Biomedical subjects

H Zhou

Publications and source records attributed to H Zhou.

At least 307 records · Page 17Linked to original sources

PubMed1999

Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons.

The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human alpha(1)-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3-5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helper-dependent vectors with sequential delivery of vectors of different serotypes.

Adenoviridae↗

PubMed1999

Phenylalanine 138 in the second intracellular loop of human thromboxane receptor is critical for receptor-G-protein coupling.

Eicosanoid receptors exhibit a highly conserved ERY(C)XXV(I)XXPL sequence in the second intracellular loop. The carboxyl end of this motif contains a bulky hydrophobic amino acid (L,I,V, or F). In human thromboxane A2 receptor (TXA(2)R), phenylalanine 138 is located at the carboxyl end of this highly conserved motif. This study examined the function of the F138 in G protein coupling. F138 was mutated to aspartic acid (D) and tyrosine (Y), respectively. Both mutants F138D and F138Y showed similar ligand binding activity to that of the wild type TXA(2)R. The Kd and Bmax values of either mutant were comparable to those of the wild type receptor. However, both mutants showed significant impairment of agonist induced Ca(2+) signaling and phospholipase C activation. These results suggest that the F138 plays a key role in G protein coupling.

Amino Acid Sequence↗

PubMed1999

Characterization of recombinant human CXCR4 in insect cells: role of extracellular domains and N-glycosylation in ligand binding.

The cDNA of the human CXCR4/fusin was isolated from a human HeLa cell cDNA library by PCR and functionally expressed in Sf9 insect cells. The recombinant receptor was found to bind its natural ligand SDF-1alpha with an affinity comparable to that of the native receptor. Sequence-specific antibodies against each of the four extracellular domains were generated and used to investigate the interactions between the different domains of the receptor and the ligand. Each of the four antibodies was found to be able to inhibit ligand binding. CXCR4 was shown to be a glycoprotein. The role of N-glycosylation of CXCR4 in ligand binding was investigated in the insect cells overexpressed with recombinant CXCR4. Two potential N-glycosylation sites (Asn-11 and Asn-176) were either singly or doubly mutated to a leucine residue. Both single mutant receptors exhibited a significant decrease in ligand binding activity and affinity. The double mutant receptor showed little binding activity. Our data suggest that all of the extracellular domains are involved in ligand-receptor interactions and that N-glycosylation is required to maintain high-affinity ligand binding.

Animals↗

PubMed1999

Excited-state electronic asymmetry of the special pair in photosynthetic reaction center mutants: absorption and Stark spectroscopy.

The electronic absorption line shape and Stark spectrum of the lowest energy Q(y)() transition of the special pair in bacterial reaction centers contain a wealth of information on mixing with charge transfer states and electronic asymmetry. Both vary greatly in mutants that perturb the chemical composition of the special pair, such as the heterodimer mutants, and in mutants that alter interactions between the special pair and the surrounding reaction center protein, such as those that add or remove hydrogen bonds. The conventional and higher-order Stark spectra of a series of mutants are presented with the aim of developing a systematic description of the electronic structure of the excited state of the special pair that initiates photosynthetic charge separation. The mutants L168HF, M197FH, L131LH and L131LH/M160LH/M197FH are known to have different hydrogen-bonding patterns to the special pair; however, they exhibit Stark effects that are very similar to wild type. By contrast, the addition of a hydrogen bond to the M-side keto carbonyl group of the special pair in M160LH greatly affects both the absorption and Stark spectra. The heterodimer special pairs, L173HL and M202HL, exhibit much larger Stark effects than wild type, with the greatest effect in the M-side mutant. Double mutants that combine the M-side heterodimer and a hydrogen-bond addition to the L-side of the special pair decrease the magnitude of the Stark effect. These results suggest that the electronic asymmetry of the dimer can be perturbed either by the formation of a heterodimer or by adding or deleting a hydrogen bond to a keto carbonyl group. From the pattern observed, it is concluded that the charge transfer state P(L)(+)P(M)(-) has a larger influence on the excited state of the dimer in wild type than the P(L)(-)P(M)(+)charge transfer state. Furthermore, asymmetry can be varied continuously, from extreme cases in which the heterodimer and hydrogen-bond effects work together, to cases in which hydrogen bonding offsets the effects of the heterodimer, to cases in which the homodimer is perturbed by hydrogen bonds. This leads to a unified model for understanding the effects of perturbations on the electronic symmetry of the special pair, and this can be connected with perturbations on the properties of many other systems such as donor-acceptor-substituted polyenes.

Dimerization↗

PubMed1999

Distinct domains of MuSK mediate its abilities to induce and to associate with postsynaptic specializations.

