Biomedical subjects

D B Wilson

Publications and source records attributed to D B Wilson.

At least 91 records · Page 5Linked to original sources

PubMed1997

Kinetics of Cellulose Digestion by Fibrobacter succinogenes S85.

Growing cultures of Fibrobacter succinogenes S85 digested cellulose at a rapid rate, but nongrowing cells and cell extracts did not have detectable crystalline cellulase activity. Cells that had been growing exponentially on cellobiose initiated cellulose digestion and succinate production immediately, and cellulose-dependent succinate production could be used as an index of enzyme activity against crystalline cellulose. Cells incubated with cellulose never produced detectable cellobiose, and cells that were preincubated for a short time with thiocellobiose lost their ability to digest cellulose (competitive inhibition [K(infi)] of only 0.2 mg/ml or 0.56 mM). Based on these results, the crystalline cellulases of F. succinogenes were very sensitive to feedback inhibition. Different cellulose sources bound different amounts of Congo red, and the binding capacity was HCl-regenerated cellulose > ball-milled cellulose > Sigmacel > Avicel > filter paper. Congo red binding capacity was highly correlated with the maximum rates of metabolism of cellulose digestion and inversely related to K(infm). Congo red (250 (mu)g/ml) did not inhibit the growth of F. succinogenes S85 on cellobiose, but this concentration of Congo red inhibited the rate of ball-milled cellulose digestion. A Lineweaver-Burk plot of ball-milled cellulose digestion rate versus the amount of cellulose indicated that Congo red was a competitive inhibitor of cellulose digestion (K(infi) was 250 (mu)g/ml).

Journal Article↗

PubMed1997

Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg(2+)-contaminated environments.

Escherichia coli strains were genetically engineered to express an Hg2+ transport system and metallothionein. Overexpression of a glutathione S-transferase fusion protein of Saccharomyces cerevisiae or pea metallothionein significantly increased the bioaccumulation of Hg2+ transported by MerT and MerP and protected the cells from the accumulated Hg2+. The recombinant strains have excellent properties for bioremediation of Hg(2+)-contaminated environments.

Bacterial Proteins↗

PubMed1997

Expression and hormonal regulation of transcription factors GATA-4 and GATA-6 in the mouse ovary.

Two members of the GATA-binding family of transcription factors, GATA-4 and GATA-6, are expressed in the vertebrate ovary. To gain insight into the role of these factors in ovarian cell differentiation and function, we used in situ hybridization to determine the patterns of expression of GATA-4 and GATA-6 in mouse ovary during development and in response to hormonal stimulation. GATA-4 messenger RNA (mRNA) was first evident in the ovary around the time of birth. In the adult ovary, abundant GATA-4 mRNA was detected in granulosa cells of primary and antral follicles, with lesser amounts of GATA-4 message detected in theca cells, germinal epithelium, and interstitial cells. Little or no GATA-4 mRNA was found in corpus luteum. GATA-6 message exhibited a different distribution in the ovary, with abundant expression evident in both granulosa cells and corpora lutea. Stimulation of 3-week-old females with PMSG or estrogen enhanced follicular expression of GATA-4 and GATA-6 transcripts. Subsequent induction of ovulation with human CG resulted in a decrease in GATA-4 mRNA expression in granulosa cells, whereas GATA-6 mRNA expression persisted in granulosa cells after ovulation and in corpora lutea. Moreover, follicular apoptosis was associated with a decrease in the expression of GATA-4 but not GATA-6 message. Stimulation of cultured gonadal cell lines with FSH resulted in increased expression of GATA-4 message, whereas GATA-6 mRNA expression was not affected. In light of these findings, the established role of other GATA-binding proteins in hematopoetic cell differentiation and apoptosis, and the presence of conserved GATA motifs in the promoters of genes expressed selectively in ovary, we propose that GATA-4 and GATA-6 play distinct roles in follicular development and luteinization.

Animals↗

PubMed1997

Cardiomyocyte differentiation by GATA-4-deficient embryonic stem cells.

