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Biomedical subjects

T Wang

Publications and source records attributed to T Wang.

At least 91 records · Page 5Linked to original sources
PubMed2001

Provitamins and vitamins D₂and D₃in Cladina spp. over a latitudinal gradient: possible correlation with UV levels.

Provitamin D2, vitamin D2 and vitamin D3 were identified in the thallus of a lichen species, Cladina arbuscula (Wallr.) Hale and W.L. Culb. The identification of vitamin D3 was supported by: (1) co-chromatography in both reverse and straight phase HPLC (high performance liquid chromatography), (2) ultraviolet absorption spectrum, and (3) molecular ion peaks demonstrated by ESI (electrospray ionisation) mass spectrometry. The contents of vitamin D3 range from 0.67 to 2.04 μg g⁻¹ dry matter in the thalli of C. arbuscula specimens grown under different natural conditions, while provitamin D3 could not be detected. The ranges for provitamin D2 and vitamin D2 were 89-146 and 0.22-0.55 μg g⁻¹ dry matter, respectively, while the contents of provitamin D3 were below the detection limit (0.01 microg g(-1) dry matter). When C. arbuscula thalli collected at different latitudes from northern Finland to Greece were compared, a positive correlation of vitamin D2 and D3 contents with modelled UV-B radiation at the collection sites was found. A single sample of C. rangiferina from northern Finland gave much higher values for the vitamins. A possible reason could be the lower content of UV-B absorbing pigment in the latter species.

Ascomycota↗
PubMed2001

Solid-state NMR characterization of amylose tris(3,5-dimethylphenylcarbamate) chiral stationary-phase structure as a function of mobile-phase composition.

Solid-state NMR (1H/13C CPMAS) was utilized to identify structural differences in amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase (Chiralpak AD), as a function of mobile-phase composition. Dry Chiralpak AD stationary phase displayed an amorphous CPMAS NMR spectrum. However, CPMAS spectra of Chiralpak AD flushed with organic mobile phases clearly displayed evidence of solvent complexes. Chiralpak AD flushed with nonpolar hexane exhibited solvent complexes with minimal structural perturbation. For Chiralpak AD flushed with hexane containing alcohol modifiers, however, solvent incorporation caused significant difference in conformation distribution as evidenced by increased resolution of 13C peaks in the CPMAS spectrum of the stationary phase. 2-Propanol modifier displayed more efficient displacement of incorporated hexane while forming relatively more distinct/ordered solvent complexes with Chiralpak AD in comparison to ethanol modifier. Reversed elution order and unusual retention behavior on Chiralpak AD as a function of mobile-phase modifier was reported earlier. These chromatographic behaviors are believed to be due to different alterations of the steric environment of the chiral cavities in the CSP by the different mobile-phase modifiers. In addition, on the basis of the chemical shift of C-1 carbon on the amylose backbone, it is possible that Chiralpak AD's structure is a helix with a number of fold less than six.

Journal Article↗
PubMed2001

Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair.

Studies of recombination-dependent replication (RDR) in the T4 system have revealed the critical roles played by mediator proteins in the timely and productive loading of specific enzymes onto single-stranded DNA (ssDNA) during phage RDR processes. The T4 recombination mediator protein, uvsY, is necessary for the proper assembly of the T4 presynaptic filament (uvsX recombinase cooperatively bound to ssDNA), leading to the recombination-primed initiation of leading strand DNA synthesis. In the lagging strand synthesis component of RDR, replication mediator protein gp59 is required for the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA. Together, uvsY and gp59 mediate the productive coupling of homologous recombination events to the initiation of T4 RDR. UvsY promotes presynaptic filament formation on 3' ssDNA-tailed chromosomes, the physiological primers for T4 RDR, and recent results suggest that uvsY also may serve as a coupling factor between presynapsis and the nucleolytic resection of double-stranded DNA ends. Other results indicate that uvsY stabilizes uvsX bound to the invading strand, effectively preventing primosome assembly there. Instead, gp59 directs primosome assembly to the displaced strand of the D loop/replication fork. This partitioning mechanism enforced by the T4 recombination/replication mediator proteins guards against antirecombination activity of the helicase component and ensures that recombination intermediates formed by uvsX/uvsY will efficiently be converted into semiconservative DNA replication forks. Although the major mode of T4 RDR is semiconservative, we present biochemical evidence that a conservative "bubble migration" mode of RDR could play a role in lesion bypass by the T4 replication machinery.

Bacteriophage T4↗
PubMed2001

Force measurement and inhibitor binding assay of monomer and engineered dimer of bovine carbonic anhydrase B.

