Biomedical subjects
Publications and source records attributed to R Wang.
Quinoline-4-acetamides were designed as potential phospholipase A(2) inhibitors by structural based method and synthesized. The chemical structures of the obtained compounds were confirmed by elemental analyses, 1H NMR and MS. Preliminary bioassay study shows that quinoline-4-acetamides display certain inhibition to sPLA(2).
Epidemiological surveys and animal experimental studies suggest that exposure to 2-bromopropane (2-BP) could result in reproductive and hematopoietic disorders. The objectives of this study were to investigate the role of apoptosis in 2-BP-induced testicular toxicity and whether this process involves Bcl-2 family genes and the Fas signaling system. Rats were injected percutaneously with 1350 mg/kg 2-BP for 1 to 5 days and then were euthanized at 6 or 12 h after one dose, 6 h after two, three, or five doses, and 2 or 9 days after the final treatment. Light and electron microscopic analyses, TUNEL staining of DNA fragments, agarose gel electrophoresis of low-molecular-weight DNA, and Western blotting analysis of Bcl-2 family proteins and Fas receptor and ligand were conducted. Two-day treatment resulted in selective degeneration of spermatogonia with marked nuclear chromatin condensation. DNA ladder formation on the agarose gel further validated the findings of TUNEL-stained apoptotic cells. The percentage of apoptotic-positive tubules and apoptotic cell index increased time dependently. 2-BP treatment resulted in two distinct morphological changes: an immediate effect on spermatogonia and secondary apoptosis of spermatocytes 9 days after treatment. Downregulation of Bcl-2 after the first or second injection of 2-BP and upregulation of Bax after the first treatment contributed to the initiation of primary apoptosis of spermatogonia. Expression of FasL was inhibited while expression of Fas increased after the 2-BP treatment and remained at levels about two times of the control. However, it increased about sixfold of the control by day 9 after final injection, which contributed to the induction of secondary apoptosis of spermatocytes. Our results indicate that 2-BP resulted in apoptotic death of testicular germ cells and that this process involves the Bcl-2 family genes and the Fas signaling system.
Gene expression patterns were examined in lens epithelial cells conditioned to grow in 125 microM hydrogen peroxide in order to define the protective mechanisms that may be involved in survival during oxidative stress. RNA was extracted from normal and hydrogen peroxide-resistant alphaTN4 mouse lens epithelial cells. Gene expression was evaluated using Differential Display (DD) and RT-PCR. Upregulation of mRNAs for antioxidant and cellular defense enzymes was observed. The highest elevation detected was a 14-fold increase in catalase in the hydrogen peroxide-resistant cells. Glutathione peroxidase, ferritin, and alphaB-crystallin were upregulated 2-fold, and reticulocalbin was upregulated 6-fold in the resistant cells. alphaA-crystallin was downregulated 5-fold, while aldose reductase and mitochondrial gene products were unchanged. Thus, in the alphaTN4 mouse lens cell line, long-term exposure to high levels of hydrogen peroxide elicited an upregulation of transcripts for enzymes involved in hydrogen peroxide degradation, metal binding, and chaperone function. Since mitochondrial gene transcription is sensitive to hydrogen peroxide, the presence of normal levels of mitochondrial transcripts, in this study, demonstrates the effectiveness of the antioxidant defense systems.
We have isolated and determined the coding sequences of human and mouse orthologs of the rat orphan G-protein-coupled receptor GPR54. Mouse and rat GPR54 are nearly 95% identical to each other, and both are approximately 85% identical to human GPR54 at the amino acid level. Screening of agonists for GPR54 identified several invertebrate neuropeptides of the RFamide and RWamide family that were able to activate GPR54 at microM range through the G(alpha)q pathway. Substitution analysis showed that the C-terminal optimal sequence of GPR54-activating peptides is Gly-Leu-Arg-Trp-NH2. Northern analysis of human GPR54 detected expression in several peripheral tissues and many regions of the central nervous system.
