Advances in refolding of proteins produced in E. coli

Guanabenz (Wytensin) was shown to inactivate nitric oxide synthase (NOS) activity in vitro and in vivo. In in vitro studies with the use of a cytosolic fraction from penile tissue, the inactivation was found to depend on NADPH, time, and the concentration of guanabenz. The L-, but not the D-, isomer of arginine could protect from the inactivation, suggesting an active site-directed event. The kinetics of inactivation could be described by an apparent dissociation constant for the initial reversible complex (Ki) and a pseudo first-order inactivation constant (kinact) of 38.5 microM and 0.179 min-1, respectively. In in vivo studies, guanabenz was shown to inhibit penile cytosolic NOS activity in a dose- and time-dependent manner. Treatment of rats with guanabenz (5 mg/kg/day) for 4 days caused a decrease of approximately one-half in the NOS activity of the penile cytosolic fraction with a concomitant loss in the amount of immunodetectable NOS protein. Treatment for 4 days at a dose of 0. 5 mg/kg/day showed a similar decrease in activity, whereas a dose of 0.05 mg/kg/day showed no effects. Due to the multitude of processes that are regulated by NO, the inactivation of NOS is a potential mechanism to be considered in a variety of biological effects associated with drugs.     

哈萨克族食管癌中MMP-1、MMP-2、MMP-7、TIMP-1和MTA1的表达及其临床病理意义

目的:探讨在新疆哈萨克族食管癌组织中基质金属蛋白酶1、2、7 (MMP-1、MMP-2、MMP-7)、基质金属蛋白酶抑制因子1(TIMP-1)和肿瘤转移相关基因1(MTA1)mRNA的表达及其临床意义.方法:采用RT-PCR方法检测75例哈萨克族食管癌标本中MMP-1、MMP-2、MMP-7、TIMP-1和MTA1 mRNA的表达水平,分析其表达与临床病理特征的关系.结果:MMP-1、MMP-2、MMP-7、TIMP-1和MTA1 mRNA在哈萨克族食管癌组织中的表达较正常组织增高,差异均具有统计学意义(P均<0.05);且MMP-2、MMP-7、MTA1 mRNA的表达与淋巴结转移有关(P<0.05),MMP-1、MMP-7 mRNA的表达与临床分期有关(P<0.05);TIMP-1与MMP-1、MMP-7、MTA1的表达呈正相关(r=0.446、0.458、0.333,P均<0.01).结论:MMP-1、MMP-2、MMP-7、TIMP-1和MTA1 mRNA表达上调共同参与了哈萨克族食管癌的发生发展过程;MMP-2、MMP-7和MTA1可能是哈萨克族食管癌发生侵袭、转移的主导因素.     

Circulating levels of matrix metalloproteinases MMP-3 and MMP-1, tissue inhibitor of metalloproteinases 1 (TIMP-1), and MMP-1/TIMP-1 complex in rheumatic disease. Correlation with clinical activity of rheumatoid arthritis versus other surrogate markers

