Biomedical subjects

L Wang

Publications and source records attributed to L Wang.

At least 1,387 records · Page 77Linked to original sources

PubMed1998

New advances and validation of knowledge management tools for critical care using classifier techniques.

An earlier version (2.0) of the case-based reasoning (CBR) tool, called IDEAS for ICU's, allowed users to compare the ten closest matching cases to the newest patient admission, using a large database of intensive care patient records, and physician-selected matching-weights [1,2]. The new version incorporates matching-weights, which have been determined quantitatively. A faster CBR matching engine has also been incorporated into the new CBR. In a second approach, a back-propagation, feed-forward artificial neural network estimated two classes of the outcome "duration of artificial ventilation" for a subset of the database used for the CBR work. Weight-elimination was successfully applied to reduce the number of input variables and speed-up the estimation of outcomes. New experiments examined the impact of using a different number of input variables on the performance of the ANN, measured by correct classification rates (CCR) and the Average Squared Error (ASE).

Critical Care↗

PubMed1997

IL-11 regulates macrophage effector function through the inhibition of nuclear factor-kappaB.

Recombinant human IL-11 (rhIL-11) is an anti-inflammatory cytokine that can reduce the production of inflammatory mediators such as TNF-alpha, IL-1beta, IL-12, IL-6, and nitric oxide. Inhibition of proinflammatory cytokine production from activated macrophages was associated with a reduction in the levels of LPS-induced TNF-alpha, IL-1beta, IL-6, and IL-12 p40 mRNA. Analysis of rhIL-11 effects on transcription factors that activate proinflammatory cytokines demonstrated that the level of LPS-induced NF-kappaB binding activity in the nucleus of rhIL-11-treated peritoneal macrophages was significantly reduced. The block to NF-kappaB nuclear translocation correlated with the ability of rhIL-11 to maintain or increase protein levels of the inhibitors of NF-kappaB, IkappaB-alpha, and IkappaB-beta following LPS treatment. Furthermore, rhIL-11-treatment of LPS macrophages resulted in significant elevation of IkappaB-alpha and IkappaB-beta mRNA levels. These results suggest that the anti-inflammatory activity of rhIL-11 is mediated in part by inhibition of NF-kappaB-dependent transcriptional activation. Furthermore, these studies demonstrate for the first time the regulation of IkappaB-beta by an anti-inflammatory cytokine. Given the finding that inappropriate activation of NF-kappaB contributes to multiple inflammatory conditions, the ability of rhIL-11 to inhibit the binding activity of this pleiotropic transcription factor indicates that rhIL-11 has therapeutic potential in a wide range of diseases.

Animals↗

PubMed1997

Hyperinsulinemia but no diabetes in transgenic mice homozygously expressing the tyrosine kinase-deficient human insulin receptor.

We generated transgenic mice homozygous for the tyrosine kinase-deficient human insulin receptor (hIRK1030M(+/+)) under control of the insulin receptor promoter. Similar growth patterns and results of glucose tolerance tests were observed among normal, heterozygous, and homozygous mice. Insulin tolerance test indicated no significant difference in the hypoglycemic response to insulin among the three genotypes. However, the serum insulin levels of the homozygous mice before and after glucose loading (201.42 +/- 58.15 pg/ml to 578.57 +/- 49.03 pg/ml) were significantly higher than in the control mice (100.92 +/- 19.55 pg/ml to 356.36 +/- 55.08 pg/ml; p < 0.01 and p < 0.01, respectively) and heterozygous mice (74.46 +/- 18.55 pg/ml to 352.33 +/- 52.43 pg/ml; p < 0.005 and p < 0.01, respectively). Immunohistological evidence of pancreatic islets showed no significant difference among the three genotypes. Taken together, these results suggest that the tyrosine kinase-deficient insulin receptor causes hyperinsulinemia but not diabetes in these homozygous transgenic mice.

Animals↗

PubMed1997

An ultraviolet crosslink in the hammerhead ribozyme dependent on 2-thiocytidine or 4-thiouridine substitution.