Agrin released from motor nerve terminals activates a muscle-specific receptor tyrosine kinase (MuSK) in muscle cells to trigger formation of the skeletal neuromuscular junction. A key step in synaptogenesis is the aggregation of acetylcholine receptors (AChRs) in the postsynaptic membrane, a process that requires the AChR-associated protein, rapsyn. Here, we mapped domains on MuSK necessary for its interactions with agrin and rapsyn. Myotubes from MuSK(-/)- mutant mice form no AChR clusters in response to agrin, but agrin-responsiveness is restored by the introduction of rat MuSK or a Torpedo orthologue. Thus, MuSK(-/)- myotubes provide an assay system for the structure-function analysis of MuSK. Using this system, we found that sequences in or near the first of four extracellular immunoglobulin-like domains in MuSK are required for agrin responsiveness, whereas sequences in or near the fourth immunoglobulin-like domain are required for interaction with rapsyn. Analysis of the cytoplasmic domain revealed that a recognition site for the phosphotyrosine binding domain-containing proteins is essential for MuSK activity, whereas consensus binding sites for the PSD-95/Dlg/ZO-1-like domain-containing proteins and phosphatidylinositol-3-kinase are dispensable. Together, our results indicate that the ectodomain of MuSK mediates both agrin- dependent activation of a complex signal transduction pathway and agrin-independent association of the kinase with other postsynaptic components. These interactions allow MuSK not only to induce a multimolecular AChR-containing complex, but also to localize that complex to a primary scaffold in the postsynaptic membrane.

Adenosine Triphosphate↗

PubMed1999

Essential role for oncogenic Ras in tumour maintenance.

Advanced malignancy in tumours represents the phenotypic endpoint of successive genetic lesions that affect the function and regulation of oncogenes and tumour-suppressor genes. The established tumour is maintained through complex and poorly understood host-tumour interactions that guide processes such as angiogenesis and immune sequestration. The many different genetic alterations that accompany tumour genesis raise questions as to whether experimental cancer-promoting mutations remain relevant during tumour maintenance. Here we show that melanoma genesis and maintenance are strictly dependent upon expression of H-RasV12G in a doxycycline-inducible H-Ras12G mouse melanoma model null for the tumour suppressor INK4a. Withdrawal of doxycycline and H-RasV12G down-regulation resulted in clinical and histological regression of primary and explanted tumours. The initial stages of regression involved marked apoptosis in the tumour cells and host-derived endothelial cells. Although the regulation of vascular endothelial growth factor (VEGF) was found to be Ras-dependent in vitro, the failure of persistent endogenous and enforced VEGF expression to sustain tumour viability indicates that the tumour-maintaining actions of activated Ras extend beyond the regulation of VEGF expression in vivo. Our results provide genetic evidence that H-RasV12G is important in both the genesis and maintenance of solid tumours.

Animals↗

PubMed1999

Use of a liver-specific promoter reduces immune response to the transgene in adenoviral vectors.

Previous studies using adenoviral (Ad) vectors expressing human alpha1-antitrypsin (hAAT) under the control of ubiquitous promoters (RSV, mPGK) elicited the production of antibodies to hAAT in some mouse strains (C3H/HeJ and BALB/c) but not in others (C57BL/6J). In contrast, when a helper-dependent Ad vector (AdSTK109) with all viral coding sequences deleted and expressing hAAT from human genomic DNA with the endogenous promoter was used, C3H/HeJ mice failed to develop antibodies and demonstrated long-term expression. These results suggested that promoter choice and/or properties of the vector itself might influence the host immune response to the transgene product. Direct comparison of first-generation vectors expressing the hAAT cDNA from a ubiquitous mouse PGK promoter rather than from a liver-specific mouse albumin promoter demonstrated that an antibody response to hAAT occurred with the mPGK promoter but not with the albumin promoter in C3H/HeJ mice. As expected, neither vector elicits an antibody response in C57BL/6J mice. Coinjection of the two first-generation vectors containing the mPGK and albumin promoter in C3H/HeJ mice induced an antibody response with resulting loss of detectable hAAT from the sera of the injected mice in 3-4 weeks. From these data, we conclude that under certain conditions, the choice of promoter with its associated liver-specific expression can modulate the host immune response to the transgene independent of viral backbone.

Adenoviridae↗

PubMed1999

Molecular oxygen oxidizes the porphyrin ring of the ferric alpha-hydroxyheme in heme oxygenase in the absence of reducing equivalent.

