Biomedical subjects

W Lin

Publications and source records attributed to W Lin.

At least 163 records · Page 9Linked to original sources

PubMed1998

Replication and integration of a Vibrio cholerae cryptic plasmid linked to the CTX prophage.

We identified a 4.7kb cryptic plasmid in all ctxAB+ Vibrio cholerae strains we tested. An isolate of the V. cholerae classical biotype strain 0395 that harbours the cryptic plasmid at high copy number was found. Hybridization analysis demonstrated that sequences highly related or identical to this plasmid exist in all toxigenic strains of V. cholerae but were notably absent in all non-toxigenic environmental isolates that lacked the genes for toxin-co-regulated pili and the filamentous CTX prophage. Accordingly, we have named the cryptic plasmid pTLC for toxin-linked cryptic. The complete nucleotide sequence of pTLC from the high-copy-number isolate was determined. The largest open reading frame in the plasmid is predicted to encode a protein similar to the replication initiation protein (pII) of Escherichia coli F-specific filamentous phages. The nucleotide sequence of pTLC also facilitated the structural characterization of the DNA homologous to pTLC in other strains of V. cholerae. pTLC-related DNA exists in these strains as both low-copy-number, covalently closed circular DNA and tandemly duplicated, chromosomally integrated DNA. Remarkably, the chromosomally integrated form of pTLC is adjacent to the CTX prophage. The strain distribution, chromosomal location and DNA sequence of pTLC suggests that it may be a genetic element that plays some role in the biology of CTXphi, perhaps facilitating either its acquisition or its replication.

Amino Acid Sequence↗

PubMed1998

Experimental hypoxemic hypoxia: effects of variation in hematocrit on magnetic resonance T2*-weighted brain images.

T2*-weighted gradient echo magnetic resonance images of rat brain were obtained dynamically during acute hypoxemic hypoxia to investigate the relations between changes in cerebral blood oxygen saturation (deltaYb), blood hematocrit (Hct), and R2* (deltaR2*). Images from hypoxemic rats with normal Hct (42.8%+/-2.33%; n=12) were compared with those from hypoxemic rats with mild (33.4%+/-1.88%; n=8) or moderate (27.14%+/-2.7%; n=10) reduction of Hct. A linear relation between deltaYb and deltaR2* was obtained for all three groups. However, the slopes of the linear regressions were statistically different from one another (P < 0.001), with the slopes of the regression lines increasing inversely with Hct; that is, the slope for normal Hct is less than the slope for mildly reduced Hct, which is less than the slope for moderately reduced Hct. These data suggest that for any given reduction in the oxygen saturation of cerebral blood, the deltaR2* will be of a lesser magnitude when the hemoglobin concentration is reduced; the data are consistent with existing theoretical models of deoxyhemoglobin content-dependent effects in T2*-weighted magnetic resonance imaging.

Animals↗

PubMed1998

Stop-flow studies of distribution of filtration in rat lungs.

The stop-flow approach was used to investigate where filtration occurs in the pulmonary vasculature after elevation of left atrial pressure and aspiration of HCl. Rat lungs were perfused for 11 min at zero left atrial pressures, and then flow was stopped for 10 min and left atrial pressures were increased to 20 cmH2O. Thereafter, 3HOH was instilled into the air spaces, and the pulmonary vasculature was flushed by perfusing it from the pulmonary artery to left atrium (anterograde flush) or in the opposite direction (retrograde flush). Increases in fluorescein isothiocyanate (FITC)-dextran (molecular weight 2,000,000) indicated filtration, and these preceded increases in 3HOH after anterograde but not retrograde flushes. This suggests that some filtration occurred through vessels that were relatively venous compared with those through which 3HOH exchange had occurred. Filtration increased fivefold after instillation of 0.1 N HCl in isotonic saline into the air spaces before perfusion. Increases in Evans blue-labeled albumin concentrations were < 40% those of FITC-dextran, indicating loss from the vasculature, but increases in unlabeled albumin and FITC-albumin were comparable.

Animals↗

PubMed1998

Earliest steps in primary tumor formation and micrometastasis resolved with histochemical markers of gene-tagged tumor cells.

