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S Huang

Publications and source records attributed to S Huang.

At least 505 records · Page 28Linked to original sources
PubMed1994

Crystal structure of fructose-1,6-bisphosphatase complexed with fructose 2,6-bisphosphate, AMP, and Zn2+ at 2.0-A resolution: aspects of synergism between inhibitors.

The crystal structure of fructose-1,6-bisphosphatase (Fru-1,6-Pase; EC 3.1.3.11) complexed with Zn2+ and two allosteric regulators, AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) has been determined at 2.0-A resolution. In the refined model, the crystallographic R factor is 0.189 with rms deviations of 0.014 A and 2.8 degrees from ideal geometries for bond lengths and bond angles, respectively. A 15 degrees rotation is observed between the upper dimer C1C2 and the lower dimer C3C4 relative to the R-form structure (fructose 6-phosphate complex), consistent with that expected from a T-form structure. The major difference between the structure of the previously determined Fru-2,6-P2 complex (R form) and that of the current quaternary T-form complex lies in the active site domain. A zinc binding site distinct from the three binding sites established earlier was identified within each monomer. Helix H4 (residues 123-127) was found to be better defined than in previously studied ligated Fru-1,6-Pase structures. Interactions between monomers in the active site domain were found involving H4 residues from one monomer and residues Tyr-258 and Arg-243 from the adjacent monomer. Cooperativity between AMP and Fru-2,6-P2 in signal transmission probably involves the following features: an AMP site, the adjacent B3 strand (residues 113-118), the metal site, the immediate active site, the short helix H4 (residues 123-127), and Tyr-258 and Arg-243 from the adjacent monomer within the upper (or lower) dimer. The closest distance between the immediate active site and that on the adjacent monomer is only 5 A. Thus, the involvement of H4 in signal transmission adds another important pathway to the scheme of the allosteric mechanism of Fru-1,6-Pase.

Adenosine Monophosphate↗
PubMed1994

Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma.

A transgenic mouse strain that expresses the hepatitis B virus (HBV) large envelope protein in the liver was used to determine the extent of oxidative DNA damage that occurs during chronic HBV infection. This mouse strain develops a chronic necroinflammatory liver disease that mimics the inflammation, cellular hyperplasia, and increased risk for cancer that is evident in human chronic active hepatitis. When perfused in situ with nitroblue tetrazolium, an indicator for superoxide formation, the liver of transgenic mice displayed intense formazan deposition in Kupffer cells, indicating oxygen radical production, and S-phase hepatocytes were commonly seen adjacent to the stained Kupffer cells. Similar changes were not observed in nontransgenic control livers. To determine whether these events were associated with oxidative DNA damage, genomic DNA from the livers of transgenic mice and nontransgenic controls was isolated and examined for 8-oxo-2'-deoxyguanosine, an oxidatively modified adduct of deoxyguanosine. Results showed a significant, sustained accumulation in steady-state 8-oxo-2'-deoxyguanosine that started early in life exclusively in the transgenic mice and increased progressively with advancing disease. The most pronounced increase occurred in livers exhibiting microscopic nodular hyperplasia, adenomas, and hepatocellular carcinoma. Thus, HBV transgenic mice with chronic active hepatitis display greatly increased hepatic oxidative DNA damage. Moreover, the DNA damage occurs in the presence of heightened hepatocellular proliferation, increasing the probability of fixation of the attendant genetic and chromosomal abnormalities and the development of hepatocellular carcinoma.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1994

Toward a mechanism for the allosteric transition of pig kidney fructose-1,6-bisphosphatase.

We examine structural aspects of the allosteric transition of pig kidney fructose-1,6-bisphosphatase (Fru-1,6-Pase) by analyzing the X-ray structures of the R and T form enzymes. The results show a hierarchical structural change during the R to T transition. Upon binding of AMP, a cascade of structural changes occurs starting from the AMP site: expansion of the AMP site, local conformational changes of helices H1 and H2, independent rotations and translation of helices H1, H2 and H3 (and loops connecting them), reorganization of the AMP domain as a whole and its 1.9 degrees rotation relative to the fructose-1,6-bisphosphate domain, and conformational changes at the C1-C2 and C1-C4 interfaces leading to the quaternary conformational change of a 17 degrees rotation between dimers. The AMP inhibition results from the relative movement between the AMP and FBP domains which distorts the active site during the transition by shifting the metal binding sites to unfavourable positions. Communication that ensures cooperativity during R to T transition relies on changes in positions of helices H1, H2 and H3, loops 127-131, 168-170 and 187-192, and on N-terminal residues. All of these features are close to the C1-C4 and symmetry equivalent C2-C3 interfaces and the relatively small C1-C3 interface of the T form. These secondary structures form the framework along which structural changes due to AMP binding can propagate to other parts of the monomers as well as across monomer interfaces. Future dynamics studies may be useful to analyze initiation, propagation and completion of the quaternary conformational change of Fru-1,6-Pase upon AMP binding. Also, site directed mutagenesis experiments are expected to provide more detailed descriptions of the importance of each of the residues that has been identified here in the proposed mechanisms.

