Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.     

Species distribution and <Emphasis Type="Italic">in vitro</Emphasis> antifungal susceptibility of oral yeast isolates from Tanzanian HIV-infected patients with primary and recurrent oropharyngeal candidiasis

Background  

In Tanzania, little is known on the species distribution and antifungal susceptibility profiles of yeast isolates from HIV-infected patients with primary and recurrent oropharyngeal candidiasis.     

Functional Analysis of Glycoprotein L (gL) from Rhesus Lymphocryptovirus in Epstein-Barr Virus-Mediated Cell Fusion Indicates a Direct Role of gL in gB-Induced Membrane Fusion

Glycoprotein L (gL), which complexes with gH, is a conserved herpesvirus protein that is essential for Epstein-Barr virus (EBV) entry into host cells. The gH/gL complex has a conserved role in entry among herpesviruses, yet the mechanism is not clear. To gain a better understanding of the role of gL in EBV-mediated fusion, chimeric proteins were made using rhesus lymphocryptovirus (Rh-LCV) gL (Rh gL), which shares a high sequence homology with EBV gL but does not complement EBV gL in mediating fusion with B cells. A reduction in fusion activity was observed with chimeric gL proteins that contained the amino terminus of Rh gL, although they retained their ability to process and transport gH/gL to the cell surface. Amino acids not conserved within this region in EBV gL when compared to Rh gL were further analyzed, with the results mapping residues 54 and 94 as being functionally important for EBV-mediated fusion. All chimeras and mutants displayed levels of cell surface expression similar to that of wild-type gL and interacted with gH and gp42. Our data also suggest that the role of gL involves the activation or recruitment of gB with the gH/gL complex, as we found that reduced fusion of Rh gL, EBV/Rh-LCV chimeras, and gL point mutants could be restored by replacing EBV gB with Rh gB. These observations demonstrate a distinction between the role of gL in the processing and trafficking of gH to the cell surface and a posttrafficking role in cell-cell fusion.Epstein-Barr virus (EBV) is a member of the lymphocryptovirus (LCV) subgroup of gammaherpesviruses and primarily infects epithelial and B lymphocytes, where it establishes productive and latent infections, respectively (8, 40). EBV is extremely prevalent in humans, with more than 90% of the population being infected. Following primary infection, EBV persists in a latent state in memory B lymphocytes, where it can remain indefinitely (3, 43). The expansion and proliferation of these cells can lead to the development of a number of EBV-related malignancies, including tumors of lymphoid tissues, such as Hodgkin''s lymphoma, Burkitt''s lymphoma, and some T-cell lymphomas, as well as tumors associated with epithelial tissues, such as nasopharyngeal carcinoma and gastric carcinoma (5, 32, 36, 48). EBV is also associated with lymphoproliferative disorders in immunocompromised patients, such as oral hairy leukoplakia and posttransplant lymphoproliferative disorders (12, 15, 43, 45).Herpesvirus entry is a multistep process that includes the initial binding of the virus to the cell surface, interaction with a cellular entry receptor, membrane-virion fusion, and internalization of the virion (40). The enveloped membrane of EBV contains numerous glycoproteins that are necessary for mediating attachment and the subsequent fusion between viral and cell membranes, although the required glycoproteins differ for epithelial and B-cell infection (13, 22). Infection of B cells is initiated upon the binding of EBV glycoprotein gp350/220 to the CD21/CR2 cellular receptor expressed on target cells (9, 27, 41). Following the initial binding, viral glycoprotein gp42 binds to human leukocyte antigen class II (HLA class II) and triggers fusion, which is mediated by the concerted actions of glycoproteins gB, gH, and gL (21, 41). Glycoproteins gH and gL form a heteromeric complex that is conserved among the herpesvirus family despite a low degree of sequence homology in gH and gL across herpesviruses (33, 40). The gH/gL complex has an essential role in entry of herpesviruses, yet the exact role of each protein is not well understood. The role of gH is thought to be in cell fusion, while gL mediates processing and transport of gH to the cell surface (34, 35, 47, 51). Since gL is generally essential for the proper processing and transport of gH in all herpesviruses, it has been difficult to examine the role of gL separate from gH.While little is known about the role of gL in EBV-mediated fusion, data from studies of other herpesviruses have suggested a more direct role of gL in fusion. Antibodies specific for the C terminus of herpes simplex virus (HSV) gL, mapped to residues 168 to 224, were found to inhibit fusion with some strains of HSV type 1 (HSV-1), suggesting a role of this region in fusion (28). Deletion analysis of HSV-1 gL determined that the first 161 amino acids of gL are sufficient for binding gH, and further deletion identified the region between amino acids 155 and 161 of HSV-1 gL as critical for gH transport and cell fusion (19, 20, 33). Together, the results of these studies suggest that the inhibition of fusion observed with gL antibodies (Abs) is not due to an inhibition of gH/gL complex formation. Another study found that mutations to the amino-terminal region of HSV-2 gH permitted transport of the glycoprotein to the cell surface independent of gL but that gL was still required for fusion, suggesting a role of gL in fusion after the complex has trafficked to the plasma membrane (6). Most recently, the results of an investigation of the functional homology of the primate rhesus LCV (Rh-LCV) gL (Rh gL) suggest that gL may have a functional role in EBV fusion with B cells. Rh gL shares a high degree of sequence similarity with EBV (81.6%), and yet, the glycoprotein was unable to mediate fusion with human B cells when expressed with either EBV gH or Rh gH, even though the gH/gL complex was detected on the cell surface and Rh gL could associate with gH and gp42 (31). The LCV that infects rhesus primates is biologically similar to EBV in that it shares a similar genome organization, repertoire of lytic and latent genes, and pathogenesis (7, 25, 37, 46).To gain a better understanding of the role of gL in EBV-mediated fusion with B cells, we constructed chimeric gL proteins that included portions of Rh gL inserted into the sequence of EBV gL. We identified the amino terminus of EBV gL as being functionally necessary in mediating fusion with B cells, and this role is distinguishable from the role of gL in the processing and transport of gH to the cell surface. Single and double point mutations were constructed in both EBV and Rh gL, and our results indicated that residues 54 and 94 are functionally critical in mediating fusion with B cells. Interestingly, our studies also identified a species-specific reliance between gL and gB, suggesting a possible association between these two glycoproteins that is necessary in mediating fusion. Thus, our studies demonstrated that EBV gL has a more substantial role in mediating fusion with B cells that is distinct from its role in the processing and trafficking of gH.     