To facilitate detection of tumor cells at the highest resolution in any organ in athymic nude mouse model systems, a histochemical marker gene [bacterial lacZ or human placental alkaline phosphatase (ALP)] was transfected into specified transformed/tumor cells (fibrosarcoma or neuroblastoma). The fates of tumor cells were followed qualitatively and quantitatively by histochemical staining of whole organs or organ sections. Primary tumors developed initially via formation of "curly-haired" complexes of cells in the subcutis or dermis, followed by division of a large fraction of cells. When two tumor classes were mixed before injection, outgrowth occurred in regional concentrations of the primary tumor. Blood microvessels were detectable within 72 hr of injection, growing into tumor regions. iv injection routinely yielded multicellular foci in the lungs within minutes as precursors of experimental metastases. Micrometastasis was further resolved with cells "inactivated" by different treatments and by co-injection of two different tagged cell types. These approaches using different histochemical marker genes to "tag" different tumor cell classes, along with more advanced molecular biological approaches, permit us to characterize gene expression and its reversibility during the earliest stages of primary tumor formation and micrometastasis to virtually any organ in the recipient animal.

Animals↗

PubMed1998

Pentobarbital antagonism of morphine analgesia mediated by spinal cholecystokinin.

Pentobarbital administered intracerebroventricularly to mice has been shown previously to inhibit the analgesic action of morphine given intrathecally. The purpose of the present study was to examine the proposal that this antianalgesic action was mediated spinally by cholecystokinin. First, intrathecal coadministration of cholecystokinin-B sulfate (CCK8s) with morphine inhibited the analgesic action of morphine in the mouse tail-flick test. This rightward shift of the morphine dose-response curve was reversed by the intrathecal administration of either the CCKA receptor antagonist, lorglumide, or the CCKB receptor antagonist, PD135, 158. Second, lorglumide and PD135, 158 given intrathecally also eliminated the antianalgesic effect of intracerebroventricularly administered pentobarbital against intrathecal morphine. Third, intrathecal pretreatment with CCKB antiserum eliminated the effect of pentobarbital. Thus, the results indicated that pentobarbital antianalgesia was obtained through activation of a descending system to the spinal cord where cholecystokinin inhibited the spinal analgesic action of morphine.

Animals↗

PubMed1998

Functional homodimeric glycoprotein hormones: implications for hormone action and evolution

Background:. Human chorionic gonadotropin (hCG), lutropin, follitropin, and thyrotropin act as alphabeta heterodimers to control reproduction and thyroid function. The alpha and beta subunits of these proteins are divided into three loops (alpha1, alpha2,alpha3; beta1,beta2,beta3) by cysteine knots and the heterodimer is stabilized by 20 beta-subunit residues wrapped around alpha2 like a seatbelt. Understanding how these hormones interact with their receptors, a matter of considerable dispute, would facilitate design of pro- and anti-fertility agents. Results:. By swapping alpha2 for beta2 and vice versa and, in some cases, adding an amino-terminal coiled-coil dimerization domain, we prepared homodimeric analogs that have the conformation found in each 'half' of hCG. Homodimers containing loops beta1,alpha2,beta3 and none, part, or all of the seatbelt stimulated signal transduction to the same extent as hCG, albeit with lower potency. Those containing alpha1,beta2,alpha3 were inactive. Conclusions:. The activities of homodimers containing the beta1,alpha2,beta3 groove exceed those of other minimized analogs more than 100-1000-fold, suggesting this portion of the hormone forms the major receptor contact. The discovery that glycoprotein hormone heterodimers can be converted to functional homodimers supports the proposal that this protein family evolved from an active homodimeric ancestor by gene duplication and acquisition of mutations to loop 2 that prevent homodimerization. This approach to protein minimization should be applicable to other proteins composed of architecturally related subunits, including those that might have arisen by gene duplication.

Journal Article↗

PubMed1998

Functional homodimeric glycoprotein hormones: implications for hormone action and evolution.