We applied atomic force microscopy (AFM) to study the intramolecular mechanics of the globular protein molecule, bovine carbonic anhydrase B. The immobilized protein on an amino-functionalized silicon wafer was pulled from its N- and C-termini after being covalently cross-linked to the AFM tip, and the relationship between the tensile force applied on the protein and its extension was recorded. The native enzyme (having 261 residues with two Cys added at its ends, and in a theoretical stretching length of 96 nm) was extended only to 13 +/- 2 nm under physiological conditions before disruption of the covalent cross-linking system. Contrary to the above observation, an engineered dimer was extended to about 110 nm even in the absence of the denaturant. The difference was ascribed to the presence or presumed absence of a "knot" structure at the C-terminal end of the two forms, respectively. When a specific inhibitor was added to the experimental solution, native monomers (sp activity = 88% of the wild type enzyme) were extended to 28 +/- 4 nm, whereas dimers (sp activity = 46%) were extended to about 56 +/- 3 nm, suggesting that both monomeric units in the dimer could bind inhibitor molecules, which was further corroborated by a titration experiment using a fluorescent inhibitor. Thus, one of the monomeric units in the engineered dimer was concluded to be enzymatically inactive but capable of binding inhibitors.

Animals↗
PubMed2001

First examples of oxidizing secondary alcohols to ketones in the presence of the disulfide functional group: synthesis of novel diketone disulfides.

The disulfide functionality is present in a number of organic compounds of interest in the fields of both chemistry and biology. Because the disulfide group is known to be highly susceptible to further oxidation by a wide range of agents, performing a chemoselective oxidation without further oxidizing the disulfide moiety poses a synthetic challenge. Reported herein are the first examples of such a chemoselective oxidation in which a series of novel secondary alcohol disulfides 2a-f have been converted to the corresponding symmetrical diketones 3a-f utilizing a modified Swern oxidation.

Alcohols↗
PubMed2001

Action of low calcemic 1alpha,25-dihydroxyvitamin D3 analogue EB1089 in head and neck squamous cell carcinoma.

BACKGROUND: 1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and its analogues inhibit growth of various types of cancer cells. Although the therapeutic potential of 1,25(OH)(2)D(3) is limited by its tendency to induce hypercalcemia, analogues such as EB1089 are potent inhibitors of cell growth and exhibit reduced calcemic effects. We analyzed the antiproliferative and calcemic effects of EB1089 in tissue culture and animal models of head and neck squamous cell carcinoma (SCC) to investigate its potential as a chemotherapeutic/chemopreventive agent. METHODS: The effects of 1,25(OH)(2)D(3) and EB1089 on cell growth and expression of p21(WAF1/CIP1) and p27(KIP1), which encode cyclin-dependent kinase inhibitors, and a novel target, gadd45alpha, a growth-arrest and DNA-damage gene, were monitored in cultured murine AT-84 SCC cells. The effects of these agents on AT-84 cell growth in vitro and on growth of AT-84 tumors in syngeneic C3H mice were monitored; treatment started at the time of tumor implantation (early tumor model) or after 12 days (late tumor model). Weight and serum calcium levels were also monitored in these animals. All P values were two-sided. RESULTS: Both 1,25(OH)(2)D(3) and EB1089 arrested proliferation of AT-84 cells in G(0)/G(1) phase, inhibited p21(WAF1/CIP1) expression, and induced expression of p27(KIP1) protein. 1,25(OH)(2)D(3) also enhanced the expression of gadd45alpha, apparently by a p53-independent mechanism. There was a statistically significant decrease in tumor growth for 1,25(OH)(2)D(3)-treated mice (P<.001 for early tumor model) and EB1089-treated mice (P<.001 and P =.001 for early and late tumor models, respectively). Unlike 1,25(OH)(2)D(3), EB1089 did not induce cachexia or hypercalcemia. The effects of 1,25(OH)(2)D(3) and EB1089 on expression of p21(WAF1/CIP1) and GADD45alpha were similar in tumors and in vitro. CONCLUSIONS: EB1089 completely inhibited growth of AT-84 SCC cells at nanomolar concentrations, reduced tumor growth, and did not have calcemic effects. Our results support continued investigation of EB1089 as a chemopreventive/chemotherapeutic agent for head and neck SCC.

Animals↗
PubMed2001

Diallyl sulfide inhibition of CYP2E1 does not rescue diabetic rats from thioacetamide-induced mortality.