OBJECTIVE: To investigate whether plasma levels of matrix metalloproteinases 3 (MMP-3, stromelysin), MMP-1 (collagenase), tissue inhibitor of metalloproteinases 1 (TIMP-1), and MMP1/TIMP-1 complex (MT complex) are specifically elevated in erosive joint diseases compared to nonerosive rheumatic diseases, and to assess how these markers reflect the clinical activity of rheumatoid arthritis (RA) compared to circulating cytokines and markers of connective tissue turnover as well as established variables [C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and rheumatoid factor titer]. METHODS: Plasma levels of MMP-3, MMP-1, TIMP- 1, and MT complex were determined by ELISA. One hundred fifteen patients with RA, 20 with osteoarthritis (OA), 28 with psoriasis arthritis (PsA), 24 with ankylosing spondylitis (AS), 3 groups with systemic autoimmune diseases, and 30 healthy controls were analyzed. In patients with RA routine laboratory variables, circulating inflammatory cytokines [interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and IL-6], collagen degradation products, and markers of bone formation were determined in parallel and were correlated to 4 variables of clinical activity. RESULTS: MMP-3 levels were markedly elevated in RA compared to controls and OA, but also in all other groups, including 26 patients with systemic lupus erythematosus (SLE). MMP-1 levels were significantly elevated in RA, but also in OA, PsA, SLE, and mixed connective tissue disease. In contrast, MT complex was elevated in RA only. TIMP-1 was not different from controls. CRP levels, MMP-3, and ESR correlated best with clinical activity of RA. In contrast, there was no correlation of IL-1 and TNF-alpha and only a weak correlation of IL-6 with clinical measures. Among variables of connective tissue turnover, only pyridinoline and deoxypyridinoline crosslinks were weakly correlated with disease activity. CONCLUSION: Elevated MMP-3 and MMP-1 levels are not specific for RA or for erosive joint diseases in general. In contrast, elevated MT complex levels were observed in patients with RA. However, the correlation of MT-1 with clinical data was weaker than that of MMP-3. Elevated MMP-3 levels reflected disease activity of RA better than cytokine levels or markers of connective tissue turnover. However, MMP-3 levels do not exceed the association of CRP with clinical activity.     

Gamma Irradiation for Inactivation of C. parvum, E. coli, and Coliphage MS-2

Wastewater and drinking water disinfection are typically achieved via chlorination, ozonation, or UV irradiation. However, there has been increased interest in recent years in alternative disinfectants. This interest came about primarily as a result of concerns over the toxicological effects of disinfection by-products created by conventional disinfection processes and the resistance of some recalcitrant microorganisms to these disinfectants. The work reported herein represents an investigation of the effect of an alternative disinfectant, gamma radiation, on the viability of three important waterborne microorganisms. Escherichia coli, coliphage MS-2, and Cryptosporidium parvum were chosen for this investigation as representative bacterial, viral, and protozoan microorganisms, respectively. A 60Co irradiator was used to expose test microorganisms to a controlled radiation dose. Experiments were performed for each of the test microorganisms to evaluate the effect of dissolved oxygen concentration and carbonate alkalinity on inactivation efficiency. For each microorganism, a strong effect of dissolved oxygen was observed, regardless of alkalinity. A subtle effect of alkalinity was observed for E. coli and coliphage MS-2, but only in air-saturated solutions. No significant alkalinity effect was observed for Cryptosporidium parvum. Inactivation kinetics were modeled for E. coli and coliphage MS-2 using single-target theory to calculate an inactivation rate constant. Multitarget theory was used to represent the inactivation response of Cryptosporidium parvum. The inactivation models based on target theory were found to provide suitable representations of experimental observations.     

膀胱癌组织中MMP-2与TIMP-2 mRNA的表达及临床意义

Objective:The aim of the study was to investigate the expressions of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of MMP-2 (TIMP-2) mRNA in transitional cell carcinomas of bladder and discuss their clinical significances.Methods:Using RT-PCR and real time quantitative PCR (RQ-PCR) technique, the expressions of MMP-2 and TIMP-2 mRNA of 45 cases of bladder carcinoma (tumor group) and 10 cases of normal bladder tissue (control group) were analyzed. Results:MMP-2 and TIMP-2 were not expressed in control group. MMP-2 was expressed in 30 cases tumor samples and TIMP2 was expressed in 26 cases. The expression of MMP-2 mRNA in non-muscle invasive bladder cancers and grade I cancers significant (P < 0.05). The expression of MMP-2 in recurrent patients was higher than that in incipient patients. TIMP-2 mRNA expression decreased with grades and stage. The expression of TIMP-2 in non-muscle invasive bladder cancers and statistical difference between two groups (P < 0.05). TIMP-2 expression in incipient patients was higher than that in recurrent patients, but the difference was not significant (P > 0.05). Conclusion:These results suggest that MMP-2 and TIMP-2 play an important role in the invasion step of transitional cell carcinoma of bladder. MMP-2 may become a new approach to the diagnosis of bladder carcinoma.     