BACKGROUND: Human chorionic gonadotropin (hCG), lutropin, follitropin, and thyrotropin act as alpha beta heterodimers to control reproduction and thyroid function. The alpha and beta subunits of these proteins are divided into three loops (alpha 1,alpha 2,alpha 3; beta 1,beta 2,beta 3) by cysteine knots and the heterodimer is stabilized by 20 beta-subunit residues wrapped around alpha 2 like a seatbelt. Understanding how these hormones interact with their receptors, a matter of considerable dispute, would facilitate design of pro- and anti-fertility agents. RESULTS: By swapping alpha 2 for beta 2 and vice versa and, in some cases, adding an amino-terminal coiled-coil dimerization domain, we prepared homodimeric analogs that have the conformation found in each 'half' of hCG. Homodimers containing loops beta 1,alpha 2,beta 3 and none, part, or all of the seatbelt stimulated signal transduction to the same extent as hCG, albeit with lower potency. Those containing alpha 1,beta 2,alpha 3 were inactive. CONCLUSIONS: The activities of homodimers containing the beta 1,alpha 2,beta 3 groove exceed those of other minimized analogs more than 100-1000-fold, suggesting this portion of the hormone forms the major receptor contact. The discovery that glycoprotein hormone heterodimers can be converted to functional homodimers supports the proposal that this protein family evolved from an active homodimeric ancestor by gene duplication and acquisition of mutations to loop 2 that prevent homodimerization. This approach to protein minimization should be applicable to other proteins composed of architecturally related subunits, including those that might have arisen by gene duplication.

Amino Acid Sequence↗

PubMed1998

Guanine nucleotides protect Rho proteins from endogenous proteolytic degradation in renal membranes.

Purified membrane fractions have been widely used for the study of the factors regulating the functions of Rho small GTP-binding proteins. Using brush border membranes from the rat kidney as a model, we observed that in vitro incubation of these membranes resulted in time- and temperature-dependent proteolytic degradation of Cdc42 and RhoA. Treatment of kidney brush border membranes with various nucleotides showed that GDP and GTP weakly protected Cdc42 but not RhoA and that their nonhydrolyzable counterparts, guanosine 5'-O-[beta-thio] diphosphate (GDP beta S) and guanosine 5'-O-[gamma-thio]triphosphate (GTP gamma S), were highly efficient in protecting both proteins from endogenous proteolytic activity whereas ADP and ATP were without effect. GTP gamma S also protected Cdc42 and RhoA from proteolytic degradation in crude cell membranes from several rat tissues including intestine, kidney, liver, and testis. In addition, Cdc42 and RhoA associated with brush border membranes were largely resistant to increased proteolytic degradation induced by membrane treatment with the denaturing reagent urea as well as to added trypsin when incubated in the presence of GTP gamma S. In brush border membranes, the resistance to endo- and exo-genous proteolytic activity conferred by GTP gamma S was usually lower for RhoA than for Cdc42. GTP gamma S also protected recombinant Cdc42 and RhoA from the action of proteases associated with brush border membranes. The only protease inhibitor protecting Cdc42 but not RhoA from proteolytic degradation in brush border membranes was the synthetic peptide acetyl-Tyr-Val-Ala-Asp-aldehyde, a selective inhibitor of interleukin-1 beta-converting enzyme. This latter result showed that different proteases cleaved the two Rho proteins. Taken together, these results suggest that the GTP gamma S-bound forms of Cdc42 and RhoA are maintained in a conformation that protects them from proteases found in many cell membranes.

Adenosine Diphosphate Ribose↗

PubMed1997

Development of membrane properties in taste cells of fungiform papillae: functional evidence for early presence of amiloride-sensitive sodium channels.

Behavioral and physiological studies have demonstrated a reduced sensitivity to several taste stimuli early in development. It has been suggested that this reduced sensitivity results from a late maturation of underlying transduction mechanisms. Little is known, however, about maturation of membrane properties of taste cells early in development. We have obtained whole-cell recordings from single fungiform taste cells of rat pups to examine the development of the NaCl transduction system. Although taste buds undergo a considerable increase in size during development, membrane capacitance measurements revealed no change in membrane surface area of individual taste cells, suggesting that the increase in size results from an increase in the total number of cells per bud. Whole-cell recordings showed that taste cells from very young pups [postnatal day 2 (PND2)] already possessed voltage-activated Na+ and K+ currents with no apparent differences in size or kinetics compared with adults. Surprisingly, amiloride-sensitive Na+ responses, important for Na+ transduction, were found as early as PND2. The magnitude of responses to amiloride and the percentage of amiloride-sensitive cells remained the same throughout all age groups. Furthermore, the similarity of amiloride inhibition constants suggested that the channel in neonates is the same channel that is expressed in adult taste buds. Our results indicate that taste cells at PND2 already have acquired the transduction elements necessary for signaling NaCl responses to the afferent nerve. We hypothesize that complete functionality of the salt taste transduction system, however, may not be reached until amiloride-sensitive Na+ channels become selectively localized at the apical membrane. This would explain previous studies indicating that amiloride sensitivity cannot be detected before PND12 in the intact tongue. Apical clustering of channels along with the opening of the taste pore and an increase in the total number of taste cells per bud likely constitute additional important steps toward a fully functional sensory system.

