Membrane Topology and Functional Analysis of Methylobacillus sp. 12S Genes epsF and epsG,Encoding Polysaccharide Chain-Length Determining Proteins

The EpsF and EpsG of the methanol-assimilating bacterium Methylobacillus sp. 12S are involved in the synthesis of a high molecular weight exopolysaccharide, methanolan. These proteins share homology with chain-length determiners in other polysaccharide-producing bacteria. The N- and C-termini of EpsF were found to locate to the cytoplasm, and EpsF was predicted to have two transmembrane regions. EpsG showed both ATPase and autophosphorylation activities.     

Unraveling the function of glycosyltransferases in Streptococcus thermophilus Sfi6.

Streptococcus thermophilus Sfi6 produces a texturizing exopolysaccharide (EPS) consisting of a -->3)[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->3)-alpha-D-GalpNAc-(1--> 3)-beta-D-Galp-(1--> repeating unit. We previously identified and analyzed a 14.5-kb gene cluster from S. thermophilus Sfi6 consisting of 13 genes responsible for its EPS production. Within this gene cluster, we found a central region of genes (epsE, epsF, epsG, and epsI) that showed similarity to glycosyltransferases. In this study, we investigated the sugar specificity of these enzymes. EpsE catalyzes the first step in the biosynthesis of the EPS repeating unit. It exhibits phosphogalactosyltransferase activity and transfers galactose onto the lipophilic carrier. The second step is fulfilled by EpsG, which transfers an alpha-N-acetylgalactosamine onto the first beta-galactoside. The activity of EpsF was determined by characterizing the EPS produced by an S. thermophilus epsF deletion mutant. This EPS consisted of the monosaccharides Gal, Glc, and GalNAc in an approximately equimolar ratio, thus suggesting that epsF codes for the branching galactosyltransferase. epsI probably codes for the beta-1,3-glucosyltransferase, since it is the only glycosyltransferase to which no gene has been assigned and it exhibits similarity to other beta-glycosyltransferases. EpsE shows the conserved features of phosphoglycosyltransferases, whereas EpsF and EpsG exhibit the primary structure of alpha-glycosyltransferases, belonging to glycosyltransferase family 4, whose members are conserved in all major phylogenetic lineages, including the Archaea and Eukaryota.     

Exopolysaccharide Biosynthesis in Lactococcus lactis NIZO B40: Functional Analysis of the Glycosyltransferase Genes Involved in Synthesis of the Polysaccharide Backbone

We used homologous and heterologous expression of the glycosyltransferase genes of the Lactococcus lactis NIZO B40 eps gene cluster to determine the activity and substrate specificities of the encoded enzymes and established the order of assembly of the trisaccharide backbone of the exopolysaccharide repeating unit. EpsD links glucose-1-phosphate from UDP-glucose to a lipid carrier, EpsE and EpsF link glucose from UDP-glucose to lipid-linked glucose, and EpsG links galactose from UDP-galactose to lipid-linked cellobiose. Furthermore, EpsJ appeared to be involved in EPS biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier.     

The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae

The type 2 secretion system (T2SS), a multi-protein machinery that spans both the inner and the outer membranes of Gram-negative bacteria, is used for the secretion of several critically important proteins across the outer membrane. Here we report the crystal structure of the N-terminal cytoplasmic domain of EpsF, an inner membrane spanning T2SS protein from Vibrio cholerae. This domain consists of a bundle of six anti-parallel helices and adopts a fold that has not been described before. The long C-terminal helix α6 protrudes from the body of the domain and most likely continues as the first transmembrane helix of EpsF. Two N-terminal EpsF domains form a tight dimer with a conserved interface, suggesting that the observed dimer occurs in the T2SS of many bacteria. Two calcium binding sites are present in the dimer interface with ligands provided for each site by both subunits. Based on this new structure, sequence comparisons of EpsF homologs and localization studies of GFP fused with EpsF, we propose that the second cytoplasmic domain of EpsF adopts a similar fold as the first cytoplasmic domain and that full-length EpsF, and its T2SS homologs, have a three-transmembrane helix topology.     