In situ hybridization studies, promoter analyses and antisense RNA experiments have implicated transcription factor GATA-4 in the regulation of cardiomyocyte differentiation. In this study, we utilized Gata4-/- embryonic stem (ES) cells to determine whether this transcription factor is essential for cardiomyocyte lineage commitment. First, we assessed the ability of Gata4-/- ES cells form cardiomyocytes during in vitro differentiation of embryoid bodies. Contracting cardiomyocytes were seen in both wild-type and Gata4-/- embryoid bodies, although cardiomyocytes were observed more often in wild type than in mutant embryoid bodies. Electron microscopy of cardiomyocytes in the Gata4-/- embryoid bodies revealed the presence of sarcomeres and junctional complexes, while immunofluorescence confirmed the presence of cardiac myosin. To assess the capacity of Gata4-/- ES cells to differentiate into cardiomyocytes in vivo, we prepared and analyzed chimeric mice. Gata4-/- ES cells were injected into 8-cell-stage embryos derived from ROSA26 mice, a transgenic line that expresses beta-galactosidase in all cell types. Chimeric embryos were stained with X-gal to discriminate ES cell- and host-derived tissue. Gata4-/- ES cells contributed to endocardium, myocardium and epicardium. In situ hybridization showed that myocardium derived from Gata4-/- ES cells expressed several cardiac-specific transcripts, including cardiac alpha-myosin heavy chain, troponin C, myosin light chain-2v, Nkx-2.5/Csx, dHAND, eHAND and GATA-6. Taken together these results indicate that GATA-4 is not essential for terminal differentiation of cardiomyocytes and suggest that additional GATA-binding proteins known to be in cardiac tissue, such as GATA-5 or GATA-6, may compensate for a lack of GATA-4.

Animals↗

PubMed1997

T-cell receptor peptides as immunotherapy for autoimmune disease.

The observations in both mouse and rat models of experimental allergic encephalomyelitis (EAE) demonstrating restricted T-cell receptor (TCR) usage among pathogenic T cells has led to the generation of a new class of therapeutic vaccines composed of TCR V region peptides. Whether a similar approach will be of use in the treatment of human autoimmune disorders is still unclear. The experiments performed in our laboratory over the past several years have focused on two aspects of TCR peptide immunoregulation, namely, (1) how to identify the critical T-cell populations involved in the pathology of autoimmune disease, and (2) how to identify biologically relevant TCR peptides--those endogenous TCR peptides presented in association with MHC molecules on the surface of pathogenic T cells that are recognized by immunoregulatory T-cell populations. Results of our recently completed clinical studies regarding TCR V beta expression among CD4+ T cells in the cerebral spinal fluid (CSF) of patients with multiple sclerosis suggests that these cells may be an appropriate T-cell population to be targeted for TCR peptide therapy. In addition, our studies on the immune response to autologous, soluble TCR heterodimers may provide a strategy for the identification of new TCR peptide candidate vaccines.

Animals↗

PubMed1996

Erythropoiesis and vasculogenesis in embryoid bodies lacking visceral yolk sac endoderm.

During mouse embryogenesis the first hematopoietic and endothelial cells form in blood islands located between layers of visceral endoderm and mesoderm in the yolk sac. The role of visceral endoderm in primitive hematopoiesis and vasculogenesis is not well understood. We have assessed the consequences of a lack of visceral endoderm on blood cell and vessel formation using embryoid bodies derived from mouse embryonic stem (ES) cells deficient in GATA-4, a transcription factor expressed in yolk sac endoderm. When differentiated in vitro, these mutant embryoid bodies do not develop an external visceral endoderm layer. We found that Gata4-/- embryoid bodies, grown either in suspension culture or attached to a substratum, are defective in primitive hematopoiesis and vasculogenesis as evidenced by a lack of recognizable blood islands and vascular channels and a reduction in the expression of the primitive erythrocyte marker epsilon y-globin. Expression of the endothelial cell transcripts FIk-1, FIt-1, and platelet-endothelial cell adhesion molecule (PECAM) was not affected in the mutant embryoid bodies. Gata4-/- ES cells retained the capacity to differentiate into primitive erythroblasts and endothelial cells when cultured in methylcellulose or matrigel. Analysis of chimeric mice, generated by injecting Gata4-/- ES cells into 8-cell stage embryos of ROSA26 transgenic animals, showed that Gata4-/- ES cells can form blood islands and vessels when juxtaposed to visceral endoderm in vivo. We conclude that the visceral endoderm is not essential for the differentiation of primitive erythrocytes or endothelial cells, but this cell layer plays an important role in the formation and organization of yolk sac blood islands and vessels.

Animals↗

PubMed1996

Analysis of T cell receptor beta chains in the rat: I. Allelic polymorphism of V beta 8.2 is not a predisposing genetic factor in susceptibility to experimental allergic encephalomyelitis.

