Search PubMed⌕ Search

Biomedical subjects

A Zhou

Publications and source records attributed to A Zhou.

At least 109 records · Page 6Linked to original sources
PubMed1996

Peptide biosynthetic processing: distinguishing prohormone convertases PC1 and PC2.

To determine whether manipulation of time, temperature and intragranular pH could be used to distinguish the actions of two subtilisin-related endoproteases, PC1 and PC2, in peptide biosynthesis, the biosynthetic processing of proneuropeptide Y (proNPY) and proopiomelanocortin (POMC) was examined in pituitary cell lines. AtT-20 cells express PC1 and POMC endogenously; stably transfected AtT-20 lines expressing NPY or PC2 were studied. GH3 cells express PC2 endogenously; NPY-expressing GH3 transfectants were investigated. PC1 mediated rapid processing of NPY and POMC; PC1-dependent cleavages were relatively insensitive to 20 degrees C blockade (which arrests secretory pathway transport at the trans-Golgi network) and do not require an acidic intracellular compartment (as in secretory granules). PC2 mediated much slower processing of proNPY and POMC which was totally blocked at 20 degrees C and required an acidic intracellular compartment. Thus, kinetics, abolition of intracellular pH gradients, and incubation at reduced temperatures can be used to distinguish PC1 and PC2 actions in neuroendocrine cells.

Animals↗
PubMed1996

Chromogranin A processing and secretion: specific role of endogenous and exogenous prohormone convertases in the regulated secretory pathway.

Chromogranins A and B and secretogranin II are a family of acidic proteins found in neuroendocrine secretory vesicles; these proteins contain multiple potential cleavage sites for proteolytic processing by the mammalian subtilisin-like serine endoproteases PC1 and PC2 (prohormone convertases 1 and 2), and furin. We explored the role of these endoproteases in chromogranin processing in AtT-20 mouse pituitary corticotropes. Expression of inducible antisense PC1 mRNA virtually abolished PC1 immunoreactivity on immunoblots. Chromogranin A immunoblots revealed chromogranin A processing, from both the NH2 and COOH termini, in both wild-type AtT-20 and AtT-20 antisense PC1 cells. After antisense PC1 induction, an approximately 66-kD chromogranin A NH2-terminal fragment as well as the parent chromogranin A molecule accumulated, while an approximately 50 kD NH2-terminal and an approximately 30 kD COOH-terminal fragment declined in abundance. Chromogranin B and secretogranin II immunoblots showed no change after PC1 reduction. [35S]Methionine/cysteine pulse-chase metabolic labeling in AtT-20 antisense PC1 and antisense furin cells revealed reciprocal changes in secreted chromogranin A COOH-terminal fragments (increased approximately 82 kD and decreased approximately 74 kD forms, as compared with wild-type AtT-20 cells) indicating decreased cleavage, while AtT-20 cells overexpressing PC2 showed increased processing to and secretion of approximately 71 and approximately 27 kD NH2-terminal chromogranin A fragments. Antisense PC1 specifically abolished regulated secretion of both chromogranin A and beta-endorphin in response to the usual secretagogue, corticotropin-releasing hormone. Moreover, immunocytochemistry demonstrated a relative decrease of chromogranin A in processes (where regulated secretory vesicles accumulate) of AtT-20 cells overexpressing either PC1 or PC2. These results demonstrate that chromogranin A is a substrate for the endogenous endoproteases PC1 and furin in vivo, and that such processing influences its trafficking into the regulated secretory pathway; furthermore, lack of change in chromogranin B and secretogranin II cleavage after diminution of PCl suggests that the action of PC1 on chromogranin A may be specific within the chromogranin/secretogranin protein family.

Animals↗
PubMed1996

IFN-alpha beta reconstitutes the deficiency in lipid A-activated AKR macrophages for nitric oxide synthase.