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Huperzine A, a promising therapeutic agent for Alzheimer's disease, was examined for its potential to antagonize the deleterious neurochemical, structural, and cognitive effects of infusing beta-amyloid protein-(1-40) into the cerebral ventricles of rats. Daily intraperitoneal administration of huperzine A for 12 consecutive days produced significant reversals of the beta-amyloid-induced deficit in learning a water maze task. This treatment also reduced the loss of choline acetyltransferase activity in cerebral cortex, and the neuronal degeneration induced by beta-amyloid protein-(1-40). In addition, huperzine A partly reversed the down-regulation of anti-apoptotic Bcl-2 and the up-regulation of pro-apoptotic Bax and P53 proteins and reduced the apoptosis that normally followed beta-amyloid injection. The present findings confirm that huperzine A can alleviate the cognitive dysfunction induced by intracerebroventricular infusion of beta-amyloid protein-(1-40) in rats. The beneficial effects are not confined to the cholinergic system, but also include favorable changes in the expression of apoptosis-related proteins and in the extent of apoptosis in widespread regions of the brain.
Tetranuclear lanthanide-hydroxo complexes of the general formula [Ln(4)(mu(3)-OH)(4)(AA)(x)(H(2)O)(y)](8+) (1, Ln = Sm, AA = Gly, x = 5, y = 11; 2, Ln = Nd, AA = Ala, x = 6, y = 10; 3, Ln = Er, AA = Val, x = 5, y = 10) have been prepared by alpha-amino acid controlled hydrolysis of lanthanide ions under near physiological pH conditions (pH 6-7). The core component of these compounds is a cationic cluster [Ln(4)(mu(3)-OH)(4)](8+) whose constituent lanthanide ions and triply bridging hydroxo groups occupy the alternate vertexes of a distorted cube. The amino acid ligands coordinate the lanthanide ions via bridging carboxylate groups. Utilizing L-glutamic acid as the supporting ligand, a cationic cluster complex (4) formulated as [Er(4)(mu(3)-OH)(4)(Glu)(3)(H(2)O)(8)](5+) has been obtained. Its extended solid-state structure is composed of the cubane-like [Er(4)(mu(3)-OH)(4)](8+) cluster building units interlinked by the carboxylate groups of the glutamate ligands. All compounds are characterized by using a combination of spectroscopic techniques and microanalysis (CHN and metal). Infrared spectra of the complexes suggest the coordinated amino acids to be zwitterionic. The presence of mass (MALDI-TOF) envelopes corresponding to the [Ln(4)(mu(3)-OH)(4)](8+) (Ln = trivalent Sm, Nd, or Er) core containing fragments manifests the integrity of the cubane-like cluster unit. Magnetic studies using Evans' method suggest that exchange interactions between the lanthanide ions are insignificant at ambient temperature. The structural identities of all four compounds have been established crystallographically. The tetranuclear cluster core has been demonstrated to be a common structural motif in these complexes. A mechanism responsible for its self-assembly is postulated.
Highly ordered hexagonal mesoporous aluminosilicates (MAS-5) with uniform pore sizes have been successfully synthesized from assembly of preformed aluminosilcate precursors with cetyltrimethylammonium bromide (CTAB) surfactant. The aluminosilicate precursors were obtained by heating, at 100--140 degrees C for 2--10 h, aluminasilica gels at the Al(2)O(3)/SiO(2)/TEAOH/H(2)O molar ratios of 1.0/7.0--350/10.0--33.0/500--2000. Mesoporous MAS-5 shows extraordinary stability both in boiling water (over 300 h) and in steam (800 degrees C for 2 h). Temperature-programmed desorption of ammonia shows that the acidic strength of MAS-5 is much higher than that of MCM-41 and is comparable to that of microporous Beta zeolite. In catalytic cracking of 1,3,5-triisopropylbenzene and alkylation of isobutane with butene, MAS-5 exhibits greater catalytic activity and selectivity, as compared with MCM-41 and HZSM-5. The MAS-5 samples were characterized with infrared, UV--Raman, and NMR spectroscopy and numerous other techniques. The results suggest that MAS-5 consists of both mesopores and micropores and that the pore walls of MAS-5 contain primary and secondary structural building units, similar to those of microporous zeolites. Such unique structural features might be responsible for the observed strong acidity and high thermal stability of the mesoporous aluminosilicates with well-ordered hexagonal symmetry.