The hammerhead domain is one of the smallest known ribozymes. Like other ribozymes it catalyzes site-specific cleavage of a phosphodiester bond. The hammerhead ribozyme has been the subject of a vast number of biochemical and structural studies aimed at determining the structure and mechanism of cleavage. Recently crystallographic analysis has produced a structure for the hammerhead. As the hammerhead is capable of undergoing cleavage within the crystal, it would appear that the crystal structure is representative of the catalytically active solution structure. However, the crystal structure conflicts with much of the biochemical data and reveals a catalytic metal ion binding site expected to be of very low affinity. Clearly, additional studies are needed to reconcile the discrepancies and provide a clear understanding of the structure and mechanism of the hammerhead ribozyme. Here we demonstrate that a unique crosslink can be induced in the hammerhead with 2-thiocytidine or 4-thiouridine substitution at different locations within the conserved core. Generation of the same crosslink with different modifications at different positions suggests that the structure trapped by the crosslink may be relevant to the catalytically active solution structure of the hammerhead ribozyme. As this crosslink appears to be incompatible with the crystal structure, this provides yet another indication that the active solution and crystal structures may differ significantly.

Base Sequence↗

PubMed1997

The enzymatic and non-enzymatic roles of protein-disulfide isomerase in apolipoprotein B secretion.

UNLABELLED: Secretion of apolipoprotein B (apoB) from mammalian cells requires the presence of functional microsomal triglyceride transfer protein (MTP). We previously reported that co-expressing the human intestinal form of apoB, B48, with both subunits of human MTP in oleate-treated Sf21 cells led to a dramatic induction of B48 secretion. Deletion mutagenesis studies showed that the cysteine-enriched amino terminus of apoB was necessary for the MTP responsiveness (Gretch, D. G., Sturley, S. L., Wang, L., Dunning, A., Grunwald, K. A. A., Wetterau, J. R., Yao, Z., Talmud, P., and Attie, A. D. (1996) J. Biol. Chem. 271, 8682-8691). We therefore hypothesized that the small subunit of MTP, protein-disulfide isomerase (PDI), plays a role in apoB secretion by facilitating correct disulfide bond formation. To determine whether the enzymatic activities of PDI are important for MTP-stimulated apoB secretion, the wild type PDI subunit was replaced with an active site mutant, mPDI (Cys36 --> Ser/Cys380 --> Ser), lacking both disulfide shuffling and redox activities. MTP containing mPDI was fully functional in promoting apoB and triglyceride secretion. Therefore, the shufflase and redox activities of PDI are not necessary for the function of MTP. Since PDI exists in large molar excess over the other subunit of MTP, the role of free PDI (independent of the MTP complex) was investigated. PDI or mPDI was co-expressed with B48 and B17, a fragment encompassing the amino-terminal 17% of apoB. Mutant PDI significantly and specifically reduced the accumulation of the B17 and B48 both intracellularly and in the culture medium. The reduction was partially eliminated by the protease inhibitor N-acetyl-leucyl-leucyl-norleucinal, consistent with rapid co- or post-translational degradation of apoB in the presence of mPDI. Treating the cells with oleate reversed the effect of mPDI on B48 secretion in a dose-dependent manner, but had no effect on B17. IN CONCLUSION: 1) the role of PDI in the MTP complex involves functions other than its known enzymatic activities; 2) one or both of the enzymatic activities of free PDI is/are important for the MTP-independent steps of apoB secretion; 3) oleate can affect apoB secretion at high physiological concentrations and compensate for the insufficiency of PDI activities.

Animals↗

PubMed1997

A factor IX-deficient mouse model for hemophilia B gene therapy.

We have generated a mouse where the clotting factor IX (FIX) gene has been disrupted by homologous recombination. The FIX nullizygous (-/-) mouse was devoid of factor IX antigen in plasma. Consistent with the bleeding disorder, the factor IX coagulant activities for wild-type (+/+), heterozygous (+/-), and homozygous (-/-) mice were 92%, 53%, and <5%, respectively, in activated partial thromboplastin time assays. Plasma factor IX activity in the deficient mice (-/-) was restored by introducing wild-type murine FIX gene via adenoviral vectors. Thus, these factor IX-deficient mice provide a useful animal model for gene therapy studies of hemophilia B.