Previously we have shown that hepatotoxicity of thioacetamide (TA) was increased in streptozotocin (STZ)-induced diabetic (DB) rats due to combined effects of enhanced bioactivation-based liver injury of TA and compromised liver tissue repair response. We have also shown that TA is primarily bioactivated by hepatic CYP2E1. The present study was done to further investigate the importance of liver tissue repair in determining the final outcome of hepatotoxicity. STZ-induced DB rats were pretreated with a CYP2E1 inhibitor, diallyl sulfide (DAS), to decrease the bioactivation-based liver injury of TA. The treatments were as follows: DB/DAS/TA, DB/corn oil/TA, and DB/DAS/saline. Nondiabetic (non-DB) rats received the same treatments as controls. A dose of TA (300 mg/kg ip), which was nonlethal in non-DB rats, caused 92 and 90% mortality in DB/DAS/TA and DB/corn oil/TA groups, respectively. At various times (0--60 h) after treatment, liver injury was assessed by plasma alanine aminotransferase and histopathology. Cell proliferation was evaluated by [(3)H]thymidine incorporation and immunohistochemical staining of proliferating cell nuclear antigen (PCNA). In the DB/DAS/TA rats, DAS pretreatment markedly reduced the CYP2E1-dependent liver injury of TA compared to that in DB/corn oil/TA rats. However, subsequent hepatic DNA synthesis in both DB groups was inhibited approximately 50%. PCNA analysis showed a corresponding decrease in cell-cycle progression. This compromised tissue repair response in DB rats was insufficient to compensate for cell loss, resulting in progression of liver injury and culminating in high mortality in both DB groups. Furthermore, non-DB rats were pretreated with a CYP2E1 inducer, isoniazid, to increase the bioactivation-based TA liver injury equal to the liver injury observed in DB/DAS/TA rats. Despite equal injury up to 36 h following TA treatment, the tissue repair response in the non-DB rats was highly stimulated to compensate for liver injury and led to 70% survival in this group. These studies underscore the importance of adequate and timely tissue repair in compensating for liver injury and protecting from lethality.

Alanine Transaminase↗
PubMed2001

Intersection of interferon and hypoxia signal transduction pathways in nitric oxide-induced tumor apoptosis.

Activated macrophages play a central role in antitumor immunity. However, the stimuli that activate macrophages to kill tumor cells are not completely understood. Because the center of solid tumors can be hypoxic, we hypothesized that hypoxia may be an important signal in activating macrophages to kill tumor cells. Hypoxia stimulates IFN-primed macrophages to express the inducible nitric oxide synthase (NOS2) and to synthesize nitric oxide (NO). We show that this synergy between IFN and hypoxia is mediated by the direct interaction of the hypoxia inducible factor-1 (HIF-1) and IFN regulatory factor-1 (IRF-1), which are both required for the hypoxic transcription of NOS2. This interaction between HIF-1 and IRF-1 may explain the mechanism by which macrophages infiltrating into tumors are activated to express NOS2 and to produce NO, a mediator of tumor apoptosis.

Animals↗
PubMed2001

Role of the LTR region between the enhancer and promoter in mink cell focus-forming murine leukemia virus pathogenesis.

Long terminal repeat (LTR) sequences are important determinants of mink cell focus-forming (MCF) murine leukemia virus pathogenesis. These sequences include the enhancer and sequences between the enhancer and promoter (DEN). In a previous study we showed that a virus missing the DEN region in its LTR was severely attenuated in its ability to induce thymic lymphoma. In this study we observed that a virus with an LTR consisting of DEN but no enhancer sequences was pathogenic. We compared the pathogenicity of this DEN virus with other LTR mutant MCF13 viruses that contained a single enhancer (1R) or a single enhancer plus DEN (1R + DEN). All LTR mutant viruses generated thymic lymphoma, however, at a much lower incidence and with a longer latency compared with wild-type (WT) MCF13 virus. DEN virus replication in the thymus was the lowest compared with the 1R and 1R + DEN viruses. Viral replication in a different thymic subpopulation could not explain the decreased pathogenicity of the LTR mutant viruses compared with WT virus. However, lower levels of mutant virus replication in the thymus compared with WT during the preleukemic period may contribute to the attenuation of pathogenicity. The phenotype of tumors induced by the mutant viruses was similar and differed from tumors induced by WT virus by the presence of CD3(-)CD4(-)CD8(-) cells. Analysis of LTR sequences of infectious virus rescued from tumors induced by the 1R and 1R + DEN viruses showed that amplification of enhancer sequences had occurred during tumor development. The lack of DEN virus expression by tumor cells led us to propose that DEN sequences may play a role at an early step in tumorigenesis.

Animals↗
PubMed2001

Synthesis of novel 3'-C-methylene thymidine and 5-methyluridine/cytidine H-phosphonates and phosphonamidites for new backbone modification of oligonucleotides.