Overexpression is the most common abnormality of receptor tyrosine kinases (RTKs) in human tumors. It is presumed that overexpression leads to constitutive activation of RTKs, but the mechanism of that activation has been uncertain. Here we show that overexpression of the Met RTK allows activation of the receptor by cell attachment and that this form of activation can be tumorigenic. Transgenic mice that overexpressed Met in hepatocytes developed hepatocellular carcinoma (HCC), one of the human tumors in which Met has been implicated previously. The tumorigenic Met was activated by cell attachment rather than by ligand. Inactivation of the transgene led to regression of even highly advanced tumors, apparently mediated by apoptosis and cessation of cellular proliferation. These results reveal a previously unappreciated mechanism by which the tumorigenic action of RTKs can be mediated, provide evidence that Met may play a role in both the genesis and maintenance of HCC, and suggest that Met may be a beneficial therapeutic target in tumors that overexpress the receptor.
Linear interaction energy/molecular dynamics calculations have been used to compute steroid/antibody binding energies. The absolute binding affinities of 10 steroids to antibody DB3 and of a hapten to catalytic antibody 1E9 are computed and compared to experiment. A detailed analysis of the molecular origins of the observed binding patterns is provided. The binding energy of an untested steroid is predicted.
Alzheimer's Disease (AD) is characterized by cerebral accumulation of beta-amyloid peptides (Abeta), which are proteolytically derived from beta-amyloid precursor protein (betaAPP). betaAPP metabolism is highly regulated via various signal transduction systems, e.g., several serine/threonine kinases and phosphatases. Several growth factors known to act via receptor tyrosine kinases also have been demonstrated to regulate sbetaAPP secretion. Among these receptors, insulin and insulin-like growth factor-1 receptors are highly expressed in brain, especially in hippocampus and cortex. Emerging evidence indicates that insulin has important functions in brain regions involved in learning and memory. Here we present evidence that insulin significantly reduces intracellular accumulation of Abeta and that it does so by accelerating betaAPP/Abeta trafficking from the trans-Golgi network, a major cellular site for Abeta generation, to the plasma membrane. Furthermore, insulin increases the extracellular level of Abeta both by promoting its secretion and by inhibiting its degradation via insulin-degrading enzyme. The action of insulin on betaAPP metabolism is mediated via a receptor tyrosine kinase/mitogen-activated protein (MAP) kinase kinase pathway. The results suggest cell biological and signal transduction mechanisms by which insulin modulates betaAPP and Abeta trafficking in neuronal cultures.
Total syntheses of the cytotoxic marine natural products bengamides B and E are described. Both bengamides are prepared via amide coupling of a protected polyhydroxylated lactone intermediate 9 with a suitably substituted aminocaprolactam intermediate. Lactone 9 is prepared in five steps from commercially available alpha-D-glucoheptonic gamma-lactone. The key reactions are a selective deprotection of a 1,2-acetonide in the presence of a 1,3-acetonide and an (E)-selective olefination of an unstable aldehyde using a gem-dichromium reagent. The bengamide B lactam intermediate 10 is prepared in seven steps from commercially available (5R)-5-hydroxy-L-lysine (12). The desired S-configuration at the gamma-OH lactam position is established using the Mitsunobu reaction.