Previous studies of experimental allergic encephalomyelitis (EAE) in the LER rat have suggested that amino acid differences present in the LER TCR V beta 8.2 chain may be associated with disease resistance. We report here that LEW rats bred to express a V beta 8.2 gene from DA rats, identical to that found in LER, are susceptible to EAE induction. Furthermore, T cells infiltrating the spinal cord of diseased animals primarily utilized V beta 8.2 and the associated AspSer CDR3 motif, typical of TCR V beta 8.2 chains expressed by pathogenic, anti-MBP responsive T cells in the LEW rat.

Alleles↗

PubMed1996

The gene for transcription factor GATA-6 resides on mouse chromosome 18 and is expressed in myocardium and vascular smooth muscle.

We report the mapping and developmental expression pattern of the gene encoding mouse GATA-6, a member of a family of transcription factors involved in tissue-specific gene expression. Using backcross analysis, the Gata6 gene was localized to mouse chromosome 18, linked to the gene encoding transthyretin. RNase protection analysis showed that Gata6 is abundantly expressed in the heart, stomach, intestine, and ovaries of the adult mouse. The developmental expression patterns of Gata6 and the closely related gene Gata4 were directly compared using in situ hybridization. Both genes were found to be highly expressed in the myocardium, stomach epithelium, and small intestinal epithelium throughout mouse development. Of the two genes, however, only Gata6 was expressed in vascular smooth muscle. The overlapping distributions of GATA-4 and GATA-6 transcripts in the heart support the possibility of functional redundancy or interplay between these two transcription factors in this tissue. The presence of GATA-6 mRNA in vascular smooth muscle suggests that this transcription factor may play a distinctive role in gene expression in this cell type.

Animals↗

PubMed1996

Identification of two functionally different classes of exocellulases.

There are two classes of synergism in cellulase mixtures: synergism between endocellulases and exocellulases, and synergism between certain exocellulases. Exocellulases have been defined traditionally as releasing cellobiose from the nonreducing ends of cellulose, but this definition is inadequate to explain exo/exo synergism. Several recent reports indicate that some exocellulases are capable of hydrolyzing cellulose from the reducing end. The existence of two exocellulase classes with different specificities could provide an explanation for exo/exo synergism. In this paper, we report the substrate specificity of three Thermomonospora fusca (E3, E4, and E6) and two Trichoderma reesei (CBH I and CBH II) exocellulases on labeled cellooligosaccharides. We describe a new nonradioactive technique for determining substrate specificity, in which ion-spray mass spectrometry was used to analyze the products of enzymatic digests of cellopentaose labeled with 18O at the reducing end. Exocellulase reactivity was also investigated on cellopentaose labeled at the nonreducing end with 14C, and cellooligosaccharides reduced with NaBH4. The distribution of label in the reaction products supports the existence of two functional classes of exocellulases. One class (containing CBH I, E4, and E6) preferentially cleaves cellooligosaccharides from the reducing end, while the other (containing E3 and CBH II) preferentially cleaves from the nonreducing end. This classification of exocellulases is consistent with exo/exo synergism experiments, and with published cellulase crystallographic data.

Actinomycetales↗

PubMed1996

Analysis of hindbrain neural crest migration in the long-tailed monkey (Macaca fascicularis).

Neural crest cells make a substantial contribution to normal craniofacial development. Despite advances made in identifying migrating neural crest cells in avian embryos and, more recently, rodent embryos, knowledge of crest cell migration in primates has been limited to what was obtained by conventional morphological techniques. In order to determine the degree to which the nonhuman primate fits the mammalian pattern, we studied the features of putative neural crest cell migration in the hindbrain of the long-tailed monkey (Macaca fascicularis) embryo. Cranial crest cells were identified on the basis of reported distributional and morphological criteria as well as by immunocytochemical detection of the neural cell adhesion molecule (N-CAM) that labels a subpopulation of these cells. The persistent labeling of a sufficient number of crest cells with antibodies to N-CAM following their exit from the rostral, pre-otic and post-otic regions of the hindbrain facilitated tracking them along subectodermal pathways to their respective destinations in the first, second and third pharyngeal arches. Peroxidase immunocytochemistry was also employed to localize laminin and collagen-IV in neuroepithelial basement membranes. At stage 10 (8-11 somites), crest emigration occurred in areas of unfused neural folds through focal disruptions in the neuroepithelial basement membrane in both the rostral and pre-otic regions, although there was little evidence of crest migration in the post-otic hindbrain. By stage 11 (16-17 somites), the neural folds were fused (pre- and post-otic hindbrain) or in the process of fusing (rostral hindbrain), yet crest cell emigration was apparent in all three areas through discontinuities in the basement membrane. Emigration was essentially complete at stage 12 (21 somites) as indicated by nearly continuous cranial neural tube basement membranes. At this stage the pre-ganglia (trigeminal, facioacoustic and glossopharyngeal) were consistently stained with N-CAM. The current study has provided new information on mammalian neural crest in a well-established experimental model for normal and abnormal human development, including its use as a model for the retinoic acid syndrome. In this regard, the current results provide the basis for probing the mechanisms of retinoid embryopathy which may involve perturbation of hindbrain neural crest development.