Novel 5'-O-DMT- and MMT-protected 3'-C-methylene-modified thymidine, 5-methyluridine, and 5-methylcytidine H-phosphonates 1-7 with O-methyl, fluoro, hydrogen, and O-(2-methoxyethyl) substituents at the 2'-position have been synthesized by a new effective strategy from the corresponding key intermediates 3'-C-iodomethyl nucleosides and intermediate BTSP, prepared in situ through the Arbuzov reaction. The modified reaction conditions for the Arbuzov reaction prevented the loss of DMT- and MMT-protecting groups, and directly provided the desired 5'-O-DMT- and/or MMT-protected 3'-C-methylene-modified H-phosphonates 1-6 although some of them were also prepared through the manipulation of protecting groups after the P-C bond formation. The modified Arbuzov reaction of 3'-C-iodomethyl-5-methylcytidine 53, prepared from its 5-methyluridine derivative 42, with BTSP provided the 5-methylcytidine H-phosphonate 54, which was further transferred to the corresponding 4-N-(N-methylpyrrolidin-2-ylidene)-protected H-phosphonate monomer 7. 5'-O-MMT-protected 3'-C-methylene-modified H-phosphonates 5, 3, and 7 were converted to the corresponding cyanoethyl H-phosphonates 50, 51, and 56 using DCC as a coupling reagent. One-pot three-step reactions of 50, 51, and 56 provided the desired 3'-C-methylene-modified phosphonamidite monomers 8-10. Some of these new 3'-methylene-modified monomers 1-10 have been successfully utilized for the synthesis of 3'-methylene-modified oligonucleotides, which have shown superior antisense properties including nuclease resistance and binding affinity to the target RNA.

Anti-Infective Agents↗
PubMed2001

Inhibition effects of di(2-ethylhexyl)phthalate on mouse-liver lysosomal vacuolar H(+)-ATPase.

We investigated the effects of di(2-ethylhexyl)phthalate (DEHP) on mouse-liver lysosomes. After 2 weeks of oral administration in mice, a reduction in vacuolar H(+)-ATPase (V-ATPase) was observed, and after 3 weeks, the liver lysosomal compartment was completely negative for V-ATPase, as determined by immunocytochemical analysis. When the mice were subsequently fed a normal diet for 1 week, V-ATPase levels recovered to normal values. According to Northern blot analysis, V-ATPase subunit A mRNA decreased gradually with DEHP treatment. Enzyme cytochemical staining showed acid phosphatase (AcPase) to be present in lysosomes and late autophagosomes (autolysosomes) in normal animals as well as in DEHP-treated animals. But the number of late autophagosomes containing AcPase increased clearly after DEHP treatment. These results suggest that: (1) DEHP causes marked V-ATPase reduction in the liver lysosomal compartment and the effect of DEHP is reversible; and (2) the effect of DEHP on protein expression is likely to be exerted at the transcriptional level.

Animals↗
PubMed2001

A novel series of highly potent benzimidazole-based microsomal triglyceride transfer protein inhibitors.

A series of benzimidazole-based analogues of the potent MTP inhibitor BMS-201038 were discovered. Incorporation of an unsubstituted benzimidazole moiety in place of a piperidine group afforded potent inhibitors of MTP in vitro which were weakly active in vivo. Appropriate substitution on the benzimidazole ring, especially with small alkyl groups, led to dramatic increases in potency, both in a cellular assay of apoB secretion and especially in animal models of cholesterol lowering. The most potent in this series, 3g (BMS-212122), was significantly more potent than BMS-201038 in reducing plasma lipids (cholesterol, VLDL/LDL, TG) in both hamsters and cynomolgus monkeys.

Administration, Oral↗
PubMed2001

Comparative binding energy (COMBINE) analysis of influenza neuraminidase-inhibitor complexes.