Adenosine Monophosphate↗
PubMed1994

How a protein binds B12: A 3.0 A X-ray structure of B12-binding domains of methionine synthase.

The crystal structure of a 27-kilodalton methylcobalamin-containing fragment of methionine synthase from Escherichia coli was determined at 3.0 A resolution. This structure depicts cobalamin-protein interactions and reveals that the corrin macrocycle lies between a helical amino-terminal domain and an alpha/beta carboxyl-terminal domain that is a variant of the Rossmann fold. Methylcobalamin undergoes a conformational change on binding the protein; the dimethylbenzimidazole group, which is coordinated to the cobalt in the free cofactor, moves away from the corrin and is replaced by a histidine contributed by the protein. The sequence Asp-X-His-X-X-Gly, which contains this histidine ligand, is conserved in the adenosylcobalamin-dependent enzymes methylmalonyl-coenzyme A mutase and glutamate mutase, suggesting that displacement of the dimethylbenzimidazole will be a feature common to many cobalamin-binding proteins. Thus the cobalt ligand, His759, and the neighboring residues Asp757 and Ser810, may form a catalytic quartet, Co-His-Asp-Ser, that modulates the reactivity of the B12 prosthetic group in methionine synthase.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1994

Expression of CD43 on murine and human pluripotent hematopoietic stem cells.

To understand regulation of hemopoiesis, it would be helpful to identify physiologically relevant function-associated molecules on stem cells. Here, we report the detailed examination of CD43 expression on murine and human pluripotent hemopoietic stem cells. Mouse stem cells were found within the Ly6+Lin-CD43high subpopulation of bone marrow. These cells, upon transfer into SCID mice, caused rapid repopulation of thymus, spleen, and bone marrow. Retransfer of bone marrow cells from primary SCID recipients of Ly6+Lin-CD43high cells into secondary recipients resulted in repopulation of lymphohemopoietic cells. All Ly6+Lin-CD43high cells were found to express high levels of c-kit. In contrast, Ly6+Lin-CD43-/low cells caused limited and variable thymic and splenic repopulation. These cells failed to repopulate the marrow cavity and did not contain retransplantable stem cells. These data indicate that murine pluripotent stem cells express high levels of CD43. Examination of human fetal bone marrow cells revealed a population of CD34+CD38-CD43+ cells. When single sorted cells with this phenotype were cultured in vitro, they were able to produce colonies with a dispersed growth pattern. Cells with this growth pattern have previously been shown to have myeloid and lymphoid growth potentials and extensive self-renewal capacity. Furthermore, CD34+CD38-HLA-DR+ cells, recently shown to be highly enriched in stem cell activity, expressed relatively high levels of CD43. Because CD43 has recently been shown to bind to intercellular adhesion molecule-1, these data suggest a possible role for CD43 in the regulation of hemopoiesis.

ADP-ribosyl Cyclase↗
PubMed1994

Expression of antisense fibronectin RNA in human colon carcinoma cells disrupts the regulation of carcinoembryonic antigen by transforming growth factor beta 1.

Transforming growth factor beta 1 regulates the expression of extracellular matrix adhesion molecules and the carcinoembryonic antigen gene family of glycoproteins in the Moser colon carcinoma cell line. Expression of fibronectin antisense RNA in the Moser cells down-regulated fibronectin mRNA expression and blocked the ability of transforming growth factor beta 1 to stimulate fibronectin secretion. Cells expressing antisense fibronectin RNA exhibited a higher rate of proliferation, changed their morphology, and produced more laminin in response to treatment with transforming growth factor beta 1. The stimulation of carcinoembryonic antigen secretion by transforming growth factor beta 1 (which normally lagged 10-16 h behind that of fibronectin secretion stimulated by transforming growth factor beta 1) was blocked by the expression of antisense fibronectin RNA. Thus, the stimulation of fibronectin secretion by transforming growth factor beta 1 was a prerequisite for subsequent stimulation of carcinoembryonic antigen secretion. In addition, the stimulation of the cellular expression of carcinoembryonic antigen and three carcinoembryonic antigen cross-reactive species of glycoproteins by transforming growth factor beta 1 was down-modulated in cells expressing antisense fibronectin RNA. We therefore conclude that fibronectin may play an important role in the mechanisms of transforming growth factor beta 1 regulation of the expression of carcino-embryonic antigen gene family of glycoproteins.

