Biomedical subjects

H Zhou

Publications and source records attributed to H Zhou.

At least 397 records · Page 22Linked to original sources

PubMed1998

Incidence and time course of thromboembolic outcomes following total hip or knee arthroplasty.

BACKGROUND: Little is known about the incidence and time course of clinical thromboembolic events after total hip or knee arthroplasty, particularly after hospital discharge. METHODS: We used a linked hospital discharge database provided by the State of California to identify cases diagnosed as having deep vein thrombosis or pulmonary embolism within 3 months of unilateral total hip or knee arthroplasty. Also, we surveyed orthopedic surgeons to estimate the frequency of postoperative thromboprophylaxis during July 1991 through June 1993. Medical charts were audited to determine the accuracy of the coded records. RESULTS: Among 19,586 primary hip and 24,059 primary knee arthroplasties, the cumulative incidence of deep vein thrombosis or pulmonary embolism within 3 months of surgery was 556 (2.8%) after hip arthroplasty and 508 (2.1%) after knee arthroplasty. The diagnosis of thromboembolism was made after hospital discharge in 76% and 47% of the total hip and total knee arthroplasty cases, respectively (P<.001), with a median time of diagnosis of 17 days and 7 days after surgery, respectively (P<.001). Questionnaire results indicated that 95% of all cases received thromboprophylaxis and that the frequency, type, and duration of thromboprophylaxis was virtually identical after hip and knee arthroplasty. CONCLUSIONS: There is a difference in the temporal patterns of clinically symptomatic thromboembolic complications after total hip and total knee arthroplasty, suggesting differences in pathogenesis or natural history. The findings suggest that to further reduce thromboembolic outcomes, earlier, more intense prophylaxis may be needed for total knee arthroplasty, and more prolonged prophylaxis may be required after total hip arthroplasty.

Aged↗

PubMed1998

Toxicological comparison of E2a-deleted and first-generation adenoviral vectors expressing alpha1-antitrypsin after systemic delivery.

Second-generation adenoviral vectors, mutated in E2a, have been proposed to decrease host immune responses against transduced cells, reduce toxicity, and increase duration of expression as compared with first-generation vectors deleted only in E1. To test these hypotheses further, we have developed an E2a-deleted adenoviral vector expressing human alpha1-antitrypsin (hAAT). Toxicity of first-generation and E2a-deleted vectors, as determined by hematological indices, liver function tests, and histological analyses, was evaluated in C3H mice for 21 days after vector administration at increasing doses starting at 1 x 10(12) particles/kg. Both vectors induced dose-dependent abnormalities including transient thrombocytopenia, elevated ALT levels in serum, and increased hepatocyte proliferation followed by inflammation and then hypertrophy. Differences in the ratio of particles to plaque-forming units among vector preparations led to differences in hAAT expression at similar particle doses. There were no differences in toxicity between the two vectors when measured at matching levels of hAAT expression. However, the E2a-deleted vector was demonstrated to have slightly reduced hepatocyte toxicity at an intermediate particle dose. This suggests that hepatocyte toxicity is related primarily to viral entry and expression, rather than to the presence of noninfectious particles, and implies that vectors with complete elimination of viral gene expression, such as vectors with all viral coding sequences deleted, are likely to have substantial advantages in terms of safety and toxicity.

Adenoviridae↗

PubMed1998

Construction of a modular yeast two-hybrid cDNA library from human EST clones for the human genome protein linkage map.

Identification of all human protein-protein interactions will lead to a global human protein linkage map that will provide important information for functional genomics studies. The yeast two-hybrid system is a powerful molecular genetic approach for studying protein-protein interactions. To apply this technology to generate a human protein linkage map, the first step is to construct two-hybrid cDNA libraries that cover the entire human genome. With a homologous recombination-mediated approach, we have constructed a modular human EST-derived yeast two-hybrid library in the Gal4 activation domain-based vector, pACT2. Quality analysis of this library indicated that the approach of constructing two-hybrid cDNA libraries from individually arrayed human EST clones is feasible, and such a two-hybrid library is suitable for detecting protein-protein interactions. This is also the first time that a comprehensive two-hybrid system cDNA library has been constructed from a collection of individually arrayed EST clones.