Amiloride↗

PubMed1997

Differential responses of extracellular GABA to intrastriatal perfusions of 3-nitropropionic acid and quinolinic acid in the rat.

Although both quinolinic acid and 3-nitropropionic acid destroy medium sized, GABAergic, spiny projection neurons after direct perfusion of neurotoxin into the rat striatum, changes in extracellular GABA concentration in the striatum within the first 90 min reflect different toxic mechanisms in these two animal models for Huntington's disease. Since quinolinic acid acts as a potent excitotoxin, the early depolarizing response in GABAergic neurons results in an early increase in extracellular GABA activity (peak at 40 min) whereas the more indirect action of 3-nitropropionic acid on mitochondrial energy metabolism results in a delayed increase in extracellular GABA activity (peak at 60 min) with a pattern of gradual increase and decline. The localized delivery of cytotoxin provides an opportunity for kinetic comparisons of direct and indirect cytotoxic mechanisms that can be useful in developing neuroprotective treatment strategies in Huntington's disease.

Animals↗

PubMed1997

A euryarchaeal lysyl-tRNA synthetase: resemblance to class I synthetases.

The sequencing of euryarchaeal genomes has suggested that the essential protein lysyl-transfer RNA (tRNA) synthetase (LysRS) is absent from such organisms. However, a single 62-kilodalton protein with canonical LysRS activity was purified from Methanococcus maripaludis, and the gene that encodes this protein was cloned. The predicted amino acid sequence of M. maripaludis LysRS is similar to open reading frames of unassigned function in both Methanobacterium thermoautotrophicum and Methanococcus jannaschii but is unrelated to canonical LysRS proteins reported in eubacteria, eukaryotes, and the crenarchaeote Sulfolobus solfataricus. The presence of amino acid motifs characteristic of the Rossmann dinucleotide-binding domain identifies M. maripaludis LysRS as a class I aminoacyl-tRNA synthetase, in contrast to the known examples of this enzyme, which are class II synthetases. These data question the concept that the classification of aminoacyl-tRNA synthetases does not vary throughout living systems.

Acylation↗

PubMed1997

A humanized form of a CD4-specific monoclonal antibody exhibits decreased antigenicity and prolonged plasma half-life in rhesus monkeys while retaining its unique biological and antiviral properties.

Certain monoclonal antibodies (MAbs) directed against CD4 can efficiently block HIV-1 replication in vitro. To explore CD4-directed passive immunotherapy for prevention or treatment of AIDS virus infection, we previously examined the biological activity of a nondepleting CD4-specific murine MAb, mu5A8. This MAb, specific for domain 2 of CD4, blocks HIV-1 replication at a post-gp120-CD4 binding step. When administered to normal rhesus monkeys, all CD4+ target cells were coated with antibody, yet no cell clearance or measurable immunosuppression occurred. However, strong anti-mouse Ig responses rapidly developed in all monkeys. In the present study, we report a successfully humanized form of mu5A8 (hu5A8) that retains binding to both human and monkey CD4 and anti-AIDS virus activity. When administered intravenously to normal rhesus monkeys, hu5A8 bound to all target CD4+ cells without depletion and showed a significantly longer plasma half-life than mu5A8. Nevertheless, an anti-hu5A8 response directed predominantly against V region determinants did eventually appear within 2 to 4 weeks in most animals. However, when hu5A8 was administered to rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques, anti-hu5A8 antibodies were not detected. Repeated administration of hu5A8 in these animals resulted in sustained plasma levels and CD4+ cell coating with humanized antibody for 6 weeks. These studies demonstrate the feasibility of chronic administration of CD4-specific MAb as a potential means of treating or preventing HIV-1 infection.