Heme oxygenase catalyzes the regiospecific oxidative degradation of iron protoporphyrin IX (heme) to biliverdin, CO and Fe, utilizing molecular oxygen and electrons donated from the NADPH-cytochrome P-450 reductase. The catalytic conversion of heme proceeds through two known heme derivatives, alpha-hydroxyheme and verdoheme. In order to assess the requirement of reducing equivalents in the second stage of heme degradation, from alpha-hydroxyheme to verdoheme, we have prepared the alpha-hydroxyheme complex with rat heme oxygenase isoform-1 and examined its reactivity with molecular oxygen in the absence of added electrons. Upon reaction with oxygen, the majority of the alpha-hydroxyheme in heme oxygenase is altered to a species which exhibits an optical absorption spectrum with a broad Soret band, along with the minority which is converted to verdoheme. The major product species, which is electron paramagnetic resonace-silent, can be recovered to the original alpha-hydroxyheme by addition of sodium dithionite. We have also found that oxidation of the alpha-hydroxyheme-heme oxygenase complex by ferricyanide or iridium(IV) chloride yields a species which exhibits an optical absorption spectrum and reactivity similar to those of the main product of the oxygen reaction. We infer that the oxygen reaction with the ferric alpha-hydroxyheme-heme oxygenase complex forms a ferric-porphyrin cation radical. We conclude that in the absence of reducing agents, the oxygen molecule functions mainly as an oxidant for the porphyrin ring and has no role in the oxygenation of alpha-hydroxyheme. This result corroborates our previous conclusion that the catalytic conversion of alpha-hydroxyheme to verdoheme by heme oxygenase requires one reducing equivalent along with molecular oxygen.

Electron Spin Resonance Spectroscopy↗

PubMed1999

Attenuation of morphine tolerance and dependence in scopolamine-treated rats.

Morphine tolerance and dependence were investigated in scopolamine-treated rats. The results showed that scopolamine treatment (up to 2 mg/kg) did not affect basal line or morphine-induced latency in the tail-flick test but significantly increased the escape latency in the Morris water-maze task. Co-administration of scopolamine could considerably attenuate development of morphine tolerance and naloxone-precipitated withdrawal syndrome. Pretreatment of scopolamine for 7 days prior to morphine administration also significantly reduced the tolerance and withdrawal symptoms. Scopolamine treatment was further shown to attenuate morphine-induced conditioned place preference, an indicator of morphine psychological dependence. The present study demonstrated the attenuation of morphine tolerance and dependence in scopolamine-treated animals, probably related to scopolamine-produced learning and memory impairment.

Analgesics, Opioid↗

PubMed1999

Tyrosine kinase inhibitors. 15. 4-(Phenylamino)quinazoline and 4-(phenylamino)pyrido[d]pyrimidine acrylamides as irreversible inhibitors of the ATP binding site of the epidermal growth factor receptor.

A series of 6- and 7-acrylamide derivatives of the 4-(phenylamino)quinazoline and -pyridopyrimidine classes of epidermal growth factor receptor (EGFR) inhibitors were prepared from the corresponding amino compounds by reaction with either acryloyl chloride/base or acrylic acid/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. All of the 6-acrylamides, but only the parent quinazoline 7-acrylamide, were irreversible inhibitors of the isolated enzyme, confirming that the former are better-positioned, when bound to the enzyme, to react with the critical cysteine-773. Quinazoline, pyrido[3,4-d]pyrimidine, and pyrido[3,2-d]pyrimidine 6-acrylamides were all irreversible inhibitors and showed similar high potencies in the enzyme assay (likely due to titration of the available enzyme). However the pyrido[3,2-d]pyrimidine analogues were 2-6-fold less potent than the others in a cellular autophosphorylation assay for EGFR in A431 cells. The quinazolines were generally less potent overall toward inhibition of heregulin-stimulated autophosphorylation of erbB2 (in MDA-MB-453-cells), whereas the pyridopyrimidines were equipotent. Selected compounds were evaluated in A431 epidermoid and H125 non-small-cell lung cancer human tumor xenografts. The compounds showed better activity when given orally than intraperitoneally. All showed significant tumor growth inhibition (stasis) over a dose range. The poor aqueous solubility of the compounds was a drawback, requiring formulation as fine particulate emulsions.