Carcinoembryonic Antigen↗
PubMed1994

The allosteric site of human liver fructose-1,6-bisphosphatase. Analysis of six AMP site mutants based on the crystal structure.

The molecular structure of human liver fructose-1,6-bisphosphatase complexed with AMP was determined by x-ray diffraction using molecular replacement, starting from the pig kidney enzyme AMP complex. Of the 34 amino acid residues which differ between these two sequences, only one interacts with AMP; Met30 in pig kidney is Leu30 in human liver. From this analysis, six sites in which side chains of amino acid residues are in contact with AMP, Ala24, Leu30, Thr31, Tyr113, Arg140, and Met177, were mutated by polymerase chain reaction. The wild-type and mutant forms were expressed in Escherichia coli, purified, and their kinetic properties determined. Circular dichroism spectra of the mutants were indistinguishable from that of the wild-type enzyme. Kinetic analyses revealed that all forms had similar turnover numbers, Km values for fructose 2,6-bisphosphate, and inhibition constants for fructose 2,6-bisphosphate. Apparent Ki values for AMP inhibition of the Leu30 --> Phe and Met177 --> Ala mutants were similar to those of the wild-type enzyme, but the apparent Ki values for the Arg140 --> Ala and Ala24 --> Phe mutants were 7-to 20-fold higher, respectively. The Thr31 --> Ser mutant exhibited a 5-fold increase in apparent Ki for AMP, while mutation of Thr31 to Ala increased the apparent Ki 120-fold. AMP inhibition of the Tyr113 --> Phe mutant was undetectable even at millimolar AMP concentrations. Fructose 2,6-bisphosphate potentiated AMP inhibition of the mutants to the same extent as for the wild-type enzyme, except in the case of the Thr31 --> Ala and Tyr113 --> Phe mutants. Thus, the Met177 --> Ala mutant suggests that the side chain beyond C alpha is not needed for AMP binding, and that the Leu30 --> Phe mutant preserves the AMP contacts with these side chains. Thr31, Tyr113, and Arg140 form key hydrogen bonds to AMP consistent with strong side chain interactions in the wild-type enzyme. Finally, the absence of any effect of fructose 2,6-bisphosphate on AMP inhibition observed in the Thr31 --> Ala mutant may be an important clue relating to the mechanism of synergism of these two inhibitors.

Adenosine Monophosphate↗
PubMed1994

A genetic and molecular characterization of the recA gene from Staphylococcus aureus.

Previous studies have identified mutant strains of Staphylococcus aureus that have deficiencies in genetic recombination and DNA repair. Although these phenotypes were tentatively attributed to mutations within the S. aureus recA gene, experimental evidence to confirm this has never been reported. To characterize recA from S. aureus, we first isolated transposon insertion mutations that were in close proximity to the recA-like mutation (uvs-568) in strain 112 UVS-1. This allowed for the mobilization of the uvs-568 mutation into strain RN4220, the common laboratory strain of S. aureus. Next, using Bacillus subtilis recA as a probe, we cloned S. aureus recA and determined its nucleotide sequence. The deduced amino acid (aa) sequence of RecA contained 347 aa and was 74% identical to B. subtilis RecA. Using a cloned DNA fragment originating from within S. aureus recA, we then constructed a recA null mutant strain, designated KB103, which exhibited the same phenotypic characteristics imposed by the uvs-568 mutation in the same background. Furthermore, genetic and physical mapping of S. aureus recA placed it in the same region as the uvs-568 mutation. These data strongly suggest that these mutations represent different alleles of the same recA gene.

Amino Acid Sequence↗
PubMed1994

H+ is involved in the activation of calcineurin by calmodulin.