Chromosome Mapping↗

PubMed1998

Adenosine regulates tissue factor expression on endothelial cells.

The aim of this study was to evaluate the inhibitory activity of adenosine on tumor necrosis factor-alpha (TNF), thrombin-, or phorbol 12-myristate 13-acetate (PMA)-induced tissue factor (TF) expression on human umbilical vein endothelial cells (HUVECs). This inhibitory effect of adenosine was found to be counteracted by the non-selective adenosine receptor (AR) antagonist, 8-(p-sulfophenyl) theophylline. To clarify the role of ARs (A1, A2a, A2b, and A3) in this regulation, we evaluated the effect of several agonists and antagonists specific for AR-subclass on TF expression. The selective A2aAR agonist, 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride (CGS 21680), the A3AR agonist, N6-2-(4-aminophenyl) ethyladenosine (APNEA), and the A1AR antagonist, 1,3-dipropyl-8-(2-amino-4-chlorophenyl) xanthine (PACPX) each inhibited TF activity expression induced by TNF, thrombin, or PMA on HUVECs. In contrast, the selective A1AR agonist, chloro-N6-cyclopentyladenosine (CCPA) and the A2AR antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX) enhanced each stimulant-induced TF activity expression. All agonist or antagonist alone did not alter the basal TF expression on HUVECs. Our results suggest that stimulation of A2aAR and A3AR down-regulates and that of A1AR up-regulates the endothelial cell TF expression induced by TNF, PMA, or thrombin. Thus, it appears that adenosine itself may exert anticoagulant activity on vascular endothelial cells via its A2a and A3 receptors, particularly during ischemic or atherosclerotic processes which are known to be associated with local increased levels of adenosine.

Adenosine↗

PubMed1998

Cloning of cDNAs encoding xenopus neuregulin: expression in myotomal muscle during embryo development.

Neuregulin has diverse functions in neural development, and one of them is the up regulation of acetylcholine receptors (AChRs) at the muscle fiber during the formation of neuromuscular junctions. Although the primary source of neuregulin is derived from motor neuron, the expression in muscle has also been demonstrated. The precise role of neuron-derived and muscle-derived neuregulin during the early stages of development is not known. In order to study the role of neuregulin during early embryo development, we isolated the cDNAs encoding Xenopus neuregulin by cross-hybridization with its chick homologue. The amino acid sequence of Xenopus protein is 50 to 70% identical to members of the neuregulin family. The cDNAs encoding different isoforms of Xenopus neuregulin were identified, and these isoforms have two variation sites: (i) the spacer domain with either 0 or 43 amino acid insertion; and (ii) the C-terminus of EGF-like domain to derive either alpha or beta isoform. When the EGF-like domain of Xenopus neuregulin was expressed in mammalian cells, the recombinant protein was able to induce the expression of AChR and the tyrosine phosphorylation of erbB receptors in cultured myotubes. An approximately 6.5 kb transcript corresponding to neuregulin was detected in RNA isolated from brain and muscle. Various splicing variants were expressed in different Xenopus tissues. In situ hybridization showed a strong expression of neuregulin in developing brain and spinal cord of Xenopus embryo. In addition, it was also prominently expressed in the myotomal muscle. These data suggest that in addition to motor neurons, the postsynaptic muscle cells can also contribute neuregulin for synaptogenesis.

Amino Acid Sequence↗

PubMed1998

Inhibition of Akt kinase by cell-permeable ceramide and its implications for ceramide-induced apoptosis.

Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. However, mechanisms responsible for ceramide-induced apoptosis remain unclear. We investigated the possibility that ceramide may decrease antiapoptotic signaling in cells by inhibiting Akt kinase activity. Our data show that C2-ceramide induces apoptosis in HMN1 motor neuron cells and decreases both basal and insulin- or serum-stimulated Akt kinase activity 65-70%. These results are consistent with decreased Akt kinase activity being involved in the apoptotic effects of ceramide. This possibility is further supported by studies showing that constitutively active Akt kinase decreases C2-ceramide-induced death of HMN1 cells as well as COS-7 cells. Decreased Akt activity is not due to ceramide activating the ceramide-activated protein phosphatase or to a direct inhibition of Akt kinase by ceramide, suggesting that ceramide acts upstream of Akt kinase to decrease its activity. Treating cells with C2-ceramide does not affect phosphorylation of insulin receptor substrate-1, interactions between insulin receptor substrate-1 and p85, or insulin-stimulated phosphatidylinositol 3-kinase activity, suggesting that the effects of C2-ceramide on Akt kinase are not mediated through modulating phosphatidylinositol 3-kinase. In sum, our results suggest that inhibition of the key antiapoptotic kinase, Akt, may play an important role in ceramide-induced apoptosis.

Animals↗

PubMed1998

Length of hospital stay for treatment of deep venous thrombosis and the incidence of recurrent thromboembolism.

BACKGROUND: Current guidelines suggest that all patients with acute deep venous thrombosis should be treated with intravenous heparin for at least 5 days, overlapping with warfarin sodium for 4 to 5 days. METHODS: Using linked state of California hospital discharge records from 1991 to 1994 we identified patients with acute deep venous thrombosis without pulmonary embolism, and determined the 6-month cumulative incidence of rehospitalization for recurrent thromboembolism. Coding was validated by reviewing the charts of 218 patients matched with the statewide data from 4 local hospitals. RESULTS: A total of 36924 linked records met study criteria. In the validation group, objectively confirmed thrombosis that was treated with intravenous heparin followed by warfarin was noted in 20%, 65%, 94%, and 95% of the patients who were hospitalized for 2 or fewer days or 3, 4, or 5 or more days, respectively. Statewide, among patients hospitalized for 3, 4, 5, and 6 days, the 6-month cumulative incidence of hospitalization for recurrent thromboembolism was 5.4%, 5.1%, 5.4%, and 6.0%, respectively. Multivariate modeling of patients hospitalized for 3 to 10 days revealed that recurrent thromboembolism was associated with the length of hospitalization (odds ratio [OR], 1.06 each additional day; 95% confidence interval [CI], 1.04-1.08), presence of malignancy (OR, 1.58; 95% CI, 1.46-1.68), age (OR, 0.85 each 10 years; 95% CI, 0.84-0.86), dementia (OR, 0.38; 95% CI, 0.26-0.49), hospitalization for multiple injuries within 3 months (OR, 0.46; 95% CI, 0.32-0.60), and surgery within 3 months (OR, 0.84; 95% CI, 0.78-0.90). CONCLUSIONS: We found no evidence that a stay of 4 days for treatment of deep venous thrombosis was associated with a higher rate of recurrent thromboembolism compared with hospitalization for 5 or more days. Although the evidence was not as strong, the incidence of recurrent thromboembolism after a stay of 3 days appeared comparable with that after a stay of 5 days. These findings suggest that fewer than 5 days of intravenous heparin overlapping with warfarin may provide effective initial treatment for deep venous thrombosis among patients deemed ready for hospital discharge.

Anticoagulants↗

PubMed1998

Incidence of idiopathic deep venous thrombosis and secondary thromboembolism among ethnic groups in California.