Age Factors↗

PubMed1996

Site-directed mutagenesis of monofunctional chorismate mutase engineered from the E. coli P-protein.

Analysis of the active-site residues of a fully functional chorismate mutase representing the N-terminal 113 amino acids of the Escherichia coli P-protein suggests that Lys39 and Gln88 play critical roles in catalyzing the rearrangement of chorismate to prephenate. Five site-directed mutants at these positions have been constructed in which Lys39 was replaced with Arg, Asn, and Gln, and Gln88 was replaced with Arg and Glu. Although the Gln88Arg plasmid failed to produce detectable cross-reacting proteins in E. coli, the other four plasmids were expressed, and the mutant proteins purified to homogeneity. Their structures were similar to wild type enzyme, as indicated by circular dichroism spectra, with Lys39Gln showing a small deviation. In accordance with predictions, all mutations result in major loss of catalytic activity at pH 7.8. However, activity of the Gln88Glu mutant at pH 4.5 exceeded wild-type EcCM. Implications for the mechanism of mutase catalysis are discussed.

Bacterial Proteins↗

PubMed1996

Identification of a novel Drosophila opsin reveals specific patterning of the R7 and R8 photoreceptor cells.

The function of the compound eye is dependent upon a developmental program that specifies different cell fates and directs the expression of spectrally distinct opsins in different photoreceptor cells. Rh5 is a novel Drosophila opsin gene that encodes a biologically active visual pigment that is expressed in a subset of R8 photoreceptor cells. Rh5 expression in the R8 cell of an individual ommatidium is strictly coordinated with the expression of Rh3, in the overlying R7 cell. In sevenless mutant files, which lack R7 photoreceptor cells, the expression of the Rh5 protein in R8 cells is disrupted, providing evidence for a specific developmental signal between the R7 and R8 cells that is responsible for the paired expression of opsin genes.

Animals↗

PubMed1996

Hodgkin disease after renal transplantation in childhood.

PURPOSE: Hodgkin disease (HD) has rarely been described in organ transplant recipients. We herewith report two cases of Epstein Barr virus (EBV)-associated HD that occurred in renal transplant recipients during childhood. PATIENTS: A 19-year-old who had a renal transplant at 12 years of age developed mixed cellularity HD 1 year following diagnosis of an EBV-associated lymphoproliferative disorder. The second case is an 18-year-old man who developed nodular sclerosing HD 7 years after renal transplantation. RESULTS: EBV latent membrane protein-1 antigen was strongly expressed in the Hodgkin Reed-Sternberg cells in both cases. These patients achieved complete remission with standard chemotherapy without unusual toxicity. CONCLUSIONS: Renal transplant recipients on immunosuppressive therapy can develop Hodgkin disease. A review of literature on this subject with emphasis on recent molecular studies on EBV-associated cases and observed increase in incidence of HD in HIV seropositive individuals is presented.

Adolescent↗

PubMed1996

Use of a modified Bacteroides-Prevotella shuttle vector to transfer a reconstructed beta-1,4-D-endoglucanase gene into Bacteroides uniformis and Prevotella ruminicola B(1)4.