Adenoviridae↗

PubMed1997

A novel histidine kinase inhibitor regulating development in Bacillus subtilis.

Kinase A is the sensor histidine kinase responsible for processing postexponential phase information and providing phosphate input to the phosphorelay that activates developmental transcription via phosphorylated Spo0A. A protein inhibitor, KipI, of kinase A was discovered encoded in an operon of genes of unknown function but regulated by the availability of fixed nitrogen. KipI is a potent inhibitor of the autophosphorylation reaction of kinase A but does not inhibit phosphate transfer to the Spo0F response regulator once kinase A is phosphorylated. KipI is an inhibitor of the catalytic domain of kinase A affecting the ATP/ADP reactions and not the phosphotransferase functions of this domain. The inhibitory activity of KipI is counteracted by the product of another gene in the operon, KipA. This protein may bind to KipI, preventing its function as an inhibitor of kinase A. KipI may be the first representative of a new class of signal transduction inhibitors that function by direct interaction with the catalytic domain of histidine kinases to counteract signals influencing the "sensor" domain of such kinases. This inhibitor represents yet another way by which the phosphorelay signal transduction system is affected by negative regulators under the control of metabolic, environmental, or cell cycle influences antithetical to the initiation of developmental transcription.

Bacillus subtilis↗

PubMed1997

Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice.

Leptin is a circulating protein involved in the long-term regulation of food intake and body weight. Cholecystokinin (CCK) is released postprandially and elicits satiety signals. We investigated the interaction between leptin and CCK-8 in the short-term regulation of food intake induced by 24-hr fasting in lean mice. Leptin, injected intraperitoneally (i.p.) at low doses (4-120 microg/kg), which did not influence feeding behavior for the first 3 hr postinjection, decreased food intake dose dependently by 47-83% during the first hour when coinjected with a subthreshold dose of CCK. Such an interaction was not observed between leptin and bombesin. The food-reducing effect of leptin injected with CCK was not associated with alterations in gastric emptying or locomotor behavior. Leptin-CCK action was blocked by systemic capsaicin at a dose inducing functional ablation of sensory afferent fibers and by devazepide, a CCK-A receptor antagonist but not by the CCK-B receptor antagonist, L-365,260. The decrease in food intake which occurs 5 hr after i.p. injection of leptin alone was also blunted by devazepide. Coinjection of leptin and CCK enhanced the number of Fos-positive cells in the hypothalamic paraventricular nucleus by 60%, whereas leptin or CCK alone did not modify Fos expression. These results indicate the existence of a functional synergistic interaction between leptin and CCK leading to early suppression of food intake which involves CCK-A receptors and capsaicin-sensitive afferent fibers.

Animals↗

PubMed1997

Immunohistochemical studies on Waf1p21, p16, pRb and p53 in human esophageal carcinomas and neighboring epithelia from a high-risk area in northern China.

To better understand the roles of p53 and cell cycle-regulating protein alterations in human esophageal carcinogenesis, we investigated immunohistochemically the distribution patterns of Waf1p21, pRb, p16 and p53 in 22 cases of surgically resected esophageal cancer as well as in the neighboring non-cancerous squamous epithelia. Waf1p21 protein was detected in 13 of the 20 cases of well-differentiated squamous-cell carcinoma (SCC), where the Waf1p21-positive cells were located mainly in the interior layers of the cancer nests. Conversely, p53-positive cells were found mostly in the peripheral layers. Cells containing both Waf1p21- and p53-positive immunostaining were not observed in a double-immunostaining experiment. p16 was detected in both the nucleus and cytoplasm in 3 of the 22 cases of SCC. All of these p16-positive cancers showed an absence of pRb immunostaining; this result is consistent with the idea that expression of p16 is regulated negatively by pRb. Eleven of the 22 esophageal SCCs (50%) showed extensive pRb immunostaining cells, and the remaining 11 cases displayed a few pRb-positive cells or an absence of pRb immunostaining. In a majority of the morphologically normal squamous-cell epithelia samples, immunostaining of Waf1p21 and pRb was found in most of the cells in the parabasal layers (proliferation compartment), where PCNA-positive cells also resided. In the pre-cancerous lesions, Waf1p21 and pRb were detected in cells surrounding the top of the lesioned region, p16-positive cells were scattered in the basal cell hyperplastic and dysplastic lesions and p53-positive cells existed in 2 distinct patterns: "scattered" and "focal".