AKR mouse peritoneal macrophages (PM) were previously found to have a defect in their response to lipid A for nitric oxide (NO)-mediated tumor cytotoxicity, which was related to a lower level of C1q synthesis and reconstituted by exogenous IFN-gamma or C1q. We used AKR-PM as a model to further define the role of IFN-alpha beta in modulation of induction of macrophage nitric oxide synthase (NOS) in response to lipid A. Studies have revealed that AKR-PM produced a significantly lower level of IFN-alpha beta than responsive C3H-PM in response to lipid A. AKR-PM failed to increase NOS mRNA synthesis and NO generation when exposed to lipid A, although they had normal levels of TNF-alpha bioactivity and mRNA expression. This partial deficiency of AKR-PM to lipid A stimulation was reconstituted completely by exogenous IFN-alpha beta for both synthesis of NOS mRNA and release of NO. The failure of AKR-PM to produce NOS to lipid A stimulation appears to be related to reduced secretion of IFN-alpha beta and the resultant failure to express TNF-alpha type II receptor (TNF-RII) mRNA, which in turn decreases TNF-alpha binding to its receptor for autocrine induction of NOS. Insufficient synthesis and secretion of endogenous IFN-alpha beta may be the primary reason for AKR-PM refractoriness to induction of NOS in response to lipid A. furthermore, the close correlation between lack of IFN-alpha beta secretion and decreased TNF-RII mRNA synthesis may implicate a critical role for IFN-alpha beta in the upregulation of macrophage TNF-RII receptor expression for autocrine induction of NOS during lipid A stimulation.

Animals↗
PubMed1996

Complement subcomponent C1q modulation of TNF-alpha binding to L929 cells for enhanced TNF-mediated cytotoxicity.

Complement subcomponent C1q has been recently implicated in the modulation of autocrine binding of TNF-alpha to murine macrophages for induction of nitric oxide synthase. In the present study, the putative role of C1q in increasing TNF-alpha binding to L929 cells to mediate cytotoxicity was explored. TNF-sensitive L929 cells (L929-S) had higher total endogenous cellular and surface C1q levels and bound correspondingly more phycoerythrin-labelled rTNF-alpha (PE-TNF) than did a TNF-resistant L929 variant (L929-R). Pretreatment of L929-S with soluble C1q increased their sensitivity to TNF-mediated cytotoxicity coincident with increased binding of PE-TNF, but similar treatment of L929-R had no effect. Pretreatment of L929-S with an inhibitor of C1q secretion, 3,4 dehydro-D,L-proline (DHP), resulted in a decrease in their TNF-mediated cytotoxicity, as well as reduced binding of PE-TNF. Subsequent exposure of DHP-treated L929-S with exogenous soluble C1q restored their TNF-mediated cytotoxicity and binding of PE-TNF. These results provide evidence for the modulation of TNF-alpha binding to TNF sensitive tumour targets L929 by either endogenously synthesized or exogenously added C1q to promote TNF-mediated cytotoxicity by mechanisms which remain to be elucidated.

Animals↗
PubMed1996

High-level expression of active human plasminogen activator inhibitor type 1 (PAI-1) in E. coli.

Plasminogen activator inhibitor 1 (PAI-1) is an important regulator of plasminogen activation, which inhibits both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). The DNA sequence encoding mature PAI-1 protein was inserted into an inducible expression vector. This gene was highly expressed to produce a soluble active protein in E. coli cells. The amount of the recombinant protein was up to 20% of total cellular protein. By efficient purification with a yield of about 15-20%, the recombinant protein could be purified to homogeneity with its specific activity up to 6.1 x 10(4) (uPA) IU/mg. Its inhibitory activity declined during incubation at 37 degrees C with a half life of about 2 hr.

Escherichia coli↗
PubMed1996

Translation initiation region plays an important role in the expression of human thrombopoietin in Escherichia coli.

A mutant human thrombopoietin (TPO) gene with a modified translation initiation region (TIR) sequence was created by site-specific mutagenesis based on the PCR technique. This mutant TPO gene encoded the same amino acid sequence as wild-type TPO gene. The wild-type TPO gene was expressed in E. coli with very low efficiency. The mutant TPO gene could reach an expression level of up to 10% of total cellular proteins in E. coli, which was much higher than the wild-type gene. The recombinant protein was mainly in the form of inclusion body which could acquire in vitro activities of human thrombopoietin after refolding.