Lactobacillus helveticus glycosyltransferases: from genes to carbohydrate synthesis

Bioactive carbohydrates are crucial in mediating essential biological processes, and their biosynthesis is an essential aspect to develop for a global view of their biological functions. Lactic acid bacteria display an array of diverse and complex carbohydrates and, therefore, are of particular interest. Here we present the identification of a novel exocellular polysaccharide structure and the corresponding gene cluster from Lactobacillus helveticus NCC2745. The development of a glycosyltransferase-specific enzymatic assay allowed the assignment of sugar specificities, which as a general approach will for the future permit a faster and more direct characterization of glycosyltransferase specificities. A model of the biosynthesis of the repeating unit is proposed. EpsE is a phosphoglucosyltransferase initiating the repeating unit biosynthesis by linking a glucose residue to a membrane-associated lipophilic acceptor. EpsF elongates the carbohydrate chain by forming an alpha(1,3)-Glcp linkage onto the first Glcp, whereas EpsG adds a backbone alpha(1,6)-Galp onto alpha-Glcp and EpsH attaches a alpha(1,6)-Glcp branch onto the first glucose residue. Finally, EpsI would add a beta(1,6)-Galp linkage onto alpha-Glcp terminating the sidechain and EpsJ would terminate the synthesis of the polysaccharides' repeating unit by forming a beta(1,3)-Galp linkage onto alpha-Galp.     

In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae

The type II secretion system is a multi-protein complex that spans the cell envelope of Gram-negative bacteria and promotes the secretion of proteins, including several virulence factors. This system is homologous to the type IV pilus biogenesis machinery and contains five proteins, EpsG-K, termed the pseudopilins that are structurally homologous to the type IV pilins. The major pseudopilin EpsG has been proposed to form a pilus-like structure in an energy-dependent process that requires the ATPase, EpsE. A key remaining question is how the membrane-bound EpsG interacts with the cytoplasmic ATPase, and if this is a direct or indirect interaction. Previous studies have established an interaction between the bitopic inner membrane protein EpsL and EpsE; therefore, in this study we used in vivo cross-linking to test the hypothesis that EpsG interacts with EpsL. Our findings suggest that EpsL may function as a scaffold to link EpsG and EpsE and thereby transduce the energy generated by ATP hydrolysis to support secretion. The recent discovery of structural homology between EpsL and a protein in the type IV pilus system implies that this interaction may be conserved and represent an important functional interaction for both the type II secretion and type IV pilus systems.     

General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae.

The general secretion pathway (GSP) of Vibrio cholerae is required for secretion of proteins including chitinase, enterotoxin, and protease through the outer membrane. In this study, we report the cloning and sequencing of a DNA fragment from V. cholerae, containing 12 open reading frames, epsC to -N, which are similar to GSP genes of Aeromonas, Erwinia, Klebsiella, Pseudomonas, and Xanthomonas spp. In addition to the two previously described genes, epsE and epsM (M. Sandkvist, V. Morales, and M. Bagdasarian, Gene 123: 81-86, 1993; L. J. Overbye, M. Sandkvist, and M. Bagdasarian, Gene 132:101-106, 1993), it is shown here that epsC, epsF, epsG, and epsL also encode proteins essential for GSP function. Mutations in the eps genes result in aberrant outer membrane protein profiles, which indicates that the GSP, or at least some of its components, is required not only for secretion of soluble proteins but also for proper outer membrane assembly. Several of the Eps proteins have been identified by use of the T7 polymerase-promoter system in Escherichia coli. One of them, a pilin-like protein, EpsG, was analyzed also in V. cholerae and found to migrate as two bands on polyacrylamide gels, suggesting that in this organism it might be processed or otherwise modified by a prepilin peptidase. We believe that TcpJ prepilin peptidase, which processes the subunit of the toxin-coregulated pilus, TcpA, is not involved in this event. This is supported by the observations that apparent processing of EpsG occurs in a tcpJ mutant of V. cholerae and that, when coexpressed in E. coli, TcpJ cannot process EpsG although the PilD peptidase from Neisseria gonorrhoeae can.     

The 1.59 Å resolution structure of the minor pseudopilin EpsH of Vibrio cholerae reveals a long flexible loop

The type II secretion complex exports folded proteins from the periplasm to the extracellular milieu. It is used by the pathogenic bacterium Vibrio cholerae to export several proteins, including its major virulence factor, cholera toxin. The pseudopilus is an essential component of the type II secretion system and likely acts as a piston to push the folded proteins across the outer membrane through the secretin pore. The pseudopilus is composed of the major pseudopilin, EpsG, and four minor pseudopilins, EpsH, EpsI, EpsJ and EpsK. We determined the x-ray crystal structure of the head domain of EpsH at 1.59 Å resolution using molecular replacement with the previously reported EpsH structure, 2qv8, as the template. Three additional N-terminal amino acids present in our construct prevent an artifactual conformation of residues 160–166, present in one of the two monomers of the 2qv8 structure. Additional crystal contacts stabilize a long flexible loop comprised of residues 104–135 that is more disordered in the 2qv8 structure but is partially observed in our structure in very different positions for the two EpsH monomers in the asymmetric unit. In one of the conformations the loop is highly extended. Modeling suggests the highly charged loop is capable of contacting EpsG and possibly secreted protein substrates, suggesting a role in specificity of pseudopilus assembly or secretion function.     