Carcinoma, Squamous Cell↗

PubMed1997

Oxidative defense enzyme activity and mRNA levels in lenses of diabetic rats.

This study examines the mRNA expression and enzyme activity of oxidative defense enzymes during the course of streptozotocin-induced hyperglycemic cataract development. Diabetes was produced in 5-wk-old male Sprague-Dawley rats by administering streptozotocin ip and mRNA expression and enzyme activity were monitored on d 4, 8, 12, 16, 20, 40, 60, and 80; concomitantly, the onset and progress of cataract was followed by digital image analysis. Peak enzyme activity and mRNA expression were attained between d 20 and 40. Although catalase and glutathione peroxidase maintained high levels of mRNA expression through d 60, induction of CuZu-superoxide dismutase was transient, with the activity and mRNA levels returning to baseline values by d 40. There was a pronounced increase in aldose reductase activity, which gradually declined to basal levels by d 60; however, the mRNA levels remained unaltered. Other changes included a progressive loss of lenticular transparency, which declined to 40% of control by d 80. The role of antioxidant defense enzymes and, more interestingly, aldose reductase in combating oxidative stress in diabetic cataractogenesis is discussed.

Aldehyde Reductase↗

PubMed1997

Engineering the independent folding of the subtilisin BPN' prodomain: analysis of two-state folding versus protein stability.

In complex with subtilisin BPN', the 77 amino acid prodomain folds into a stable compact structure comprising a four-stranded antiparallel beta-sheet and two three-turn alpha-helices. When isolated from subtilisin, the prodomain is 97% unfolded even under optimal folding conditions. Traditionally, to study stable proteins, denaturing cosolvents or temperatures are used to shift the equilibrium from folded to unfolded. Here we manipulate the folding equilibrium of the unstable prodomain by introducing stabilizing mutations generated by design. By sequentially introducing three stabilizing mutations into the prodomain we are able to shift the equilibrium for independent folding from 97% unfolded to 65% folded. Spectroscopic and thermodynamic analysis of the folding reaction was carried out to assess the effect of stability on two-state behavior and the denatured state. The denatured states of single and combination mutants are not discernably different in spite of a range of DeltaGunfolding from -2.1 to 0.4 kcal/mol. Conclusions about the nature of the denatured state of the prodomain are based on CD spectral data and calorimetric data. Two state folding is observed for a combination mutant of marginal stability (DeltaG = 0). Evidence for its two-state folding is based on the observed additivity of individual mutations to the overall DeltaGunfolding and the conformity of DeltaGunfolding vs T to two-state assumptions as embodied in the Gibbs-Helmholz equation. We believe our success in stabilizing the two-state folding reaction of the prodomain originates from the selection of mutations with improved ability to fold subtilisin rather than selection for increase in secondary structure content. The fact that a small number of mutations can stabilize the independent folding of the prodomain implies that most of the folding information already exists in the wild-type amino acid sequence in spite of the fact that the unfolded state predominates.

Bacillus subtilis↗

PubMed1997

Lateralization and functional organization of the locus coeruleus projection to the trigeminal somatosensory pathway in rat.

The primary goals of this study were to (1) examine the distribution of locus coeruleus (LC) neurons, which project to cortical and subcortical sites along the trigeminal somatosensory pathway in rats, and (2) determine the extent to which different regions within this ascending sensory system receive collateral projections from the same LC neuron. Long-Evans hooded rats received unilateral pressure injections of different combinations of retrograde fluorescent tracers into whisker-related regions of primary (SI) and secondary (SII) somatosensory cortices, the ventrobasal (VB) and posterior group (POm) nuclei of the thalamus, and the principalis nucleus of the trigeminal complex (PrV). Coronal sections (40-100 microm) through the LC were examined by fluorescence microscopy, and the distribution of retrogradely labeled cells was recorded. The major finding was that whisker-related regions of the cortex receive efferent projections from neurons concentrated in the caudal portion of the ipsilateral LC, whereas subcortical trigeminal somatosensory structures receive bilateral input from both LC nuclei. Despite the bilateral nature of the LC projection to subcortical sites, the majority of LC efferents to VB and POm thalamus originate in the ipsilateral LC nucleus, whereas projections to PrV originate primarily from the contralateral LC. An additional finding was that a relatively large proportion of LC cells, which project to a single somatosensory structure, also send axon collaterals to other relay sites along the same ascending somatosensory pathway. Taken together, these results suggest that the LC-noradrenergic system maintains a more selective relationship with functionally related efferent targets than has been previously appreciated.