Voltage-dependent K+ (Kv) channels were studied in smooth muscle cells (SMCs) freshly isolated from rat mesenteric arteries. A delayed outward rectifier Kv current (IK) with a weak voltage dependence was identified. The amplitude of IK, but not its inactivation kinetics, was inhibited by 4-aminopyridine (4-AP) (IC50, 5.1 +/- 0.9 mM). The inhibitory effect of 4-AP was not use-dependent, and the unbinding of 4-AP from IK channels was complete in the absence of depolarization stimuli, suggesting the binding of 4-AP to the closed state of Kv channels. There was no change in the steady-state inactivation, but the steady-state activation curve of IK was shifted in the presence of 4-AP by +6 mV. Including 4-AP in pipette solution instantly inhibited IK upon the rupture of cell membrane, indicating that 4-AP bound to the inner mouth of Kv channel pores. Several Kv channel proteins encoding the native IK-type Kv channels, but not the transient outward A-type Kv channels, were identified. Among the identified IK-encoding gene transcripts, the expression of Kv1.5 was the most abundant. Our results elucidate the modulating mechanisms for the 4-AP-induced IK inhibition in rat mesenteric artery SMCs and suggest that the unique properties of Kv channels in these cells might be related to the heteromeric expression of the IK-encoding genes with Kv1.5 subunit playing an important role.
The serine-threonine protein kinase Akt is a direct downstream target of phosphatidylinositol 3-kinase (PI3-K). The PI3-K-generated phospholipids regulate Akt activity via directly binding to the Akt PH domain. The binding of PI3-K-generated phospholipids is critical to the relocalization of Akt to the plasma membrane, which plays an important role in the process of Akt activation. Activation of the PI3-K/Akt signaling pathway promotes cell survival. To elucidate the structural basis of the interaction of PI3-K-generated phospholipids with the Akt PH domain with the objective of carrying out structure-based drug design, we modeled the three-dimensional structure of the Akt PH domain. Comparative modeling-based methods were employed, and the modeled Akt structure was used in turn to construct structural models of Akt in complex with selected PI3-K-generated phospholipids using the computational docking approach. The model of the Akt PH domain consists of seven beta-strands forming two antiparallel beta-sheets capped by a C-terminal alpha-helix. The beta1-beta2, beta3-beta4, and beta6-beta7 loops form a positively charged pocket that can accommodate the PI3-K-generated phospholipids in a complementary fashion through specific hydrogen-bonding interactions. The residues Lys14, Arg25, Tyr38, Arg48, and Arg86 form the bottom of the binding pocket and specifically interact with the 3- and 4-phophate groups of the phospholipids, while residues Thr21 and Arg23 are situated at the wall of the binding pocket and bind to the 1-phosphate group. The predicted binding mode is consistent with known site-directed mutagenesis data, which reveal that mutation of these crucial residues leads to the loss of Akt activity. Moreover, our model can be used to predict the binding affinity of PI3-K-generated phospholipids and rationalize the specificity of the Akt PH domain for PI(3,4)P2, as opposed to other phospholipids such as PI(3)P and PI(3,4,5)P3. Taken together, our modeling studies provide an improved understanding of the molecular interactions present between the Akt PH domain and the PI3-K-generated phospholipids, thereby providing a solid structural basis for the design of novel, high-affinity ligands useful in modulating the activity of Akt.
Antigen expression in melanoma is heterogeneous. Immunophenotyping using a panel of monoclonal antibodies may facilitate immunotherapy. An immunoblot procedure was developed to detect antigens in melanoma cells. Numerous monoclonal antibodies were tested to determine if (1) antigens were detected after transfer to membranes, (2) single bands or discrete multiple bands were obtained, (3) co-incubation of multiple monoclonal antibodies had no interference, and (4) banding patterns were non-overlapping. Antigens were selected based upon their association with melanoma and the availability of respective monoclonal antibodies. Antigens were melanoma antigen recognized by T-cells (MART-1), tyrosinase, tyrosinase-related protein 1 (TRP-1), S100, vimentin, glycoprotein 130 (gp130), a carcinoembryonic antigen (CEA)-like marker, KBA-62 and NKI-C3. Actin positive controls could be assessed simultaneously. Test samples were separated by polyacrylamide gel electrophoresis in a 4-15% polyacrylamide gradient, transferred to polyvinylidine fluoride membrane, blotted using a Fast-Blot apparatus (Pierce), and developed using diaminobenzidine/metal. Melanoma cell lines were immunophenotyped using this panel immunoblot, and were compared to a standard control and to non-melanoma cells. Up to four antigens could be detected simultaneously in a single lane of the immunoblot, using a single test sample of greater than 100000 cells.