BACKGROUND: Few studies have compared the incidence of deep venous thrombosis among ethnic groups. OBJECTIVE: To determine the incidence of deep venous thrombosis among ethnic groups. DESIGN: Analysis of the linked California Patient Discharge Data Set from 1991 to 1994. SETTING: California. PATIENTS: 17991 patients with idiopathic deep venous thrombosis (thrombosis without cancer or hospitalization within preceding 6 months) and 5573 patients with secondary thromboembolism (thromboembolism occurring within 3 months of seven different events). MEASUREMENTS: Ethnicity was determined by using race as documented in the data set. For idiopathic deep venous thrombosis, standardized age- and sex-adjusted incidences were calculated. For secondary thromboembolism, proportional hazards modeling was done. RESULTS: The annual incidence of idiopathic deep venous thrombosis per 1000000 persons older than 18 years of age was 230 for white persons, 293 for African Americans (rate ratio, 1.27 [95% CI, 1.07 to 1.51]), 139 for Hispanic persons (rate ratio, 0.60 [CI, 0.54 to 0.67]), and 60 for Asians and Pacific Islanders (rate ratio, 0.26 [CI, 0.22 to 0.30]). Compared with white persons, Asians and Pacific Islanders who developed secondary thromboembolism had a significantly lower relative risk (range, 0.22 to 0.61) for all seven conditions analyzed. CONCLUSIONS: Compared with white persons, Asians and Pacific Islanders have a very low incidence of idiopathic deep venous thrombosis and a very low relative risk for secondary venous thromboembolism.

Adult↗

PubMed1998

Structure and organization of the RBMY genes on the human Y chromosome: transposition and amplification of an ancestral autosomal hnRNPG gene.

The RBMY (RNA-binding motif, Y chromosome) gene family encodes a germ-cell-specific nuclear protein implicated in spermatogenesis. It consists of approximately 30 genes and pseudogenes, found on both arms of the Y chromosome. RBMY shares high homology with an autosomal hnRNPG gene that contains an RNA-binding motif and one of the four SRGY repeats found in RBMY. One proposal is that RBMY represents an ancestral hnRNPG gene, transposed to the Y chromosome and then amplified. We characterized seven RBMY genes in interval 6 of the Y chromosome long arm. Four have the normal structure with 12 exons spanning 15 kb, whereas one lacks the first 3 exons, therefore representing a pseudogene. The remaining two genes belong to a different subfamily, resembling the autosomal hnRNPG gene with only one SRGY repeat. We also found that most RBMY genes in interval 6 are arranged in tandem. The structure and organization of the Y-linked RBMY genes support the transposition-amplification hypothesis.

Amino Acid Sequence↗

PubMed1998

Monoclonal antibody production with on-line harvesting and process monitoring.

A semi-automated system has been designed for on-line harvesting and monitoring of monoclonal antibody (mAb) production. [The antibody was directed against the peptide AGPAGTGKTTKDL.] Analytical and purification units were interfaced to the fermenter via a hollow fiber cartridge in which fermentation broth was continuously circulated through the lumen of the hollow fiber system. Permeate from the hollow fiber cartridge was pumped through either an analytical sampling loop or a preparative Protein G column where antibody species were captured. Switching between monitoring and harvesting was achieved by two 3-way toggle valves. Samples from the analytical sampling loop were transported to an analytical Protein G chromatography column for quantitation of all immunoglobulin G species in the fermenter. Data acquisition and processing was performed by the data system of the liquid chromatograph. All valves in the system except the two toggle valves were controlled by the liquid chromatograph. Antibody biosynthesis was monitored for the first 60 h of fermentation. Harvesting was initiated when mAb accumulated in the fermenter. Complete harvesting took approximately 90 h.

Amino Acid Sequence↗

PubMed1998

Identification of histidine 45 as the axial heme iron ligand of heme oxygenase-2.

A truncated, soluble, and enzymatically active form of human heme oxygenase-2 (DeltaHHO2) was expressed in Escherichia coli. To identify the axial heme ligand of HO-2, His-45 to Ala (DeltaH45A) and His-152 to Ala (DeltaH152A) mutants have been prepared using this expression system. DeltaH45A could form a 1:1 complex with hemin but was completely devoid of the heme degradation activity. A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-DeltaH45A complex. The DeltaH152A mutant was expressed as an inclusion body and was recovered from the lysis pellet by dissolution in urea followed by dialysis. The solubilized fraction obtained, however, was composed of a mixture of a functional enzyme and an inactive fraction. The inactive fraction was removed by Sephadex G-75 column chromatography since it eluted out of the column at the void volume. The gel filtration-purified DeltaH152A exhibited spectroscopic and enzymatic properties identical to those of wild-type. We conclude, in contrast to the previous reports (McCoubrey and Maines (1993) Arch. Biochem. Biophys. 302, 402-408; McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. (1997) J. Biol. Chem. 272, 12568-12574), that His-45, but not His-152, in heme oxygenase isoform-2 is the proximal ligand of the heme and is essential for the heme degradation activity of the enzyme. His-152 appears to play a structural role in stabilization of the heme oxygenase protein.