Proof-of-concept human laboratory study for protracted abstinence in alcohol dependence: effects of gabapentin

There is a need for safe medications that can effectively support recovery by treating symptoms of protracted abstinence that may precipitate relapse in alcoholics, e.g. craving and disturbances in sleep and mood. This proof-of-concept study reports on the effectiveness of gabapentin 1200 mg for attenuating these symptoms in a non-treatment-seeking sample of cue-reactive, alcohol-dependent individuals. Subjects were 33 paid volunteers with current Diagnostic and Statistical Manual of Mental Disorders-IV alcohol dependence and a strength of craving rating 1 SD or greater for alcohol than water cues. Subjects were randomly assigned to gabapentin or placebo for 1 week and then participated in a within-subjects trial where each was exposed to standardized sets of pleasant, neutral and unpleasant visual stimuli followed by alcohol or water cues. Gabapentin was associated with significantly greater reductions than placebo on several measures of subjective craving for alcohol as well as for affectively evoked craving. Gabapentin was also associated with significant improvement on several measures of sleep quality. Side effects were minimal, and gabapentin effects were not found to resemble any major classes of abused drugs. Results suggest that gabapentin may be effective for treating the protracted abstinence phase in alcohol dependence and that a randomized clinical trial would be an appropriate next step. The study also suggests the value of cue-reactivity studies as proof-of-concept screens for potential antirelapse drugs.