The crystal structure of a binary complex of two pseudopilins: EpsI and EpsJ from the type 2 secretion system of Vibrio vulnificus

Type II secretion systems (T2SS) translocate virulence factors from the periplasmic space of many pathogenic bacteria into the extracellular environment. The T2SS of Vibrio cholerae and related species is called the extracellular protein secretion (Eps) system that consists of a core of multiple copies of 11 different proteins. The pseudopilins, EpsG, EpsH, EpsI, EpsJ and EpsK, are five T2SS proteins that are thought to assemble into a pseudopilus, which is assumed to interact with the outer membrane pore, and may actively participate in the export of proteins. We report here biochemical evidence that the minor pseudopilins EpsI and EpsJ from Vibrio species interact directly with one another. Moreover, the 2.3 Å resolution crystal structure of a complex of EspI and EpsJ from Vibrio vulnificus represents the first atomic resolution structure of a complex of two different pseudopilin components from the T2SS. Both EpsI and EpsJ appear to be structural extremes within the family of type 4a pilin structures solved to date, with EpsI having the smallest, and EpsJ the largest, “variable pilin segment” seen thus far. A high degree of sequence conservation in the EpsI:EpsJ interface indicates that this heterodimer occurs in the T2SS of a large number of bacteria. The arrangement of EpsI and EpsJ in the heterodimer would correspond to a right-handed helical character of proteins assembled into a pseudopilus.     

Obstetrics and gynecology: AIDS and pregnancy

The Scientific Board of the California Medical Association presents the following inventory of items of progress in obstetrics and gynecology. Each item, in the judgment of a panel of knowledgeable physicians, has recently become reasonably firmly established, both as to scientific fact and important clinical significance. The items are presented in simple epitome and an authoritative reference, both to the item itself and to the subject as a whole, is generally given for those who may be unfamiliar with a particular item. The purpose is to assist busy practitioners, students, research workers, or scholars to stay abreast of these items of progress in obstetrics and gynecology that have recently achieved a substantial degree of authoritative acceptance, whether in their own field of special interest or another.The items of progress listed below were selected by the Advisory Panel to the Section on Obstetrics and Gynecology of the California Medical Association and the summaries were prepared under its direction.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

Neurosecretion and Salt and Water Balance in the Annelida and Crustacea

The possible role of the neurosecretory system in regulationof salt and water has been studied in the annelids and crustaceans.In the earthworm, Lumbricus terrestris, a brain factor influences'the salt and osmotic concentration of the blood and coelomicfluid. Removal of the brain results in the increase of waterinflux with a decrease in the salt and osmotic concentrationsof the body fluids. The decreases in salt and osmotic concentrationscan be prevented by the implantation of the brain or the injectionof brain homogenates. In the freshwater crayfish, Procambarus clarkii, a factor, presumablysecreted in the brain and released in the eyestalk, seems tomaintain the normal permeability of the body surfaces to water.Eyestalk removal, which eliminates the release site, resultsin the increased influx of water with a decrease in the saltconcentration of the blood. A brain factor also seems to beinvolved in maintaining the sodium and osmotic concentrationsof the blood. In the semi-terrestrial grapsid crab, Metopograpsus messor,the thoracic ganglion, under the control of an eyestalk element,secretes a factor involved in increasing the permeability ofthe body surfaces to water. The removal of the eyestalks, theimplantation of the thoracic ganglion, or the injection of extractsof thoracic ganglia, results in changes in the osmotic concentrationof the blood tending toward that of the medium. In all threespecies studied, the neuroendocrine factors seem to be involvedprimarily in the regulation of the permeability of the bodysurfaces to water.     