Animals↗

PubMed1997

Identification of highly potent retinoic acid receptor alpha-selective antagonists.

The syntheses and full retinoid receptor characterization of a novel series of retinoic acid receptor alpha (RAR alpha) antagonists, 1-5, are described. These compounds bind with high affinity to RAR alpha but were completely inactive in gene transactivation. They were also potent and effective antagonists of retinoic acid (RA) induced gene transcription at RAR alpha. Compounds 1-5 exhibited varying degrees of selectivity for RAR alpha relative to RAR beta/gamma, with compound 5 being the most selective in both binding and functional antagonism assays. These compounds will be invaluable tools in delineating the physiological roles of RAR alpha in development and in the adult animal and may themselves be useful therapeutic agents in human diseases associated with RAR alpha.

Animals↗

PubMed1997

Involvement of T-cell subsets and natural killer (NK) cells in the growth suppression of murine fibrosarcoma cells transfected with interleukin-12 (IL-12) genes.

A 3-methylcholanthrene-induced fibrosarcoma cell line of BALB/c origin, CMS5j, was co-transfected with cDNA for the p40 and p35 subunits of interleukin-12 (IL-12). Injection of transfected cells producing 5 x 10(3) U IL-12/10(6) cells/ml/day in nude mice with an established fibrosarcoma at a contralateral site efficiently eliminated tumor growth in the early phase (injection on day 0 or 4) but not later (day 8). This effect could be abrogated by simultaneous i.v. injection of antibodies against NK1.1 or ASGM1 (asialoGM1 = ganglio-N-tetraosyl-ceramide), which indicates that natural killer (NK) cells play a major role in tumor eradication or suppression when stimulated by IL-12. In wild-type mice, application of IL-12-secreting CMS5j cells abrogated growth of tumors established 8 days before but not earlier. Based on our experiments with antibody blocking in vivo, all CD4+, CD8+ and ASGM1+ cells are involved in tumor rejection. However, in our system, CD4+ cells or CD8+ cells alone, but not ASGM1+ cells alone, also could lead to tumor rejection. IL-12-engineered fibrosarcoma cells may constitute an efficient and safe system for immunotherapy of cancer.

Animals↗

PubMed1997

A large-conductance chloride channel in pigmented ciliary epithelial cells activated by GTPgammaS.

A large-conductance (or maxi-) chloride channel was identified in bovine pigmented ciliary epithelial (PCE) cells using inside-out excised patch clamp recording. The channel had a mean conductance of 293 pS when excised patches were bathed in symmetrical 130 mm NaCl although the conductance decreased to 209 pS when the solution bathing the cytoplasmic face of the patch contained only 33 mm NaCl. The channel was highly selective for chloride, with a PCl/PNa = 24. A flickery, reversible block was produced by the diuretic stilbene 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), while 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) produced a permanent block. The channel was rarely active in cell-attached patches and usually required several minutes of polarization before activity could be detected in excised patches, a process known as metagenesis. Once activated, the channel was voltage-dependent and was mainly open within the voltage range -30 to +30 mV closing when the membrane was polarized to larger values. GTPgammaS (100 microM) activated the channel with a latency of 170 sec when applied to the cytoplasmic face of patches. This activation was not reversible upon return to control solution within the duration of the experiment. We assess the available evidence and suggest a role for this channel in volume regulation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Mutated mitogen-activated protein kinase: a tumor rejection antigen of mouse sarcoma.