A novel antiporter activity catalyzing sodium and potassium transport from right-side-out vesicles of E. coli

Downhill sodium efflux from right-side-out E. coli membrane vesicles was found to be stimulated by negative electric potential, as has been reported earlier [Bassilana et al., Biochemistry 23 (1984) 1015-1022], and in agreement with the concept of electrogenic Na+/nH+ antiporters with n > 1. However, sodium efflux was much more accelerated by positive electric potential, indicating the operation of another sodium transport system. delta pH (alkaline inside), created by a pH shift from 8.5 to 6.8 in the medium was found to drive sodium efflux against its concentration gradient, but only when the vesicles had been loaded with both Na+ and K+. Efflux of K+ against the concentration gradient was also observed under these conditions. When the vesicles were loaded separately with sodium tricine or potassium tricine, no K+ efflux and insignificant Na+ efflux were observed. We propose that there are at least two different mechanisms responsible for Na+ efflux in E. coli vesicles. One is the Na+/nH+ antiporter previously described, and the other is a novel Na+,K+/mH+ antiporter.     

Crystal structure of CcdB, a topoisomerase poison from E. coli

The crystal structure of CcdB, a protein that poisons Escherichia coli gyrase, was determined in three crystal forms. The protein consists of a five-stranded antiparallel beta-pleated sheet followed by a C-terminal alpha-helix. In one of the loops of the sheet, a second small three-stranded antiparallel beta-sheet is inserted that sticks out of the molecule as a wing. This wing contains the LysC proteolytic cleavage site that is protected by CcdA and, therefore, forms a likely CcdA recognition site. A dimer is formed by sheet extension and by extensive hydrophobic contacts involving three of the five methionine residues and the C terminus of the alpha-helix. The surface of the dimer on the side of the alpha-helix is overall negatively charged, while the opposite side as well as the wing sheet is dominated by positive charges. We propose that the CcdB dimer binds into the central hole of the 59 kDa N-terminal fragment of GyrA, after disruption of the head dimer interface of GyrA.     

Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium

Age-related macular degeneration, a major cause of blindness for which no satisfactory treatments exist, leads to a gradual decrease in central high acuity vision. The accumulation of fluorescent materials, called lipofuscin, in retinal pigment epithelial cells of the aging retina is most pronounced in the macula. One of the fluorophores of retinal pigment epithelial lipofuscin has been characterized as A2E, a pyridinium bis-retinoid, which is derived from two molecules of vitamin A aldehyde and one molecule of ethanolamine. An investigation aimed at optimizing the in vitro synthesis of A2E has resulted in the one-step biomimetic preparation of this pigment in 49% yield, readily producing more than 50 mg in one step. These results have allowed for the optimization of HPLC conditions so that nanogram quantities of A2E can be detected from extracts of tissue samples. By using 5% of the extract from individual aged human eyes, this protocol has led to the quantification of A2E and the characterization of iso-A2E, a new A2E double bond isomer; all-trans-retinol and 13-cis-retinol also have been identified in these HPLC chromatograms. Exposure of either A2E or iso-A2E to light gives rise to 4:1 A2E:iso-A2E equilibrium mixtures, similar to the composition of these two pigments in eye extracts. A2E and iso-A2E may exhibit surfactant properties arising from their unique wedge-shaped structures.     