A series of (eta 6-arene)OsII complexes containing the saturated nitrogen donor ligands tmtacn, tacn, and NH3 are prepared and characterized. The electrochemical properties and photochemical reactions of these complexes are studied, and the solid-state structures for [(eta 6-p-cymene)Os(tacn)](PF6)2 (1) and [(eta 6-p-cymene)Os(tmtacn)](PF6)2 (2) are determined. Single-crystal X-ray data: 1, orthorhombic, space group Pbca-D2h15 (No. 61), with a = 14.716(3) A, b = 17.844(3) A, c = 18.350(4) A, V = 4819(2) A3, and Z = 8; 2, monoclinic, space group C2-C2(3) (No. 5), with a = 17.322(4) A, b = 10.481(3) A, c = 15.049(4) A, beta = 98.72 degrees, V = 2701(1) A3, and Z = 4.
Mutations in the sister of P-glycoprotein (Spgp) or bile salt export pump (BSEP) are associated with Progressive Familial Intrahepatic Cholestasis (PFIC2). Spgp is predominantly expressed in the canalicular membranes of liver. Consistent with in vitro evidence demonstrating the involvement of Spgp in bile salt transport, PFIC2 patients secrete less than 1% of biliary bile salts compared with normal infants. The disease rapidly progresses to hepatic failure requiring liver transplantation before adolescence. In this study, we show that the knockout of spgp gene in mice results in intrahepatic cholestasis, but with significantly less severity than PFIC2 in humans. Some unexpected characteristics are observed. Notably, although the secretion of cholic acid in mutant mice is greatly reduced (6% of wild-type), total bile salt output in mutant mice is about 30% of wild-type. Also, secretion of an unexpectedly large amount of tetra-hydroxylated bile acids (not detected in wild-type) is observed. These results suggest that hydroxylation and an alternative canalicular transport mechanism for bile acids compensate for the absence of Spgp function and protect the mutant mice from severe cholestatic damage. In addition, the spgp(-/-) mice display a significant increase in the secretion of cholesterol and phospholipids into the bile. This latter observation in spgp(-/-) mice suggests that intrahepatic, rather than intracanalicular, bile salts are the major driving force for the biliary lipid secretion. The spgp(-/-) mice thus provide a unique model for gaining new insights into therapeutic intervention for intrahepatic cholestasis and understanding mechanisms associated with lipid homeostasis.
Members of TNF superfamily are characterized by their ability to inflict apoptosis upon binding to their cognate receptors in a homotrimeric manner. These proteins are expressed on different cell types under various conditions. However, the mechanisms governing the expression of these molecules remain elusive. We have found that the TCR signal can elicit the expression of receptor activator of NF-kappaB ligand (RANKL), TNF-alpha, CD95L, and TNF-related apoptosis inducing ligand (TRAIL) in T cell hybridoma A1.1 cells, thus allowing us to examine the expression pattern of these molecules under precisely the same conditions. We have previously reported that CD95L expression requires both protein kinase C (PKC) translocation and Ca2+ mobilization and is inhibited by cyclosporin A, and dexamethasone. We demonstrate now that activation-induced expression of RANKL is mediated by Ca2+ mobilization. PKC activation does not induce RANKL expression nor does it synergize with the Ca2+ signal. Activation-induced RANKL expression is blocked by cyclosporin A, but not by dexamethasone. The expression of TNF, in contrast, is mediated by PKC, but not by Ca2+. TNF-alpha expression is not inhibited by cyclosporin A, but is sensitive to dexamethasone. A1.1 cells constitutively express TRAIL at low levels. Stimulation with anti-CD3 leads to an initial reduction and subsequent increase in TRAIL expression. TRAIL induction is not inhibited by cyclosporin A, but highly sensitive to dexamethasone. Therefore, expression of the TNF superfamily genes is regulated by distinct signals. Detailed understanding of the regulatory mechanisms could provide crucial information concerning the role of these molecules in the modulation of the immune system.