of several sequences . The pattern may contain letters from the nucleotide alphabet (A, C, G and T) including ambiguous characters (A/C, A/G, A/T; A/C/G, etc.) . This method reveals weak DNA signals on an unaligned set of DNA fragments known to be functionally related and assumes no prior information on the sequences' alignment . It determines the locations of the signals from only the information intrinsic to the sequences themselves . We have applied this method to analyze the binding sites of cAMP receptor protein (CRP) . The consensus based on these data are discussed and a comparison of the consensus with the crystal structure of CAP-DNA complex is presented . We further show that in a mixture of DNA sequences, containing binding sites for two different proteins, both classes of binding sites can be discovered simultaneously by this method . The DNA sequences of nucleosome cores from chicken erythrocyte and a set of the other known nucleosomal sequences show existence of symmetrical features in nucleosome-binding DNA sequences . We also show multi-alphabet patterns that can play a role in the phasing signal on the nucleosome DNA molecule and have compared the results with existing models of nucleosome positioning.

Biochemistry, 1995 Apr 25, 34(16), 5477 - 85
Structural determinants of the ligand-binding site of the human retinoic acid receptor alpha; Lefebvre B et al.; The ligand-dependent transactivating properties of retinoic acid receptors are controlled through a complex structure at the C-terminus of these proteins, commonly referred to as the hormone binding domain . This domain is involved not only in ligand recognition but also in protein-protein interactions such as homo- and heterodimerization processes . To identify more precisely regions of the human all-trans-retinoic acid receptor alpha (hRAR alpha) that are involved in ligand binding, we constructed a series of deletion mutants of this molecule and overexpressed them in bacteria . We found that the C-terminal part of the D domain (amino acids 186-198) was necessary for ligand binding . The F domain and the 10 C-terminal amino acids of the E domain were dispensable for high-affinity binding of various natural and synthetic retinoids . A further deletion to position 403 resulted in a moderate decrease in affinity for all-trans-(ATRA) and 9-cis-retinoic acids, whereas the binding of two RAR alpha-specific ligands (Am80 and Am580) was abolished . In addition, hRAR alpha and the minimal hormone binding domain (amino acids 186-410) bound ATRA with a positive, cooperative mechanism . This behavior was not observed with CD367, a conformationally restricted synthetic retinoid . The positive cooperativity could be correlated with stable ATRA binding to RAR homodimers, whose formation was triggered by ligand . In the same conditions, only monomeric CD367-RAR alpha complexes were detected . These data indicate that ligand binding to hRAR alpha requires the presence of part of the D domain, whereas the C-terminal end of the E domain is involved in more subtle ligand recognition processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Science, 1995 Apr 21, 268(5209), 380 - 5
Mercury-199 NMR of the metal receptor site in MerR and its protein-DNA complex; Utschig LM et al.; Structural insights have been provided by mercury-199 nuclear magnetic resonance (NMR) into the metal receptor site of the MerR metalloregulatory protein alone and in a complex with the regulatory target, DNA . The one- and two-dimensional NMR data are consistent with a trigonal planar Hg-thiolate coordination environment consisting only of Cys side chains and resolve structural aspects of both metal ion recognition and the allosteric mechanism . These studies establish 199Hg NMR techniques as useful probes of the metal coordination environment of regulatory proteins, copper enzymes, and zinc transcription factor complexes as large as 50 kilodaltons.

Nature, 1995 Apr 20, 374(6524), 693 - 700
The structure of trp RNA-binding attenuation protein; Antson AA et al.; The crystal structure of the trp RNA-binding attenuation protein of Bacclius subtilis solved at 1.8 A resolution reveals a novel structural arrangement in which the eleven subunits are stabilized through eleven intersubunit beta-sheets to form a beta-wheel with a large central hole . The nature of the binding of L-tryptophan in clefts between adjacent beta-sheets in the beta-wheel suggests that this binding induces conformational changes in the flexible residues 25-33 and 49-52 . It is argued that upon binding, the messenger RNA target forms a matching circle in which eleven U/GAG repeats are bound to the surface of the protein ondecamer modified by the binding of L-tryptophan.

FEBS Lett, 1995 Apr 17, 363(1-2), 137 - 40
15-cis-beta-carotene found in the reaction center of spinach photosystem II; Bialek-Bylka GE et al.; Solvent extraction at approximately 4 degrees C in complete darkness, and subsequent analysis by high-pressure liquid chromatography (at approximately 4 degrees C) using an apparatus equipped with a two-dimensional diode-array detector, spectroscopically identified 15-cis-beta-carotene in the reaction center (RC) of spinach photosystem II (PS II) . The result revises a previous conclusion that beta-carotene bound to the PS II RC takes the all-trans configuration {Fujiwara, H., Hayashi, H., Tasumi, M., Kanaji, M., Koyama, Y . and Satoh, K . (1987) Chem . Lett . 2005-2008}, and generalizes the concept established for purple photosynthetic bacteria that 15-cis-carotenoid is naturally selected by the photosynthetic reaction centers.

Eur J Biochem, 1995 Apr 15, 229(2), 517 - 25
The interaction between human papillomavirus type 16 E1 and E2 proteins is blocked by an antibody to the N-terminal region of E2; Hibma MH et al.; Replication of papillomavirus DNA requires two virally encoded proteins, E1 and E2 . We expressed human papillomavirus (HPV) type 16 E1 and E2 in bacteria and showed that purified full-length E2 protein interacted directly with E1, in the absence of HPV16 DNA . It was established that the first 142 amino acids of E1 were not required for binding as E2 protein was able to interact with E1 devoid of this region . The interaction of E2 with E1 could be blocked by a monoclonal antibody that bound E2 in the region of amino acids 18-41 of E2 whereas a monoclonal antibody reactive with a nearby part of the molecule (amino acids 2-17) only partially blocked this interaction . These results suggest that a region in the N-terminus of E2 around amino acids 18-41 is a site of interaction with the E1 protein.

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