IL-10 inhibition of human prostate PC-3 ML cell metastases in SCID mice: IL-10 stimulation of TIMP-1 and inhibition of MMP-2/MMP-9 expression

The molecular mechanism by which IL-10 inhibits metastases was examined using a SCID mouse model. Human PC-3 ML subclones normally metastasize to the lumbar vertebrae (approximately 70% mice injected, n = 14/20) following intravenous injection in severe combined immunodeficient (SCID) mice. IL-10 treatment of the PC-3 ML cells (15 ng/ml for 36 h) and the SCID mice (0.03 mg/kg/day for 30 days) reduced the number of metastases to 5% of the mice (n = 1/20). More importantly, following discontinuation of IL-10 treatment on day 30, the mice remained tumor-free and mouse survival rates increased dramatically (from < 30% in untreated mice) to about 85% in IL-10-treated mice. IL-10 did not appear to alter the growth rates or colony-forming ability of the PC-3 ML cells in vitro. Likewise, the growth of subcutaneous tumors and established bone marrow metastases was not inhibited by IL-10 treatment of the SCID mice. However IL-10 may inhibit the production of matrix metalloproteases (MMP) and prevent the establishment of metastasis. We therefore examined the influence of IL-10 on PC-3 ML production of MMP-2/MMP-9 and the tissue inhibitors of metalloproteinases (TIMP-1/2). Enzyme-linked immunosandwich assays (ELISAs) revealed that IL-10 (15 ng/ml for 36 h) treatment of the PC-3 ML cells down-regulated MMP-2 and MMP-9 while up-regulating TIMP-1 (not TIMP-2) expression. Likewise, IL-10-treated mice exhibited similar changes in TIMP-1 and MMP-2/MMP-9 expression. The IL-10 effects were blocked by IL-10 receptor antibodies. In comparison to IL-10, IL-4 failed to influence metastasis or the expression of TIMP-1, TIMP-2, MMP-2 and MMP-9 by PC-3 ML cells. We suggest that IL-10-regulated increases in the molar ratio of TIMP-1/MMP-9 and TIMP-2/MMP-2 might inhibit processes critical to the establishment of bone marrow metastasis.     

High expression of synthetic human interferon-gamma cDNA in E. coli

Human interferon-gamma (IFN-gamma) cDNA was synthesized, and it makes the usage of favorable codons in E. coli. The authors got 9 different expression plasmids which contain the synthetic IFN-gamma-cDNA and have different spaces between SD sequence and ATG. The free energies G0f298 in the formation of stable secondary structure in the translation initiation region (TIR) are different in various expression plasmids. One of them, pLY4-gamma 5, can highly yield INF-gamma which will be about 60%-80% of the total bacterial proteins, such a high expression was hardly noted in literature. The reasons of high expression in this work are optimal spaces between SD and ATC, favorable delta G0f298, favourable condons usage for E. coli.     

Diversity of cDNAs encoding phospholipase A2 from Agkistrodon halys pallas venom, and its expression in E. coli

As a step toward understanding the structure and function of phospholipase A2(PLA2), we isolated several novel cDNAs encoding Agkistrodon halys Pallas PLA2 isoenzymes including B-PLA2, Asn49-PLA2, A-PLA2, A'-PLA2 and BA1-PLA2 by polymerase chain reaction with oligonucleotide primers corresponding to the N- and C-terminus of these enzymes. The amino acid sequences of A-PLA2 deduced from cDNA are consistent with that isolated from venom except for four residues. Asn49-PLA2 and B-PLA2 are highly similar (> 95%), but the critical residue Asp49 in the active centre of B-PLA2 is replaced by Asn49 in Asn49-PLA2. The N-terminal residues (1-24) of BA1-PLA2 shows high similarity to that of B-PLA2 which has strong ability to hemolyze erythrocytes, while its C-terminal residues (72-125) are the same as that of A-PLA2 which can inhibit platelet aggregation. The successful cloning of these isoenzymes not only provide excellent native material to study the structure-function relationship of PLA2s, but also to disclose the genesis of structural diversity of PLA2s, namely DNA modification and gene rearrangement. The cloned cDNA for A-PLA2 has been expressed in E. coli. By Q-Sepharose column chromatography, denaturation-renaturation and FPLC, we obtained the active recombinant protein with the initiator Met. This is the first report of the production of an active recombinant PLA2 with the initiator Met.     