Escherichia coli↗
PubMed1996

Autocrine induction of macrophage synthesis of complement subcomponent C1q by endogenous interferon-alpha/beta.

Peritoneal macrophages (M phi) constitutively synthesize and secrete interferon-alpha (IFN-alpha) and IFN-beta, as well as complement subcomponent C1q. Because exogenous interferon-gamma (IFN-gamma) stimulates Mø synthesis of C1q, our purpose was to determine if endogenous secretion of IFN-alpha/beta regulated the constitutive level of endogenous C1q mRNA synthesis in an autocrine fashion. Both exogenous IFN-alpha and IFN-beta effectively substituted for IFN-gamma in stimulating M phi C1q mRNA expression in a dose-dependent fashion by northern blot analysis. Neutralizing anti-INF-alpha/beta antibodies inhibited M phi constitutive C1q mRNA synthesis by approximately twofold and abrogated the feedback stimulatory effects of exogenous C1q on C1q mRNA expression. Paraffin oil-elicited inflammatory M phi displayed distinctively different constitutive levels of C1q mRNA expression from thioglycollate brothelicited M phi, which was correlated with their relative levels of secretory IFN-alpha/beta by ELISA. Exogenous IFN-alpha/beta also restored C1q mRNA synthesis of AKR mouse M phi with low constitutive C1q mRNA expression. The cumulative results support the concept that constitutive synthesis of C1q by M phi is regulated by the endogenous synthesis and secretion of IFN-alpha/beta, which appears to act in an autocrine fashion.

Animals↗
PubMed1996

[Molecular bypass, the application of VEGF to gene therapy of limb ischemia].

OBJECTIVE: To investigate the feasibility of using VEGF gene directly for treating limb ischemia. METHODS: The human VEGF165 cDNA was cloned into the eukaryotic expression vector pcDNA3 under the control of cytomegalovirus immediate-early promoter/enhancer. When gene suture was used, the recombinant plasmid was transferred into the hindlimb's adductor of rat, of which the distal extreme of external iliac artery was ligated and the femoral artery was completely excised. With RT-PCR, Western blot, immunohistochemistry analysis, and angiography, the expression and biological effects of VEGF gene in experimental animals were investigated. RESULTS: Seven days after initiation of therapy, the transcription and expression of VEGF gene in experimental group were significantly higher than those of control groups. The results from angiography, feet temperature measuring and observation of clinical symptom showed that the transfer of VEGF gene can stimulate the formation of focal neovessles, establish colleteral circulation, augment blood perfusion, and ameliorate distal limb necrosis. CONCLUSION: These findings reveal that the "molecular bypass" using VEGF gene may represent a potential therapeutic modality for treating tissue ischemia.

Animals↗
PubMed1996

[Loss of p16 gene in bladder cancer and genitourinary carcinoma cell lines].

To determine whether p16, a novel antioncogene, is altered in human bladder cancer and urogenital carcinoma cell lines, we examined a series of 10 primary bladder cancer and 4 urogenital carcinoma cell lines (BIU-87, EJ, GRC and PC-3M) by southern blot. Two cases of 10 primary bladder cancer and EJ, BIU-87 PC-3M had homozygous deletion of p16, and 2 cases of 10 primary bladder cancer had loss of heterozygous. Loss of p16 may provide an additional growth advantage and carcinogenesis.

Carcinoma, Squamous Cell↗
PubMed1995

Structural elements that direct specific processing of different mammalian subtilisin-like prohormone convertases.