Amino Acid Sequence↗

PubMed1997

Isolation and characterization of the cDNA encoding bovine poly(ADP-ribose) glycohydrolase.

The synthesis and rapid turnover of ADP-ribose polymers is an immediate cellular response to DNA damage. We report here the isolation and characterization of cDNA encoding poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for polymer turnover. PARG was isolated from bovine thymus, yielding a protein of approximately 59 kDa. Based on the sequence of oligopeptides derived from the enzyme, polymerase chain reaction products and partial cDNA clones were isolated and used to construct a putative full-length cDNA. The cDNA of approximately 4.1 kilobase pairs predicted expression of a protein of approximately 111 kDa, nearly twice the size of the isolated protein. A single transcript of approximately 4. 3 kilobase pairs was detected in bovine kidney poly(A)+ RNA, consistent with expression of a protein of 111 kDa. Expression of the cDNA in Escherichia coli resulted in an enzymatically active protein of 111 kDa and an active fragment of 59 kDa. Analysis of restriction endonuclease fragments from bovine DNA by Southern hybridization indicated that PARG is encoded by a single copy gene. Taken together, the results indicate that previous reports of multiple PARGs can be explained by proteolysis of an 111-kDa enzyme. The deduced amino acid sequence of the bovine PARG shares little or no homology with other known proteins. However, it contains a putative bipartite nuclear location signal as would be predicted for a nuclear protein. The availability of cDNA clones for PARG should facilitate structure-function studies of the enzyme and its involvement in cellular responses to genomic damage.

Amino Acid Sequence↗

PubMed1997

Influence of subunit interactions on lutropin specificity. Implications for studies of glycoprotein hormone function.

Bovine lutropin (bLH) and human chorionic gonadotropin (hCG) are heterodimeric glycoprotein hormones required for reproduction. Both bind rat LH receptors (rLHRs), but hCG binds human LH receptors (hLHRs) 1000-10,000 fold better than bLH. We tested the premise that this difference in affinity could be used to identify lutropin receptor contacts. Heterodimers containing hCG/bLH alpha- or beta-subunit chimeras that bound hLHR like hCG (or bLH) were expected to have hCG (or bLH) residues at the receptor contact sites. Analogs containing one subunit derived from hCG bound hLHR much more like hCG than bLH, indicating that each bLH subunit contains all the residues sufficient for high affinity hLHR binding. Indeed, the presence of bovine alpha-subunit residues increased the activities of some hCG analogs. The low hLHR activity of bLH was due primarily to an interaction between its alpha-subunit and beta-subunit residue Leu95. Leu95 does not appear to contact the hLHR since it did not influence the hLHR activity of heterodimers containing human alpha-subunit. These observations show that interactions within and between the subunits can significantly influence the activities of lutropins, thereby confounding efforts to identify ligand residues that contact these receptors.

Amino Acid Sequence↗

PubMed1997

Brain magnetic resonance diffusion abnormalities in Creutzfeldt-Jakob disease.

BACKGROUND: Magnetic resonance imaging of the brain has been of limited usefulness in the diagnosis of Creutzfeldt-Jakob disease. Abnormalities on T2-weighted images have been described, but these are neither highly sensitive nor specific. OBJECTIVE: To determine whether diffusion-weighted magnetic resonance images might be useful in the evaluation of Creutzfeldt-Jakob disease. CASE PRESENTATION: A 61-year-old woman with rapidly progressive dementia was referred for cranial magnetic resonance imaging. Diffusion-weighted images were obtained as part of the examination. Brain biopsy confirmed the diagnosis of Creutzfeldt-Jakob disease histologically. FINDINGS AND CONCLUSIONS: The diffusion-weighted magnetic resonance brain images demonstrated bilaterally symmetrical marked increase in signal intensity in the caudate nuclei, putamina, thalami, cingulate gyri, and right inferior frontal cortex. The apparent diffusion coefficient map showed abnormally low diffusion in these regions (as low as 40% of normal in the caudate head). This suggests that there is restricted diffusion in these regions. The T2-weighted images demonstrated slightly increased signal bilaterally in the caudate nuclei and putamina. These findings indicate that diffusion magnetic resonance imaging might be a sensitive means of imaging the abnormalities seen in Creutzfeldt-Jakob disease.