The molecular basis of the polymorphic tumor rejection antigens of chemically induced sarcomas of inbred mice remains a mystery, despite the discovery of these antigens over 40 years ago and their critical importance to the foundation of tumor immunology. In an analysis of a panel of BALB/c 3-methylcholanthrene-induced tumors, we identified one tumor, CMS5, that elicited a strong cytotoxic T cell response with exquisite specificity for CMS5. A stable cloned line of T cells with this specificity (C18) was used to screen a CMS5 cDNA expression library. The gene encoding the C18-defined antigen was identified as a mutated form of a mouse mitogen-activated protein kinase, ERK2, and a peptide incorporating the resulting amino acid substitution (lysine to glutamine) was efficiently recognized by C18. Vaccination with this peptide elicited specific resistance to CMS5 challenge. Extensive efforts to isolate antigen-loss variants of CMS5 were unsuccessful, suggesting that the mutated mitogen-activated protein kinase is essential for maintenance of the malignant phenotype.

Amino Acid Sequence↗

PubMed1997

Transforming growth factor-beta1 inhibits type I inositol 1,4,5-trisphosphate receptor expression and enhances its phosphorylation in mesangial cells.

A potentially important cross-talk characteristic of transforming growth factor-beta (TGF-beta) is to inhibit platelet-derived growth factor-induced intracellular calcium rise (Baffy, G., Sharma, K., Shi, W., Ziyadeh, F. N., and Williamson, J. R. (1995) Biochem. Biophys. Res. Commun. 210, 378-383) in murine mesangial cells. The present study examined the possible basis for this effect by evaluating the regulation of the type I inositol 1,4,5-trisphosphate receptor (IP3R) by TGF-beta. TGF-beta1 down-regulates IP3R protein expression by >90% with maximal and half-maximal effects after 8 and 2 h, respectively. TGF-beta1 also decreased IP3R mRNA expression by 59% after 1 h. Phosphorylation of the IP3R was also demonstrated as early as 15 min after TGF-beta1 exposure. Back phosphorylation assays of IP3R from TGF-beta1-treated mesangial cells with protein kinase A (PKA), indicated that TGF-beta1-induced phosphorylation of the IP3R occurs at similar sites as for PKA. In vitro kinase assays using the known IP3R peptide substrates for PKA, RPSGRRESLTSFGNP and ARRDSVLAAS, demonstrated that TGF-beta1 induces phosphorylation of both peptides (158 and 123% of control values, respectively). TGF-beta1-induced phosphorylation was prevented by the addition of the PKA inhibitor peptide in the in vitro kinase assay. It is proposed that TGF-beta-mediated effects on the IP3R may be an important characteristic of its ability to modulate the response of cells to factors that employ IP3R-mediated calcium release.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Mutations of O6-methylguanine-DNA methyltransferase gene in esophageal cancer tissues from Northern China.

Epidemiologic studies have implicated the involvement of environmental factors in the etiology of esophageal cancer (EC). Our previous data have indicated that EC patients and their blood relatives show genomic instability and are deficient in repair of DNA damage induced by N-nitroso-compounds and related genotoxic agents. Thus, exposure to high levels of N-alkylnitrosamines, which are known animal carcinogens and which induce alkyl adducts in DNA, may be causally linked to EC. Among the alkyl adducts, O6-alkylguanine was shown to be the critical one related to carcinogenic lesions; its repair varies widely in a tissue-specific fashion. O6-methylguanine-DNA methyltransferase (MGMT) is responsible for its repair. Hence, inactivating mutations in this protein would impair the efficiency of the repair process. To screen for possible mutations in the MGMT polypeptide in EC patients, we analyzed a highly conserved region of the MGMT gene in 40 EC tissues and lymphocytes of 6 high-risk EC family members from high EC areas of Northern China by polymerase chain reaction single-strand conformation polymorphism and by direct sequencing of PCR products. Ten base substitutions (point mutations) within 8 codons of 7 EC samples were identified. However, no germline mutation was found in the high EC families studied so far. Concurrent study in 30 pairs of fresh EC tissues and their adjacent normal mucosa by Southern blot and Western blot analyses showed deletion of the MGMT gene in 2 samples. Thus, the high frequency of mutations (7/40) and deletions (2/30) of the MGMT gene may be partially responsible for the overall high mutation rate observed in EC tissues.

Base Sequence↗