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