L－亮氨酸Schiff碱Cu（Ⅱ）、Ni（Ⅱ）配合物的合成及抑菌活性

合成了Ｌ－亮氨酸Ｓｃｈｉｆｆ碱的Ｃｕ（Ⅱ）、Ｎｉ（Ⅱ）配合物,其化学结构由红外光谱、元素分析、热重分析及摩尔电导等证实。对配合物进行了抑菌测试。     

人粒细胞集落刺激因子（hG－CSF）cDNA3′－UTR对其表达的影响

利用人粒细胞集落刺激因子（ｈＧ－ＣＳＦ）ｃＤＮＡ３′端非翻译区（３′－ＵＴＲ）中存在的ＤｒａⅠ酶切位点,通过部分酶切与完全酶切,删除３′－ＵＴＲ不同长度,构建了四种ｈＧ－ＣＳＦｃＤＮＡ瞬时重组表达质粒。转染ＣＯＳ－７细胞后,生物活性测定结果提示,ｈＧ－ＣＳＦｃＤＮＡ３′－ＵＴＲ对其表达起负调控作用,其关键性序列位于紧接终止密码子ＴＧＡ下游的６５ｂｐ范围内,３′－ＵＴＲ对ｈＧ－ＣＳＦｃＤＮＡ表达的影响与转录水平的差别有一定关系。     

Specificity studies of 3-mercaptopyruvate sulfurtransferase

3-Mercaptopyruvate sulfurtransferase (E.C. 2.8.1.2; MST) is an enzyme believed to function in the endogenous cyanide (CN) detoxification system because it is capable of transferring sulfur from 3-mercaptopyruvate (3-MP) to CN, forming the less toxic thiocyanate (SCN). To date, 3-MP is the only known sulfur-donor substrate for MST. In an effort to increase the understanding of what chemical properties of 3-MP affect its utilization as a substrate, in vitro enzyme kinetic studies of MST were conducted using two mercaptic acids that are structurally related to 3-MP. Neither of these compounds was able to serve as a sulfur-donor substrate for MST. Inhibitor studies determined that 3-mercaptopropionic acid did not affect the K_m of MST for 3-MP but did decrease V_max and, thus, was determined to be a noncompetitive inhibitor. Alternatively, 2-mercaptopropionic acid 2-MPA decreased K_m and V_max and was determined to be an uncompetitive inhibitor of MST with respect to 3-MP. These data indicate that the α-keto group of 3-MP is necessary for its utilization as a substrate, and the inhibitor studies suggest that the position of the sulfur may also affect the binding of these compounds to the enzyme. These observations increase the understanding of what factors can affect the utilization of a compound as a sulfur-donor substrate for MST and may aid in the development of alternative sulfur-donor substrates for MST. © 1996 John Wiley & Sons, Inc.     

Galectin-3 mRNA level depends on transformation phenotype in ras-transformed NIH 3T3 cells

Summary— The increase in galectin-3 lectin content observed in tumours or in in vitro transformed cells suggests that this lectin is important in the transformation process. In the present study, we investigated the mRNA expression level of the galectin-3, galectin-I and macrophage mannose receptor in normal and ras-transformed NIH 3T3 cells in relation to their transformation state. The galectin3 mRNA content in ras-transformed cells is increased in fully transformed cells, with a maximum in ras-transformed cells that have lost their growth anchorage-dependence. Under the same conditions, the galectin-1 mRNA level which was high in normal cells, increased slightly in transformed cells. The mRNA for the macrophage mannose receptor was not detected in 3T3 cells or in their ras-transformed counterparts.     

Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain

The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highests levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide -II (422–434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the -II tubulin fragment, but it showed interaction with the Affigel-conjugated -I (431–444) tubulin peptide. The different MAPs componentes were characterized by western blots using specific monoclonal antibodies. A salient feature of neonatal rat brain MAP-3 was its interactions with site-directed antibodies that recognize binding epitopes on the repetitive sequences of tau and MAP-2. However, these site-specific antibodies did not interact with MAP-1B from the neonatal rat brain tissue.Abbreviations PAA poly (L-aspartic acid) - HMW-MAPs high molecular weight microtubule associated proteins     

Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA Δ3-Δ2-isomerase

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg^-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m=59 kDa/subunit), containing 2±0.2 mol FAD, 13.6±0.8 mol Fe and 10.8±1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.     