Biopsy↗

PubMed1997

Immunonephelometric quantification of group-specific component protein in patients with acute liver failure.

Serum levels of group-specific component (Gc) protein are useful in evaluating the likelihood of survival in patients with acute liver failure (ALF) who may be candidates for liver transplant surgery. Most methods for quantifying Gc protein concentration are either isotopic, manual, technically demanding, and/or time consuming to perform, and thus are not well suited for routine clinical use in a hospital setting. We modified and evaluated a recently described nonisotopic, fully automated, immunonephelometric method for quantifying serum Gc protein concentration and compared it to our previous immunoblotting method. In addition, we evaluated the effect of G-actin on the immunonephelometric measurement of Gc protein. Serum samples from 20 patients with ALF and from 20 age- and sex-matched clinic patients without liver disease were quantified by both immunoblotting and immunonephelometry. We assessed the intra-assay precision, correlation, and diagnostic accuracy of these methods in discriminating between individuals with no preexisting liver disease and those with ALF. Actin in 1.3- to 4-fold excess of Gc protein levels demonstrated minimal to no interference in the quantification of Gc protein by immunonephelometry. Immunonephelometry was more precise than immunoblotting. Gc protein values by immunonephelometry were similar to those obtained by immunoblotting, and the diagnostic accuracy of Gc protein concentration by immunonephelometry was similar to that observed by immunoblotting. Immunonephelometry provides a nonisotopic, fully automated, rapid, precise, accurate, and cost-effective method for quantifying serum levels of total Gc protein that is well suited for routine use in a hospital-based clinical laboratory.

Actins↗

PubMed1997

Quantitative regional brain water measurement with magnetic resonance imaging in a focal ischemia model.

Therapeutic approaches to cerebral edema require an understanding of both the magnitude and location of changes in brain water content. It is desirable to have a sensitive, accurate means of measuring brain water noninvasively so that effective therapies for cerebral edema in stroke, head trauma, and other conditions can be investigated. In this work, a three-dimensional magnetic resonance imaging technique that is able to provide both spin density and T1 simultaneously is described. This method was used to quantitate regional changes in brain water content in a rat model of focal cerebral ischemia. Brain water contents estimated from both relative spin density and relative T1 measurements made in vivo were compared with ex vivo measurements of relative tissue water content based on the wet-dry technique. Correlation coefficients of 0.95 and 0.98 were obtained between the wet-dry measurements and magnetic resonance measurements of T1 and spin density, respectively. Notably, the slope of the relationship between T1 and tissue water content changed dramatically after the injection of a paramagnetic contrast agent while precontrast and postcontrast spin density measurements remained essentially invariant. In addition, a plot of absolute spin density (obtained by normalizing spin density from agar gelatin phantoms of different water contents to the spin density of a sample of 100% water) was linearly related to wet-dry measurements with a slope of 0.99 (R2 = 0.99).

Animals↗

PubMed1997

Quantitative measurements of regional cerebral blood volume using MRI in rats: effects of arterial carbon dioxide tension and mannitol.

A three-dimensional (3D) T1-weighted sequence was used to acquire high spatial resolution whole brain images in rats before and after the injection of an intravascular contrast agent. These T1-weighted images were used to estimate regional cerebral blood volume (rCBV) as a percentage of blood volume in each voxel. Ventilation was manipulated to investigate the effects of altered arterial carbon dioxide tension (PaCO2) on rCBV. In addition, different doses of a hypertonic mannitol solution were used to investigate the sensitivity of the proposed method in a serial monitoring paradigm. An rCBV of 2.40% +/- 0.34% was obtained before any physiological manipulation, in good agreement with literature values using alternative techniques. Using this method, it was found that there exists a linear relationship between PaCO2 and rCBV (R2 = 0.77) and that rCBV increased in a dose and time dependent fashion in mannitol-treated rats. High signal-to-noise was available due to the substantial increase in blood signal from the intravascular contrast agent.

Animals↗