Acrylamides↗

PubMed1999

Threonine 188 is critical for interaction with NAD+ in human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in the inactivation pathway of prostaglandins. It is a member of the short-chain dehydrogenase family of enzymes. A relatively conserved threonine residue corresponding to threonine 188 of 15-PGDH is proposed to be involved in the interaction with the carboxamide group of NAD+. Site-directed mutagenesis was used to examine the important role of this residue. Threonine 188 was changed to alanine (T188A), serine (T188S) or tyrosine (T188Y) and the mutant proteins were expressed in E. coli. Western blot analysis showed that the expression levels of mutant proteins were similar to that of the wild type protein. Mutants T188A and T188Y were found to be inactive. Mutant T188S still retained substantial activity and the Km value for PGE2 was similar to the wild enzyme; however, the Km value for NAD+ was increased over 100 fold. These results suggest that threonine 188 is critical for interaction with NAD+ and contributes to the full catalytic activity of 15-PGDH.

Amino Acid Sequence↗

PubMed1999

[Cushing syndrome with occult ectopic ACTH production].

A 19-year old patient was admitted to our hospital with the typical stigmata of a prolonged Cushing's syndrome. He presented first at another hospital with elevated ACTH values and the diagnosis of pituitary Cushing's syndrome was made. A partial hypophysectomy was performed, but neither this nor a therapy with ketokonazole could influence the cortisol levels. Because of this background the diagnosis of ectopic ACTH-production was established. Since we were not able to locate a source of ACTH, we recommended a bilateral adrenalectomy, after which the symptoms of Cushing's syndrome disappeared within a few months. About one year after the operation a CT-scan demonstrated an intrapulmonary mass, which was removed surgically. There were neither signs of local infiltration nor of lymphogenic metastasis. The histological and immunhistochemical diagnosis was typical of bronchial carcinoid with ACTH-production. The follow up so far did not reveal a recurrence.

ACTH Syndrome, Ectopic↗

PubMed1999

Interaction between the skeletal muscle type 1 Na+ channel promoter E-box and an upstream repressor element. Release of repression by myogenin.

We have defined how four elements that regulate expression of the rat skeletal muscle type 1 sodium channel (SkM1) gene cooperate to yield specific expression in differentiated muscle. A basal promoter region containing within it a promoter E-box (-31/-26) is broadly expressed in many cells, including myoblasts and myotubes; mutations within the promoter E-box that disrupt binding of the myogenic basic helix-loop-helix (bHLH) factors reduce expression in all cell types only slightly. Sequential addition of upstream elements to the wild-type promoter confer increasing specificity of expression in differentiated cells, even though all three upstream elements, including a positive element (-85/-57), a repressor E-box (-90/-85), and upstream repressor sequences (-135/-95), bind ubiquitously expressed transcription factors. Mutations in the promoter E-box that disrupt the binding of the bHLH factors counteract the specificity conferred by addition of the upstream elements, with the greatest interaction observed between the upstream repressor sequences and the promoter E-box. Forced expression of myogenin in myoblasts releases repression exerted by the upstream repressor sequences in conjunction with the wild-type, but not mutant, promoter E-box, and also initiates expression of the endogenous SkM1 protein. Our data suggest that particular myogenic bHLH proteins bound at the promoter E-box control expression of SkM1 by releasing repression exerted by upstream repressor sequences in differentiated muscle cells.

Animals↗

PubMed1999

Angiopoietins 3 and 4: diverging gene counterparts in mice and humans.

The angiopoietins have recently joined the members of the vascular endothelial growth factor family as the only known growth factors largely specific for vascular endothelium. The angiopoietins include a naturally occurring agonist, angiopoietin-1, as well as a naturally occurring antagonist, angiopoietin-2, both of which act by means of the Tie2 receptor. We now report our attempts to use homology-based cloning approaches to identify new members of the angiopoietin family. These efforts have led to the identification of two new angiopoietins, angiopoietin-3 in mouse and angiopoietin-4 in human; we have also identified several more distantly related sequences that do not seem to be true angiopoietins, in that they do not bind to the Tie receptors. Although angiopoietin-3 and angiopoietin-4 are strikingly more structurally diverged from each other than are the mouse and human versions of angiopoietin-1 and angiopoietin-2, they appear to represent the mouse and human counterparts of the same gene locus, as revealed in our chromosomal localization studies of all of the angiopoietins in mouse and human. The structural divergence of angiopoietin-3 and angiopoietin-4 appears to underlie diverging functions of these counterparts. Angiopoietin-3 and angiopoietin-4 have very different distributions in their respective species, and angiopoietin-3 appears to act as an antagonist, whereas angiopoietin-4 appears to function as an agonist.

Amino Acid Sequence↗

PubMed1999

Potential for bias in estimating human fecundability parameters: a comparison of statistical models.