PC1 and PC2 are two important subtilisin-like prohormone convertases (PC) that undergo differential endoproteolytic processing steps and sequentially mediate proopiomelanocortin (POMC) processing. To investigate the structural elements directing the processing of different PCs, we constructed a series of mutant and chimeric PC proteins and expressed them in cell lines with different patterns of expression of endogenous PCs: AtT-20, hEK293, and hLoVo cells. The COOH-terminally truncated PC1 underwent efficient proregion cleavage and rapid secretion in all three cell lines, while proregion cleavage and secretion were completely blocked in an active-site mutant of PC1. The truncated PC1 produced dramatic changes in POMC processing in AtT-20 cells. PC2 with the potential oxyanion hole Asp residue changed to Asn was processed and altered several aspects of POMC processing in a manner similar to that of wild-type PC2. PC1 protein with its proregion substituted with that of furin was cleaved after its proregion, producing active PC1 enzyme. A similar furin/PC2 fusion protein underwent proregion cleavage at low efficiency. By contrast, when the proregions of PC1 and PC2 were substituted with one another, both fusion proteins failed to cleave the foreign prosequences, were unable to undergo oligosaccharide maturation, and remained in the ER. Although inactive PC mutants could theoretically function as dominant negatives, none interfered with the processing of endogenous active PCs or with POMC processing. We conclude that the COOH-terminal of PC1 plays an important role in the routing or storage of PC1, the proregions of these PC proteins are replaceable in a molecule-specific manner, removal of proregion is essential for routing and for endoproteolytic activity, and the role of the potential oxyanion hole in PC2 is still unclear.

Amino Acid Sequence↗
PubMed1995

Exogenous interferon-gamma induces endogenous synthesis of interferon-alpha and -beta by murine macrophages for induction of nitric oxide synthase.

Murine macrophages (M phi) are activated either by interferon-gamma (IFN-gamma) or interferon-alpha/beta (IFN-alpha/beta) in combination with bacterial lipopolysaccharide (LPS) to induce synthesis of tumor necrosis factor alpha (TNF-alpha) and nitric oxide synthase (iNOS) mRNA synthesis for generation of tumor cytotoxic nitric oxide (NO). In the present study, the effect of exogenous IFN-gamma on the induction of endogenous mRNA synthesis and secretion of IFN-alpha/beta by murine M phi was investigated. Neutralizing antibodies to IFN-alpha/beta reversed TNF-alpha and NOS mRNA synthesis, as well as nitric oxide (NO)-mediated tumor cytotoxicity. Quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed that treatment of M phi with IFN-gamma induced increases in both IFN-alpha and IFN-beta mRNA synthesis by approximately 2-fold and 10-fold, respectively, which corresponded to a 2-fold increase in secretion of IFN-alpha/beta by ELISA. These data indicate that exogenous IFN-gamma induces endogenous synthesis and secretion of IFN-alpha/beta by M phi, which appears to act in concert with endogenously synthesized TNF-alpha for the autocrine induction of NOS mRNA synthesis.

Animals↗
PubMed1995

Expression of human plasminogen activator inhibitor-2 in the baculovirus expression system.

A cDNA coding for human plasminogen activator inhibitor-2 (PAI-2) was transferred into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Cells infected with recombinant virus synthesized a 43-kDa PAI-2 protein, the majority of which was secreted. The recombinant PAI-2 was identical to native PAI-2 purified from human placenta with respect to interaction with polyclonal antibody and inhibition of urokinase-type plasminogen activator.

Animals↗
PubMed1995

Adenovirus mediated gene transfer of vascular smooth muscle cells and endothelial cells in vitro.

Introducing foreign gene(s) into vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) is the pre-requirement of gene therapy for cardiovascular diseases. We have explored the use of adenoviral vectors (Adv-CMV/LacZ) to transfer LacZ gene into cultured VSMCs and ECs. Our results demonstrated that adenoviral vectors transferred foreign gene into VSMCs and ECs high-efficiently with dose-dependent response pattern. The frequencies of transfection reached 100% at the viral titer of 10(9) pfu/ml. Comparing the sensitivities of VSMCs and ECs to adenoviral vectors, we found that ECs were more sensitive than VSMCs, of which the frequencies of transfection in ECs reached 80% while in VSMCs only 40% for 8 hrs after transfection. In addition, the transfection of ECs and VSMCs with adenoviral vectors was partly blocked by monoclonal antibodies to Fiber and Core protein of the adenoviral capsid, but not by monoclonal antibody to Hexon protein. It is suggested transfection of ECs and VSMCs with adenovirus vectors is mediated by Fiber or Core protein of adenoviral capsid proteins.