Pleasure and disgust: memories and associations of pleasant and unpleasant odours in Germany and Japan

The question of whether odour experiences have a function inthe cultures of the technically industrialized world was investigatedby interviews with German (166) and Japanese (88) subjects.They were asked to name pleasant and unpleasant odours frommemory and to give associations regarding them. The participantsenumerated 2040 odours and 3520 associations. Although Japanesesubjects in general gave less information than the Germans,the results for both cultures were very similar in quality aswell as quantity. The odour memories included the whole physicaland social environment and the associations showed the far-reachingeffect of odour experiences. Both pleasant and unpleasant odourswere remembered to an equal extent. Most of the odours recalledwere assessed similarly by the subjects, as either pleasantor unpleasant. Obviously, individual likings or aversions aredominated by underlying evaluations common to all subjects ofboth cultures. Uniformly judged odours were, for example, odoursof plants (pleasant) and odours of rotten and decomposed things(unpleasant). It is discussed whether this finding reflectspreprogrammed survival strategies. The few cultural differenceswhich were found seemed to reflect different life habits, normsand values in the two countries.     

Teleeducation and telepathology for open and distance education.

Our experience in creating and using telepathology system and multimedia database for education is described. This program packet currently works in the Department of Pathology of University Medical School in Poznan. It is used for self-education, tests, services and for the examinations in pathology, i.e., for dental students and for medical students in terms of self-education and individual examination services. The system is implemented on microcomputers compatible with IBM PC and works in the network system Netware 5.1. Some modules are available through the Internet. The program packet described here accomplishes the TELEMIC system for telepathology, ASSISTANT, which is the administrator for the databases, and EXAMINATOR, which is the executive program. The realization of multi-user module allows students to work on several working areas, on random be chosen different sets of problems contemporary. The possibility to work in the exercise mode will image files and questions is an attractive way for self-education. The standard format of the notation files enables to elaborate the results by commercial statistic packets in order to estimate the scale of answers and to find correlation between the obtained results. The method of multi-criterion grading excludes unlimited mutual compensation of the criteria, differentiates the importance of particular courses and introduces the quality criteria. The packet is part of the integrated management information system of the department of pathology. Applications for other telepathological systems are presented.     

Follicular dynamics and concentrations of steroids and gonadotropins in lactating cows and nulliparous heifers

Differences in follicular development and circulating hormone concentrations, between lactating cows and nulliparous heifers, that may relate to differences in fertility between the groups, were examined. Multiparous, cyclic, lactating Holstein cows (n=19) and cyclic heifers (n=20) were examined in the winter, during one estrous cycle. The examinations included ultrasound monitoring and daily blood sampling. Distributions of two-wave and three-wave cycles were similar in the two groups: 79 and 21% in cows, 70 and 30% in heifers, respectively. Cycle lengths were shorter by 2.6 days in heifers than in cows, and in two-wave than in three-wave cycles. The ovulatory follicle was smaller in heifers than in cows (13.0+/-0.3 mm versus 16.5+/-0.05 mm). The greater numbers of large follicles in cows than in heifers corresponded well to the higher concentrations of FSH in cows. The duration of dominance of the ovulatory follicle tended to be longer in cows than in heifers. Estradiol concentrations around estrus and the preovulatory LH surge were higher in heifers than in cows (20 versus 9 ng/ml). Progesterone concentrations were higher in heifers than in cows from Day 3 to Day 16 of the cycle. Circulating progesterone did not differ between two-wave and three-wave cycles. The results revealed differences in ovarian follicular dynamics, and in plasma concentrations of steroids and gonadotropins; these may account for the differences in fertility between nulliparous heifers and multiparous lactating cows.     

Azaperone and azaperone-ketamine as a neuroleptic sedative and anesthetic in rats and mice

Azaperone alone and combined with ketamine were evaluated as sedative and anesthetic agents in outbred rats and mice. Using azaperone alone the duration of immobility was 1.9 to 10.8 hours for mice and 0.9 to 2.4 hours for rats. The withdrawal reflex was not eliminated from mice receiving azaperone alone; however, the withdrawal reflex was eliminated from 0.9 to 2.4 hours in rats receiving azaperone. Azaperone produced a tachypnea in rats and male mice while a depressed respiratory rate was observed in female mice. Using azaperone combined with ketamine, the duration of immobilization was 1.1 to 8.8 hours for mice and 1.3 to 6.0 hours for rats. The duration loss of the withdrawal reflex, which was used as an indication of surgical anesthesia, was 0.9 to 1.8 hours for mice and 1.0 to 6.0 hours for rats. An increase in respiratory rate was observed in rats given the combination while mice given the combination showed transient tachypnea followed by bradypnea. Overall, azaperone alone was shown to provide sedation in mice as compared to a dose dependent anesthesia in rats. The azaperone-ketamine combination produced a surgical plane of anesthesia in both rats and mice. Azaperone and the azaperone-ketamine combination appear to be a suitable alternative to sedatives and anesthetics currently used in rats and mice.     