We have studied recently the surface hydrophobic properties of several calmodulin (CaM) target enzymes and found that a certain amphipathic domain(s) of the enzyme was exposed in response to a H+ increase within the neutral pH range. The exposed domain appeared to be related to their CaM-binding domains and associated with their activation by CaM (Huang, S., Carlson, G. M., and Cheung, W. Y. (1994) J. Biol. Chem. 269, 7631-7638). In this paper, the involvement of H+ in the activation of calcineurin (CaN), one of the CaM target enzymes, was further studied. Using dansylated CaM to monitor its conformational change, we found that the binding of Ca2+ to CaM occurred at a lower range of Ca2+ concentrations (pCa from 7 to 6) than the formation of CaM.CaN complex (pCa from 6 to 5). However, addition of H+ within the neutral pH range shifted the formation of CaM.CaN complex to the lower range of Ca2+ concentrations. Similarly, the addition of H+ shifted the Ca2+ requirement for enzyme activation to a lower Ca2+ range. These results show that the interaction of CaM with CaN could be initiated either by Ca2+ or by H+ in the presence of suboptimal Ca2+, suggesting a positive cooperative effect between Ca2+ and H+. The CaM-stimulated activity of CaN was accompanied by a time-dependent autoinactivation. Kinetic analysis showed that the autoinactivation initiated by Ca2+ or by H+ was biphasic and that the decay rate constants were comparable. In heavy water (D2O), the Ca(2+)-induced formation of CaM.CaN complex as well as the activity of CaN were decreased, indicating a solvent deuterium isotope effect. The same solvent isotope effect was observed in the H(+)-induced formation of CaM.CaN complex and in the H(+)-induced enzyme activity. When the enzyme was rendered CaM-independent by limited proteolysis, the isotope effect was abolished, suggesting that H+ was involved in the CaM-dependent and not the CaM-independent activity. Collectively, these data suggest that the interaction of CaM with CaN depends on the two cations: Ca2+ exposes an amphipathic domain on CaM, and H+ exposes the CaM-binding domain on the target enzyme.

Animals↗
PubMed1994

IFN-gamma receptor-deficient mice generate antiviral Th1-characteristic cytokine profiles but altered antibody responses.

The lymphokine IFN-gamma is a pleiotropic immunomodulator and possesses intrinsic antiviral activity. We studied its significance in the development of antiviral immune responses by using IFN-gamma receptor-deficient (IFN-gamma R-/-) mice. After inoculation with live attenuated pseudorabies virus (PRV), the mutant mice showed no infectivity titers in various tissues, and transient viral Ag expression only in the spleen, similar as in wild-type mice. However, the absence of the IFN-gamma R resulted in increased proliferative splenocyte responses. The PRV-immune animals showed a normal IFN-gamma and IL-2 production, without detectable IL-4, and with decreased IL-10 secretion in response to viral Ag or Con A. Immunohistochemically, an increased ratio of IFN-gamma:IL-4-producing spleen cells was found. After immunization with either live attenuated or inactivated PRV, IFN-gamma R-/- mice produced significantly less antiviral Ab, and more succumbed to challenge infection than the intact control animals. The reduction in Ab titers in the mutant mice correlated with lower protection by their sera in transfer experiments. Our data demonstrate that ablation of the IFN-gamma receptor surprisingly does not inhibit the generation of antiviral Th1-type and increase Th2-type cytokine responses. However, it profoundly impairs the generation of protective antiviral Ab.

Animals↗
PubMed1994

Regulation of fibronectin and laminin receptor expression, fibronectin and laminin secretion in human colon cancer cells by transforming growth factor-beta 1.

Transforming growth factor (TGF)-beta 1 modulates the expression of extracellular matrix (ECM) glycoproteins, fibronectin and laminin and the adhesion of Moser colon cancer cells to these glycoproteins. Since adhesion can be altered through expression of cell-surface receptors, binding affinities of adhesion molecules for receptors, or both, we investigated the effect of TGF-beta 1 on the binding properties of fibronectin and laminin to their cell-surface receptors by saturation binding and Scatchard analyses using radiolabeled fibronectin and laminin. Fibronectin bound to its cell-surface receptor with high affinity (Kd = 1.25 x 10(-9) M), Moser cells had approximately 7.1 x 10(4) fibronectin-binding sites per cell. TGF-beta 1 treatment rapidly up-modulated the number of cell-surface fibronectin-binding sites by 1.9-fold. The binding affinity of fibronectin for the receptor, however, was not altered. Laminin was found to bind to a higher-affinity and a lower-affinity receptor. Moser cells expressed approximately 1.1 x 10(3) higher-affinity laminin-binding sites and approximately 3.1 x 10(4) lower-affinity-binding sites per cell. TGF-beta 1 rapidly increased the expression of the higher-affinity sites 3-fold and the lower-affinity sites 5-fold. The binding affinity of both the higher-affinity and lower-affinity laminin receptors increased 3-fold after 2 and 6 hr of TGF-beta 1 treatment respectively. Concurrent with receptor modulation, TGF-beta 1 induced the secretion of fibronectin and laminin from Moser cells. Northern hybridization analyses showed a concurrent stimulation of the expression of the mRNAs for ligands (fibronectin and laminin) and the mRNAs for the integrin species of the fibronectin and laminin receptors (alpha 5 and alpha 6 subunits). Thus the production of fibronectin and laminin and the expression of their receptors were tightly co-regulated by TGF-beta 1.