Neuraminidase is a surface glycoprotein of influenza viruses that cleaves terminal sialic acids from carbohydrates. It is critical for viral release from infected cells and facilitates viral spread in the respiratory tract. The catalytic active site of neuraminidase is highly conserved in all type A and B influenza viruses, making it an excellent target for antiinfluenza drug design. Indeed, neuraminidase inhibitors have recently become available in the clinic for the treatment of influenza. Here, we describe the use of 3D structures of neuraminidase-inhibitor complexes to derive quantitative structure-activity relationships (QSARs) to aid understanding of the mechanism of inhibition and the discovery of new inhibitors. Crystal structures of neuraminidase-inhibitor complexes were used alongside modeled complexes to derive QSAR models by COMparative BINding Energy (COMBINE) analysis (Ortiz, A. R.; Pisabarro, M. T.; Gago, F.; Wade, R. C. J. Med. Chem. 1995, 38, 2681-2691). The neuraminidase proteins studied include type A subtypes N2 and N9 (which have ca. 50% sequence identity) and an active site mutant of the N9 subtype. The inhibitors include sialic acid and benzoic acid analogues with diverse frameworks and substitution groups. By considering the contributions of the protein residues and a key water molecule to the electrostatic and van der Waals intermolecular interaction energies, a predictive and robust QSAR model for binding to type A neuraminidase was obtained. In this QSAR model, 12 protein residues and 1 bound water molecule are highlighted as particularly important for inhibitory activity. This QSAR model provides guidelines for structural modification of current inhibitors and the design of novel inhibitors in order to optimize inhibitory activity.

Algorithms↗
PubMed2001

New method for analyzing the molecular weights of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Previously the method for determining protein molecular weights from SDS-PAGE depended on the accidental, only partial linearity of protein movement with the logarithm of its molecular weight. A new, mathematically rigorous method with supporting data is now described demonstrating that such movement is dependent upon the reciprocal of protein size. Experimental data, therefore, follow most closely a hyperbolic curve when plotted directly; it becomes linear and passes through the origin when movement is plotted vs the reciprocal of protein molecular weight. In the earlier method determination of the error of a measurement of molecular weight is very complex and never determined. In the method presented here such error is easily estimated and it is identical in both the hyperbolic and linear forms of data presentation. This method may eventually also allow other less-significant forces controlling movement such as protein charge to be analyzed and understood.

Animals↗
PubMed2001

Glutathione S-transferase P1-1 (GSTP1-1) inhibits c-Jun N-terminal kinase (JNK1) signaling through interaction with the C terminus.

c-Jun N-terminal kinase (JNK)-mediated cell signaling pathways are regulated endogenously in part by protein-protein interactions with glutathione S-transferase P1-1 (GSTP1-1) (). Using purified recombinant proteins, combined with fluorescence resonance energy transfer technology, we have found that the C terminus of JNK is critical to the interaction with GSTP1-1. The apparent K(d) for full-length JNK was 188 nm and for a C-terminal fragment (residues 200-424) 217 nm. An N-terminal fragment (residues 1-206) did not bind to GSTP1-1. Increased expression of the C-terminal JNK fragment in a tetracycline-inducible transfected NIH3T3 cell line produced a concentration-dependent increase in the kinase activity of JNK under normal, unstressed growth conditions indicating a dominant-negative effect. This suggests that the fragment can compete with endogenous full-length functional JNK resulting in dissociation of the GSTP1-1-JNK interaction and concomitant JNK enzyme activation. By using an antibody to hemagglutinin-tagged C-JNK, a concentration-dependent co-immunoprecipitation of GSTP1-1 was achieved. These data provide evidence for direct interactions between the C-terminal of JNK and GSTP1-1 and a rationale for considering GSTP1-1 as a critical ligand-binding protein with a role in regulating kinase pathways.

3T3 Cells↗
PubMed2001

Sorting nexin 6, a novel SNX, interacts with the transforming growth factor-beta family of receptor serine-threonine kinases.

Sorting nexins (SNX) comprise a family of proteins with homology to several yeast proteins, including Vps5p and Mvp1p, that are required for the sorting of proteins to the yeast vacuole. Human SNX1, -2, and -4 have been proposed to play a role in receptor trafficking and have been shown to bind to several receptor tyrosine kinases, including receptors for epidermal growth factor, platelet-derived growth factor, and insulin as well as the long form of the leptin receptor, a glycoprotein 130-associated receptor. We now describe a novel member of this family, SNX6, which interacts with members of the transforming growth factor-beta family of receptor serine-threonine kinases. These receptors belong to two classes: type II receptors that bind ligand, and type I receptors that are subsequently recruited to transduce the signal. Of the type II receptors, SNX6 was found to interact strongly with ActRIIB and more moderately with wild type and kinase-defective mutants of TbetaRII. Of the type I receptors, SNX6 was found to interact only with inactivated TbetaRI. SNXs 1-4 also interacted with the transforming growth factor-beta receptor family, showing different receptor preferences. Conversely, SNX6 behaved similarly to the other SNX proteins in its interactions with receptor tyrosine kinases. Strong heteromeric interactions were also seen among SNX1, -2, -4, and -6, suggesting the formation in vivo of oligomeric complexes. These findings are the first evidence for the association of the SNX family of molecules with receptor serine-threonine kinases.

Amino Acid Sequence↗