Electron Spin Resonance Spectroscopy↗

PubMed1998

The solution structure of the N-terminal domain of hepatocyte growth factor reveals a potential heparin-binding site.

BACKGROUND: Hepatocyte growth factor (HGF) is a multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. The N-terminal (N) domain of HGF, containing a hairpin-loop region, is important for receptor binding and the potent biological activities of HGF. The N domain is also the primary binding site for heparin or heparan sulfate, which enhances, receptor/ligand oligomerization and modulates receptor-dependent mitogenesis. The rational design of artificial modulators of HGF signaling requires a detailed understanding of the structures of HGF and its receptor, as well as the role of heparin proteoglycan; this study represents the first step towards that goal. RESULTS: We report here a high-resolution structure of the N domain of HGF. This first structure of HGF reveals a novel folding topology with a distinct pattern of charge distribution and indicates a possible heparin-binding site. CONCLUSIONS: The hairpin-loop region of the N domain plays a major role in stabilizing the structure and contributes to a putative heparin-binding site, which explains why it is required for biological functions. These results suggest several basic and/or polar residues that may be important for use in further mutational studies of heparin binding.

Amino Acid Sequence↗

PubMed1998

The oxygen and carbon monoxide reactions of heme oxygenase.

The O2 and CO reactions with the heme, alpha-hydroxyheme, and verdoheme complexes of heme oxygenase have been studied. The heme complexes of heme oxygenase isoforms-1 and -2 have similar O2 and CO binding properties. The O2 affinities are very high, KO2 = 30-80 microM-1, which is 30-90-fold greater than those of mammalian myoglobins. The O2 association rate constants are similar to those for myoglobins (kO2' = 7-20 microM-1 s-1), whereas the O2 dissociation rates are remarkably slow (kO2 = 0.25 s-1), implying the presence of very favorable interactions between bound O2 and protein residues in the heme pocket. The CO affinities estimated for both isoforms are only 1-6-fold higher than the corresponding O2 affinities. Thus, heme oxygenase discriminates much more strongly against CO binding than either myoglobin or hemoglobin. The CO binding reactions with the ferrous alpha-hydroxyheme complex are similar to those of the protoheme complex, and hydroxylation at the alpha-meso position does not appear to affect the reactivity of the iron atom. In contrast, the CO affinities of the verdoheme complexes are >10,000 times weaker than those of the heme complexes because of a 100-fold slower association rate constant (kCO' approximately 0. 004 microM-1 s-1) and a 300-fold greater dissociation rate constant (kCO approximately 3 s-1) compared with the corresponding rate constants of the protoheme and alpha-hydroxyheme complexes. The positive charge on the verdoporphyrin ring causes a large decrease in reactivity of the iron.

Animals↗

PubMed1998

Repair with collagen tubules linked with brain-derived neurotrophic factor and ciliary neurotrophic factor in a rat sciatic nerve injury model.