Organotin complexes with pyrrole-2-carboxaldehyde monoacylhydrazones. Synthesis, spectroscopic properties, antimicrobial activity, and genotoxicity

The Council on Quality and Leadership in Supports for People With Disabilities (The Council), formerly known as The Accreditation Council, altered its definition of quality from "compliance with organizational process" to "responsiveness to people." Council representatives conducted focus group and individual meetings with people who have disabilities to identify priority outcomes they expect from services and supports. The 1993 Outcome Based Performance Measures was used in 447 interviews as part of 54 accreditation reviews. Staff analysis and factor analysis of the outcome interviews using a principle components extraction and varimax rotation resulted in 24 variables loading onto seven major factors (Identity, Autonomy, Affiliation, Attainment, Rights, Health, and Safeguards), which form the basis of The Council 1997 Personal Outcome Measures.     

D-甘露糖异构酶在大肠杆菌中的表达及催化条件的研究

根据已知的放射性土壤杆菌体内的D-甘露糖异构酶(Fmi)氨基酸序列,设计适合在大肠杆菌内表达的Fmi基因序列(1245 bp),采用体外基因拼接合成.构建了表达Fmi的重组大肠杆菌BL21(DE3)/pET30-Fmi,诱导表达的Fmi蛋白经SDS-PAGE验证为可溶性蛋白,分子量44000.通过实验优化了Fmi的催化条件,结果表明Fmi催化的最适pH值为7.5,最适温度为45℃,催化1 h酶活达5.29 U/mL.以25%的果糖溶液为底物时,催化2 h果糖转化率达29.4%.     

Preparation, cloning, and high level expression in E. coli of interleukin 2-pseudomonas exotoxin fusion genes

Up to half of the stroke patients admitted to acute hospital wards become bed-blockers. Investigations have been carried out in an effort to identify factors related to this problem. Very little is known about options which may lead to an alleviation of this problem. We investigated to what extent, in the opinions of professional representatives of six disciplines, home care can contribute to a solution. Sixty-nine stroke patients who were actually blocking beds in an acute hospital ward were described and examined on paper by a multidisciplinary panel. These patients were all moderately to severely disabled and needed a high degree of help in activities of daily life (ADL) and household activities. Estimations of the number of patients who were judged to be suitable for home care varied, although there was a fair degree of agreement between panel members concerning those patients who could and those who certainly could not return to their homes. Concerning one-third of the patients, the opinions of the caregivers diverged. Factors relating to the judgement of each panel member are identified. A method for selecting patients to be substituted to lower levels of care is suggested and discussed.     

Induction kinetics of mutagenic DNA repair activity in E. coli following ultraviolet irradiation

In patients with psychosexual disturbances (impotence and ejaculatio praecox) the family constellation during childhood and the patients' occupations were compared with the Swedish population census. The parents of the patients were older, and the patients were more often the only son or the only child. A greater percentage of the patients had technical or office administrative professions than the inhabitants in Stockholm. The findings are discussed with references to a supposed focus on achievement in the upbringing of the oldest or only son. Focus on achievement may cause emotional restraint and subsequently psychosexual disturbances.     

Effects of prostaglandins E1, E2, F2 alpha and arachidonic acid on aryl hydrocarbon hydroxylase activity of intestinal and hepatic microsomes from male rats

The in vitro effects of prostaglandins E1, E2, F2 alpha and arachidonic acid on microsomal aryl hydrocarbon hydroxylase (AHH) activity from rat liver and intestinal mucosa were examined. PGE1 and PGE2 stimulated AHH activity of intestinal microsomes, while both prostaglandins had an inhibitory effect on AHH activity of liver microsomes. PGF2 alpha had little effect on AHH activity of intestinal and hepatic microsomes. Arachidonic acid exerted strongly inhibitory effects on AHH activity of both intestine and liver, with the greatest inhibition being exerted against intestinal AHH. Kinetic studies demonstrated that the inhibition of hepatic AHH activity by PGE1 at concentrations of 0.10 mM or less was non-competitive.