Selection, subdivision and extinction and recolonization

In a subdivided population, the interaction between natural selection and stochastic change in allele frequency is affected by the occurrence of local extinction and subsequent recolonization. The relative importance of selection can be diminished by this additional source of stochastic change in allele frequency. Results are presented for subdivided populations with extinction and recolonization where there is more than one founding allele after extinction, where these may tend to come from the same source deme, where the number of founding alleles is variable or the founders make unequal contributions, and where there is dominance for fitness or local frequency dependence. The behavior of a selected allele in a subdivided population is in all these situations approximately the same as that of an allele with different selection parameters in an unstructured population with a different size. The magnitude of the quantity N(e)s(e), which determines fixation probability in the case of genic selection, is always decreased by extinction and recolonization, so that deleterious alleles are more likely to fix and advantageous alleles less likely to do so. The importance of dominance or frequency dependence is also altered by extinction and recolonization. Computer simulations confirm that the theoretical predictions of both fixation probabilities and mean times to fixation are good approximations.     

Physical Anthropology and the dental and medical specialties

The rise of two sub-specialties in Physical Anthropology traces back to the Anatomy Departments of Schools of Medicine in Germany and France during the nineteenth century. The study of human diversity in bones and bodies was largely by medically-trained anatomists. There developed Medical Anthropology and Dental Anthropology, employing osteometry and craniometry on the skeleton, somatometry and cephalometry on the living body. As a result cross-sectional studies gave way to longitudinal studies and X-ray techniques were added to purely mensurational procedures. In Medical Anthropology the specialties most directly concerned are pediatrics, plastic surgery, endocrinology, and orthopaedics. In Dental Anthropology the specialties most directly concerned are pedodontics, orthodontics, oral surgery, and prosthodontics. The contributions of Physical Anthropology to each is discussed.     

Infant and child mortality in Pakistan--some trends and differentials

Data from reproductive histories collected in the Population, Labor Force and Migration Survey (PLM) of 1979 are used to analyze trends and differentials in infant and child mortality in Pakistan. Comparisons with the Pakistan Fertility Survey (PFS) findings are also presented. The main concern is to provide from the latest national data, the PLM, direct measures of infant and child mortality and to demonstrate the relatively static and low chances of survival for children in Pakistan. The apparent trends from the PLM and the PFS are similar and seem to confirm that infant and childhood mortality has ceased to decline, at least rapidly, since 1965-69. Neonatal mortality is higher at levels of 70-85 deaths/1000 compared to postneonatal mortality of 40-60 deaths/1000. Improvements in neonatal rates from 1950 until 1975 are only approximately 1/2 of those for postneonatal rates for that period. The relationship between maternal age and mortality in the PLM data confirms that children of youngest mothers experienced the highest rates of infant mortality; mortality is again higher for children of oldest mothers aged 35 and above. The pattern of mortality in the 2 surveys is similar except that in the PFS there was little variation among births higher than 5th order. Sex differentials in mortality are very clear in both surveys. Boys have higher chances of dying in the 1st month of life but then the probability of their surviving from age 1 to 5 years is higher, reflecting the behavioral preference for the male sex in this society. The data also demonstrate an almost monotonic decline in infant and child mortality associated with longer birth intervals. Childhood mortality shows a less clear association with preceding birth interval than does infant mortality. While neonatal mortality is much higher in rural than in urban areas, there are negligible differences in the postneonatal rate. The urban-rural differential continues into childhood, reflecting lower health care and nutrition of children in rural areas. The data confirm the importance of parental education, particularly that of mothers, as a contributor to the health and mortality of infants. Mortality between age 1 and 5 years for children of the rural educated group is lower than that for the urban uneducated indicating the strong influence that education of mothers can have in preventing child loss. The combined evidence from the PFS and PLM data stresses the importance of improving health facilities in the rural areas, in aneffort to reduce the differences in mortality by area of residence. The data from both surveys also suggest the need to restrict motherhood to between the ages of 20 and 34, when obstetrical and health risks are minimal, and indicate the definite advantages of increasing the spacing between children.