OBJECTIVE: To determine if brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) can be successfully delivered to transected and repaired peripheral nerves by cross-linking the factors to collagen tubules (CTs). METHODS: Forty-eight Sprague-Dawley rats underwent left sciatic nerve transection and repair. In the control group, CTs were implanted with no neurotrophic ligand (n=13). There were 3 experimental groups: CT with BDNF covalently linked to the collagen matrix (CT/BDNF; n=12), CT with CNTF covalently linked (CT/CNTF; n=12), and CT with both BDNF and CNTF covalently linked (CT/BDNF/CNTF; n=11). Functional outcome of neural regeneration was assessed every 10 days using walking track analysis, which was submitted to a sciatic functional index. Nerve morphometry, electrophysiologic studies, and molecular analysis for neural proteins were performed at the completion of the study at postoperative day 90. RESULTS: Animals in all 3 experimental groups achieved significantly superior maximal functional recovery, larger nerve cross-sectional areas, and a greater number of axons when compared with the control CT group (P<.001, P<.05, and P<.05, respectively). The animals in the CT/BDNF/CNTF group displayed the best functional recovery and had the largest axon diameters, greatest amplitude, and the fastest nerve conduction velocities. Molecular analysis revealed significant differences in the expression of neurofilament, neural cell adhesion molecule, myelin-associated glycoprotein, and myelin basic protein. CONCLUSIONS: We present the first evidence that CNTF covalently linked to CTs can improve functional recovery compared with CTs alone. We also support the previous finding that BDNF covalently linked to CTs significantly increases the functional recovery of transected and repaired nerves. Finally, we found that cotreatment produced the best functional recovery in our model.

Animals↗

PubMed1998

Transcriptional and posttranscriptional regulation of osteopontin gene expression in preosteoblasts by retinoic acid.

This study examines the relative importance of transcriptional and posttranscriptional actions of retinoic acid (RA) in the regulation of osteopontin gene expression in a rat clonal preosteoblastic cell line, UMR 201. Nuclear run-on analysis demonstrated constitutive expression of the osteopontin gene which was increased by threefold after 4 hr treatment with 1 microM RA, returning to a basal level by 24 hr. However, Northern blot analysis, performed concurrently, showed that RA progressively increased the steady-state osteopontin mRNA level beginning 2 hr before any increase in gene transcription and peaking at 24 hr. There was no difference in osteopontin mRNA stability between control and RA-treated cells after gene transcription was inhibited with 5,6-dichloro-1-D-ribofuranosylbenzimidazole (DRB). Total RNA was obtained from cellular subfractions (nuclear matrix, nonmatrix chromatin, nuclear membrane, and cytoplasm) and reverse transcription-polymerase chain reaction (RT-PCR) performed with primers complementary to exons 3 and 4 of the mouse osteopontin gene. Unspliced PCR product, comprising the two exons and the intervening intron, was present in the nuclear matrix fractions of control and RA-treated cells. However, RA resulted in a time-dependent accumulation of mature osteopontin mRNA in all cellular subfractions, suggesting that the proficiency of nuclear processing of primary mRNA transcripts was greatly enhanced by RA. This action depended on de novo protein synthesis. These results demonstrate that the posttranscriptional action of RA is not unique to the regulation of alkaline phosphatase gene expression.

Alkaline Phosphatase↗

PubMed1998

A pharmacokinetic study of intravenous itraconazole followed by oral administration of itraconazole capsules in patients with advanced human immunodeficiency virus infection.

A randomized, open-label, comparative study was conducted in 30 male patients with moderately advanced human immunodeficiency virus (HIV) infection to examine the pharmacokinetics of an investigational intravenous preparation of itraconazole compared with pharmacokinetics after administration of itraconazole capsules. The study also assessed whether adequate plasma concentrations of itraconazole could be rapidly achieved with the intravenous formulation and then maintained after cessation of intravenous therapy with itraconazole capsules. All patients received 200 mg intravenous itraconazole as a 1-hour infusion in 40% hydroxypropyl-beta-cyclodextrin (HP-beta-CD) vehicle twice daily for 2 days, and then 200 mg intravenously once daily for 5 days. Patients then received itraconazole capsules, either 200 mg twice daily or 200 mg once daily for 28 days. Steady-state plasma concentrations of itraconazole were reached by day 3 with intravenous infusion, a much shorter time than observed with administration of itraconazole capsules. Steady-state concentrations of itraconazole and hydroxyitraconazole were effectively maintained during the rest of the intravenous infusions of itraconazole. Oral follow-up with administration of 200-mg capsules once daily could not maintain the plasma concentrations of itraconazole and hydroxyitraconazole obtained at the end of the intravenous treatment, whereas twice-daily oral administration maintained or increased these concentrations. Mean plasma concentrations of itraconazole and hydroxyitraconazole on day 7 were similar to those on day 36 in the twice-daily group. Mean renal clearance was comparable to mean total body clearance, and approximately 93% to 101% of the HP-beta-CD was excreted unchanged in urine within 12 hours of administration. The HP-beta-CD was essentially eliminated through the kidney, and little accumulation in the body was observed in this patient population. Adverse events during the intravenous phase were most commonly associated with intravenous administration. Intravenous infusion of itraconazole for 7 days followed by administration of itraconazole capsules twice daily for 28 days is an effective dose regimen in patients with advanced HIV infection.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed1998