The effects of glycophorin A on the expression of the human red cell anion transporter (band 3) in Xenopus oocytes

The effects of human red cell glycophorin A (GPA) on the translocation to the plasma membrane and anion transport activity of the human erythrocyte anion transporter (band 3; AE1) have been examined using the Xenopus oocyte expression system. We show that band 3 accumulates steadily at the oocyte surface with time in the presence or absence of GPA, but this occurs more quickly when GPA is coexpressed. The amount of band 3 at the surface is determined by the concentrations of band 3 and GPA cRNA that are injected, with a higher proportion of total band 3 being translocated to the surface in the presence of GPA cRNA. The increased expression of DNDS-sensitive chloride transport is highly specific to GPA, and is not observed when the cRNA to the putative glycophorin E or a very high concentration of the cRNA to glycophorin C are coexpressed with band 3 in oocytes.We thank Dr. Kay Ridgwell and Charlotte Ratcliffe for supplying plasmids and Dr. David Anstee for antibodies. This work was supported by grants from the Medical Research Council.     

[3H] CGP 39653 Binding to the Agonist Site of the N-Methyl- D-Aspartate Receptor Is Modulated by Mg2+ and Polyamines Independently of the Arcaine-Sensitive Polyamine Site

Abstract: This study investigated the binding of [³H] CGP 39653, a novel high-affinity antagonist of the N-methyl-D- aspartate (NMDA) recognition site of the NMDA receptor complex. [³H] CGP 39653 bound to the NMDA receptor in well washed rat brain membranes with an affinity of about 15 nM. Other NMDA site drugs inhibited [³H] CGP 39653 binding with the following order of potency: DL-(tetrazol-5- yl)glycine > glutamate > CGS 19755 > DL-2-amino-5- phosphonovalerate (DL-AP5) > NMDA. Glycine and 5, 7- dichlorokynurenate partially inhibited binding. The poly-amines spermine and spermidine increased [³H] CGP 39653 binding (EC₅₀ values of 10 and 22 μM, respectively). This effect was mimicked by arcaine, 1, 5-diethylaminopiperidine, diaminodecane, diethylenetriamine, and Mg²⁺. The increase in [3H] CGP 39653 was a result of an increased affinity of the binding site for the ligand with very little effect on binding site density. Spermine and Mg²⁺also increased the affinity of the antagonists DL-AP5 and CGS 19755, but had only minor effects on the affinity of glutamate and NMDA. Arcaine did not reverse the enhancement of [3H] CGP 39653 binding by spermine, spermidine, or Mg²⁺. Channel-blocking dissociative anesthetics, including dizocilpine and ketamine, did not alter basal or Mg²⁺-stimulated [3H] CGP 39653 binding. Spermine did not alter either the enhancement of [³H]- dizocilpine by glutamate or the inhibition of [³H]dizocilpine by DL-AP5 or CGS 19755. These studies show that poly-amines and divalent cations selectively enhance the affinity of antagonists for the agonist binding site on the NMDA receptor complex. However, this effect is mediated by a site independent of the primary polyamine site defined using [³H] dizocilpine binding.     

Enhanced expression of group II phospholipase A2 in human hepatocellular carcinoma

Enzyme activity, protein contents, and mRNA contents of group II phospholipase A₂ (PLA₂) in hepatocellular carcinoma (HCC) surgically obtained from 8 patients were compared with those in either its neighboring liver tissues or control liver tissues. The PLA₂ specific activity towards the mixed micelles of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and cholate was significantly greater in the tumor tissues (6.62 ± 1.46 nmol/min/mg) than those in the surrounding liver tissues (1.33 ± 0.22 nmol/min/mg) and controls (0.43 ± 0.04 nmol/min/mg). The results of immunoblot analysis using a specific anti-human group II PLA₂ antibody and of Northern blot analysis using a human group II PLA₂ cDNA as a probe demonstrated that group II PLA₂ was responsible for the increased enzyme activity. The contents of immunoreactive group II PLA₂ in the tumor tissues (8.81 ± 1.24 ng/mg) were significantly higher than those in the surrounding liver tissues (1.77 ± 0.27 ng/mg); those in the control tissues were below the analytical range of the method used. The group II PLA₂ mRNA was also significantly increased in the tumor tissues, compared with that in the surrounding liver tissues, whereas it was not detectable in th controls. This indicates that group II PLA₂ in HCC is induced at the pretranslational level.