Colonic Neoplasms↗
PubMed1994

Chromium-induced early changes in renal function among ferrochromium-producing workers.

Urinary biochemical indicators of renal injury were examined in 84 male and 38 female ferrochromium-producing workers exposed to water-soluble chromium compounds [Cr(VI)]. The indicators examined included urinary chromium (U-Cr), alkaline phosphatase (ALP), gamma-glutamyl transferase (gamma-GT), glutamic-oxalacetic and glutamic-pyruvic transaminases (GOT & GPT), lactate dehydrogenase (LDH), N-acetyl-beta-D-glucosaminidase (NAG), total protein (TPr) and beta 2-microglobulin (beta 2-MG). The U-Cr levels in the exposed group were approximately 1.8 times that of the control group. Compared to controls, the activities of gamma-GT, NAG, ALP, GOT and LDH in the urine of workers were significantly increased whenever U-Cr concentration exceeded 45 microgram/g creatinine. The activities of gamma-GT, GOT and NAG were elevated in workers employed for longer than ten years. However, no clear dose-response relationships nor time-effect relationships were found. The present results suggest that long-term exposure to water-soluble chromium [Cr(VI)] produces chronic renal injury. The site of the injury appears to mainly involve the proximal tubule. U-Cr concentrations of > 15 microgram/g creatinine can be proposed as a threshold dosage for nephrotoxicity, and gamma-GT, NAG and ALP are early sensitive indicators of the most valuable for evaluating the renal injury.

Acetylglucosaminidase↗
PubMed1994

Elevated serum levels of transforming growth factor-alpha in breast cancer patients.

Previous studies suggest that transforming growth factor-alpha (TGF alpha) may have the potential of a tumor marker. Since the levels of serum TGF alpha in cancer patients and healthy individuals have not been reported, we determined the serum TGF alpha levels of 83 breast cancer patients and 74 healthy individuals by using a TGF alpha radioimmunoassay kit. All of the cancer patients' sera were positive for TGF alpha; their TGF alpha concentrations ranged from 210 to 740 pg/ml, with a mean of 353 +/- 98 pg/ml. Sixty-seven percent (50 cases) of normal sera were positive for TGF alpha; the levels ranged from 120 to 207 pg/ml, with a mean of 144 +/- 17 pg/ml. The difference in serum TGF alpha levels between cancer patients of different disease stages and healthy individuals was found to be statistically significant by Student t-test and the Mann-Whitney test. No correlation was found between serum carcinoembryonic antigen and TGF alpha levels. The potential of serum immunoreactive TGF alpha as a marker for breast cancer warrants further investigation.

Adult↗
PubMed1994

Interferon gamma receptor deficient mice are resistant to endotoxic shock.

Antibody neutralization studies have established interferon gamma (IFN-gamma) as a critical mediator of endotoxic shock. The advent of IFN-gamma receptor negative (IFN gamma R-/-) mutant mice has enabled a more direct assessment of the role of IFN-gamma in endotoxin (lipopolysaccharide [LPS]-induced shock. We report that IFN gamma R-/- mice have an increased resistance to LPS-induced toxicity, this resistance manifesting well before the synthesis and release of LPS-induced IFN-gamma. LPS-induced lymphopenia, thrombocytopenia, and weight loss seen in wild-type mice were attenuated in IFN gamma R-/- mice. IFN gamma R-/- mice tolerated 100-1,000 times more LPS than the minimum lethal dose for wild-type mice in a D-galactosamine (D-GalN)/LPS model. Serum tumor necrosis factor (TNF) levels were 10-fold reduced in mutant mice given LPS or LPS/D-GalN. Bone marrow and splenic macrophages from IFN gamma R-/- mice had a four- to sixfold decreased LPS-binding capacity which correlated with similar reduction in CD14. Serum from mutant mice reduced macrophage LPS binding by a further 50%, although LPS binding protein was only 10% reduced. The expression of TNF receptor I (p55) and II (p75) was identical between wild-type and mutant mice. Thus, depressed TNF synthesis, diminished expression of CD14, and low plasma LPS-binding capacity, in addition to blocked IFN-gamma signaling in the mutant mice, likely to combine to manifest in the resistant phenotype of IFN gamma R-/- mice to endotoxin.

Animals↗