Renaturation of 1-aminocyclopropane-1-carboxylate synthase expressed in Escherichia coli in the form of inclusion bodies into a dimeric and catalytically active enzyme.

1-Aminocyclopropane-1-carboxylate (ACC) synthase is a key enzyme regulating the biosynthesis of the plant hormone ethylene. A wound-inducible zucchini ACC synthase cDNA was isolated by reverse-transcription polymerase chain reaction (RT-PCR) and expressed in a heterologous Escherichia coli BL21(DE3)pLysS:pET30a protein expression system. A method was developed and optimized for the renaturation of the ACC synthase expressed in the form of inclusion bodies. The optimum conditions were found to be unfolding in a buffer containing 100 mM Mops, pH 9.5, 6 M urea, and 50 mM DTT, for 3 h at 4 degrees C and refolding by a combined process of dialysis and dilution in 100 mM Mops, pH 8, 30 mM Chaps, and 5 mM GSH at a protein concentration of 45 microg/ml. The purified enzyme has a specific activity of 90,000 U mg-1 and exhibits an apparent homogeneity on SDS-PAGE fractionation. Biochemical characterization of the refolded enzyme revealed a high degree of similarity to the enzyme purified from the soluble source. The refolded enzyme was found to be a dimer with a native size of 110 kDa, a Km of 23 microM, and a Vmax of 112,000 U mg-1.

Base Sequence↗

PubMed1998

Enhanced high-level expression of soluble 1-aminocyclopropane-1-carboxylase synthase and rapid purification by expanded-bed adsorption.

1-Aminocyclopropane-1-carboxylate (ACC) synthase is a key enzyme regulating the biosynthesis of the plant hormone ethylene. Expression of ACC synthase in Escherichia coli can result in the production of a large proportion of the enzyme in the form of insoluble aggregates (inclusion bodies). We investigated the effect on the soluble expression in E. coli of tomato and zucchini ACC synthases, by manipulation of the induction conditions, changing the vector, and deletions in the amino acid sequence. Manipulation of the induction conditions did not influence the soluble expression; however, soluble expression increased significantly when the enzyme was cloned into vector pET11d, in comparison to the other vector used, pET30a. It was also found that when ACC synthase with a portion of the C-terminus deleted was inserted into pET11d, the soluble expression was further enhanced in comparison to that of the full length. Structural and functional analysis of ACC synthase requires the purification of milligram quantities of protein to homogeneity. The development of a faster and simpler protocol for the purification of ACC synthase is highly desirable due to the extreme lability of the enzyme. C-terminal truncated tomato ACC synthase was overexpressed in E. coli pET11d and purified by expanded-bed adsorption and hydroxylapatite FPLC. This improved two-step purification protocol allows for rapid, high-level purification with a significantly improved yield in comparison to the multistage purification it replaces. 15.7 mg of highly purified tomato ACC synthase del-1 were obtained from 2 L of cells in comparison to 2 mg from 10 L using a multistage purification. This represents a 40-fold improvement in yield. Antibodies were raised against C-terminal deleted ACC synthase. The antibodies were purified by epitope-specific affinity chromatography and used to assess the identity and purity of the C-terminal-deleted tomato ACC synthase purified by expanded-bed adsorption.

Chromatography↗