Fecundability studies, where couples attempting pregnancy subject to 'failure' (conception) one time in each menstrual cycle, present a natural discrete failure-time scenario. Because the biologic capacity to conceive varies among couples in the population, a complication arises in choosing a method of analysis, related to the fact that the maximum follow-up time can vary from study to study, and follow-up time could potentially have different effects on parameters based on different approaches to modelling. Traditional development in fertility studies has been based on an implicit assumption that binary outcomes for different menstrual cycles are mutually independent. We contrast traditional models to a random effects model where cycle viability is modelled as subject-specific. We clarify the interpretations for different parameters from different models. We show that the traditional approach yields some regression parameters that depend on follow-up time, limiting the generalizability of inferences based on this analytic approach. By contrast, the subject-specific model consistently estimates parameters of interest, if the underlying distribution is properly specified. Data from a fecundability study carried out in North Carolina serves to illustrate these points.

Bias↗

PubMed1999

Carboxy-substituted cinnamides: a novel series of potent, orally active LTB4 receptor antagonists.

A series of carboxy-substituted cinnamides were investigated as antagonists of the human cell surface leukotriene B4 (LTB4) receptor. Binding was determined through measurement of [3H]LTB4 displacement from human neutrophils. Receptor antagonism was confirmed through a functional assay, which measures inhibition of Ca2+ release in human neutrophils. Potent antagonists were discovered through optimization of a random screening hit, a p-(alpha-methylbenzyloxy)cinnamide, having low-micromolar activity. Substantial improvement of in vitro potency was realized by the attachment of a carboxylic acid moiety to the cinnamide phenyl ring through a flexible tether, leading to identification of compounds with low-nanomolar potency. Modification of the benzyloxy substituent, either through ortho-substitution on the benzyloxy phenyl group or through replacement of the ether oxygen with a methylene or sulfur atom, produced achiral antagonists of equal or greater potency. The most potent compounds in vitro were assayed for oral activity using the arachidonic acid-induced mouse ear edema model of inflammation. Several compounds in this series were found to significantly inhibit edema formation and myeloperoxidase activity in this model up to 17 h after oral administration. Representatives of this series have been shown to be potent and long-acting orally active inhibitors of the LTB4 receptor.

Administration, Oral↗

PubMed1999

Coimmobilization of glucoamylase and glucose isomerase by molecular deposition technique for one-step conversion of dextrin to fructose.

Glucose isomerase was immobilized by itself with adsorption and coimmobilized with glucoamylase by molecular deposition technique using macroporous trimethylamine polystyrene beads. Approximately 77.5% of the enzyme added was immobilized. The pH-activity curve of the immobilized glucose isomerase was broadened, resulting in 75% retention of its maximum activity at pH 6.2. The Km of the immobilized glucose isomerase was 1.28-fold that of the soluble one. When the two enzymes were immobilized together, the system was found capable of functioning at pH 6.0 to produce fructose from starch and dextrin. At this pH, the total fructose output of the coimmobilized enzyme system after 24 h was 1.9 times that of the free enzyme system.

Aldose-Ketose Isomerases↗

PubMed1999

A cysteine-rich form of Xenopus neuregulin induces the expression of acetylcholine receptors in cultured myotubes.

Neuregulin-1 (NRG-1) has diverse functions in neural development, and one of them is to up regulate the expression of acetylcholine receptors (AChRs) at muscle fibers during the formation of neuromuscular junctions. NRG-1 has two prominent alternative splicing sites at the N-terminus; it could be an immunoglobulin (Ig)-like domain named Ig-NRG-1 or an apolar cysteine-rich domain (CRD) named CRD-NRG-1. cDNAs encoding Xenopus CRD-NRG-1 were isolated by cross-hybridization with Xenopus Ig-NRG-1 cDNA fragment. The amino acid sequence of Xenopus CRD-NRG-1 is 45 to 70% identical to the human, rat, and chick homologs. Similar to Ig-NRG-1, two variation sites within CRD-NRG-1 were identified at the spacer domain with 0 or 43 amino acids inserted and at the C-terminus of the EGF-like domain to derive either alpha or beta isoform. Two transcripts encoding CRD-NRG-1, approximately 7.5 and approximately 9.0 kb, were revealed in adult brain and spinal cord, but the expression in muscle was below the detectable level. The recombinant Xenopus CRD-NRG-1 when applied onto cultured myotubes was able to induce the tyrosine phosphorylation of ErbB receptors and the expression of AChR. The AChR-inducing activity of CRD-NRG-1 was precipitated by anti-NRG-1 antibody but not by heparin. In situ hybridization showed a strong expression of CRD-NRG-1 mRNA in developing brain, spinal cord, and myotomal muscles of Xenopus embryo. Similar to the results in other species, both CRD-NRG-1 and Ig-NRG-1 may play a role in the developing Xenopus neuromuscular junctions.

Amino Acid Sequence↗