Adenoviruses, Human↗
PubMed1995

Elevated levels of 2',5'-linked oligoadenylate-dependent ribonuclease L occur as an early event in colorectal tumorigenesis.

RNA decay in IFN-treated cells is controlled by 2'5'-linked oligoadenylate (2-5A)-dependent RNase (RNase L), a uniquely regulated endoribonuclease that requires short 5'-phosphorylated, 2-5A for its activity. Because RNase L is also implicated in the regulation of cell proliferation, we monitored its expression in colorectal adenocarcinomas and noncancerous polyps from familial adenomatous polyposis patients. Elevated levels of RNase L mRNA and activity were found in 17 of 20 tumors compared with corresponding normal mucosa. An mAb against RNase L revealed elevated amounts of this RNase in sections of the tumors, largely in the base of the villi. The occurrence of elevated levels of RNase L seems to be an early event in colorectal tumorigenesis, suggesting that control of RNA turnover is an important step in tumor progression. These data also indicate that regulating RNase L activity may be a useful strategy in treating colorectal carcinomas.

Adenocarcinoma↗
PubMed1994

Endoproteolytic processing of proopiomelanocortin and prohormone convertases 1 and 2 in neuroendocrine cells overexpressing prohormone convertases 1 or 2.

AtT-20 mouse corticotrope tumor cell lines overexpressing the prohormone convertases PC1 or PC2 were established and used to examine prohormone and prohormone convertase biosynthetic processing. On a molar basis, wild-type AtT-20 cells synthesize about 20% as much PC1 as the endogenous prohormone, proopiomelanocortin (POMC). Kinetic, oligosaccharide, and temperature blockade analyses established that proPC1 is converted to PC1 in the endoplasmic reticulum at a rate independent of the level of PC1 or PC2 expression. In contrast, proPC2 is converted to PC2 primarily in a post-trans-Golgi compartment. PC1 is further shortened from its COOH-terminal end in a post-trans-Golgi compartment in a step that is accelerated at higher levels of PC1 expression, but unaltered by PC2 overexpression. The initial steps in POMC processing are speeded up by overexpression of PC1, and overexpression of PC1 leads to more extensive cleavage of POMC to smaller products. However, even when the rate of PC1 synthesis exceeds that for POMC by 2-fold, PC1 does not cleave the Lys-Lys or Arg-Lys bonds cleaved upon overexpression of PC2.

Animals↗
PubMed1994

Intrinsic molecular activities of the interferon-induced 2-5A-dependent RNase.

2-5A-dependent RNase (RNase L), a unique endoribonuclease that requires 5'-phosphorylated 2',5'-linked oligoadenylates (2-5A), functions in the molecular mechanism of interferon action. Because this enzyme is present at very low levels in nature, characterization and analysis have been limited. The molecular cloning of human, 2-5A-dependent RNase cDNA has facilitated its expression to high levels in insect cells by infecting with recombinant baculovirus. To determine the properties of the enzyme in the absence of other proteins, the recombinant 2-5A-dependent RNase was purified to homogeneity. The purified enzyme migrated as a monomer upon gel filtration in the absence of activator and showed highly specific, 2-5A-dependent RNase activity. The precise activator requirements were determined by stimulating the purified enzyme with a variety of 2',5'-linked oligonucleotides. The activated enzyme was capable of cleaving poly(rU) and, to a lesser extent, poly(rA), to sets of discrete products ranging from between 4 and 22 nucleotides in length. Reduced rates of 2-5A-dependent RNA cleavage were observed even after removal of ATP and chelation of divalent cations. However, optimal RNA cleavage rates required the presence of either manganese or magnesium and ATP.

Adenosine Triphosphate↗