A carboxymethyl cellulase (CMCase) gene from Prevotella ruminicola B(1)4 was reconstructed by adding a cellulose binding domain from a Thermomonospora fusca cellulase and was conjugally transferred from Escherichia coli to Bacteroides uniformis 0061 by using a chloramphenicol and tetracycline resistance shuttle vector (pTC-COW). pTC-COW was specifically constructed to facilitate conjugal transfer of vectors from B. uniformis donors to P. ruminicola recipients. B. uniformis transconjugants containing CMCase constructs cloned into pTC-COW expressed Cmr, but they did not produce the reconstructed CMCase until a xylanase promoter from P. ruminicola 23 was added upstream of the CMCase (pTC-XRCMC). The xylanase promoter allowed the B. uniformis transconjugants to produce large amounts of the reconstructed CMCase, which was present on the outside surface of the cells. Although the reconstructed CMCase alone did not allow B. uniformis to grow on acid-swollen cellulose, rapid growth was observed when two exocellulases were added to the culture supernatant. Under these conditions, the reconstructed CMCase permitted faster growth than the wild-type CMCase. The frequency of transfer of pTC-XRCMC from B. uniformis to P. ruminicola B(1)4 was increased 100-fold when strictly anaerobic conditions, nitrocelluose filters (cell immobilization), and more stringent selections were employed. Although the P. ruminicola B(1)4 (pTC-XRCMC) transconjugates expressed Tcr and had DNA that hybridized with a probe to the shuttle vector, these transconjugants did not produce detectable levels of the reconstructed CMCase even when xylan was the carbon source. On the basis of these results, it appears that not all of the promoters recognized by B. uniformis and P. ruminicola 23 are functional in P. ruminicola B(1)4. However, the results with B. uniformis suggest that the introduction of a P. ruminicola B(1)4 promoter should allow expression of the reconstructed CMCase in P. ruminicola B(1)4.

Anaerobiosis↗

PubMed1996

Cloning, sequencing, and expression of a Thermomonospora fusca protease gene in Streptomyces lividans.

The major Thermomonospora fusca YX extracellular protease gene (tfpA) was cloned into Escherichia coli and Streptomyces lividans and was sequenced. The open reading frame encoded 375 residues, including a 31-residue potential signal sequence, an N-terminal prosequence containing 150 residues, and the 194-residue mature protease that belongs to the chymotrypsin family. The protease was secreted by S. lividans, but evidence suggested that it was bound to an extracellular protease inhibitor. An inhibitor-deficient mutant was selected to produce protease for purification.

Actinomycetales↗

PubMed1996

Transcription factors: regulators of gene expression in normal and pathological states.

Initiation of transcription is the major control point for gene expression. The protein factors regulating transcription, the transcription factors, are modular proteins with distinct domains responsible for different functions including DNA binding, ligand binding, nuclear localization, and protein-protein interactions. Abnormal transcription has been associated with various human diseases from neoplasia to birth defects. These can result from chromosomal translocation or transcription factor genes (e.g. Burkitt's lymphoma) or by inappropriate activation of these factors due to synthesis of fusion genes (e.g. BCR-ABL fusion gene in Philadelphia positive leukaemia). More recently, abnormalities in transcription factor genes have been linked to such human birth defects as Waardenburg syndrome, vesicoureteral reflux and craniosynostosis.

Congenital Abnormalities↗

PubMed1996

Why are ruminal cellulolytic bacteria unable to digest cellulose at low pH?

Ruminant animals depend on cellulolytic ruminal bacteria to digest cellulose, but these bacteria cannot resist the low ruminal pH that modern feeding practices can create. Because the cellulolytic bacteria cannot grow on cellobiose at low pH, pH sensitivity is a general aspect of growth and not just a limitation of the cellulases per se. Acid-resistant ruminal bacteria have evolved the capacity to let their intracellular pH decrease, maintain a small pH gradient across the cell membrane, and prevent an intracellular accumulation of VFA anions. Cellulolytic bacteria cannot grow with a low intracellular pH, and an increase in pH gradient leads to anion toxicity. Prevotella ruminicola cannot digest native cellulose, but it grows at low pH and degrades the cellulose derivative, carboxymethylcellulose. The Prevotella ruminicola carboxymethylcellulase cannot bind to cellulose, but a recombinant enzyme having the Prevotella ruminicola catalytic domain and a binding domain from Thermomonspora fusca was able to bind and had cellulase activity that was at least 10-fold higher. Based on these results, gene reconstruction offers a means of converting Prevotella ruminicola into a ruminal bacterium that can digest cellulose at low pH.

Animals↗

PubMed1995

Predicting coiled coils by use of pairwise residue correlations.

A method is presented that predicts coiled-coil domains in protein sequences by using pairwise residue correlations obtained from a (two-stranded) coiled-coil database of 58,217 amino acid residues. A program called PAIRCOIL implements this method and is significantly better than existing methods at distinguishing coiled coils from alpha-helices that are not coiled coils. The database of pairwise residue correlations suggests structural features that stabilize or destabilize coiled coils.

Algorithms↗