The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.     

Analysis of time-resolved fluorescence anisotropy in lipid-protein systems

Fluorescent probes located in heterogeneous environments give rise to anomalous time-resolved fluorescence anisotropy. A simple analytical expression of anisotropy has been derived for the case of a small difference in local fluorescence lifetimes. The expression has the diagnostic advantage that the time dependence of the fluorescence anisotropy can be predicted from the differences in fluorescence lifetimes and residual anisotropies of the probes located in different sites. Using this model, the local fluorescence anisotropy parameters and the relative contributions of the lipid probe octadecyl rhodamine B in a lipid environment and in the vicinity of bacteriophage M13 coat protein reconstituted in phospholipid bilayers, composed of 80% 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 20% 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol have been determined experimentally. At 40°C, the correlation times for bound and free probes are 2.3 and 3.0 ns, respectively, while the corresponding order parameters are 0.85 and 0.62, respectively.Abbreviations ESR electron spin resonance - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol - L/P ratio phospholipid to coat protein molar ratio - <> average fluorescence lifetime - r(0) initial anisotropy - r() residual anisotropy On leave of Shanghai Medical Equipment Research Institute, 77 Jiang Ning Rd. Shanghai, People's Republic of China Offprint requests to: M. A. Hemminga     

Application of Deuterated A-Tocopherols to the Biokinetics and Bioavailability of Vitamin E

-Tocopherol, a superior chain-breaking, peroxyl radical-trapping antioxidant and the most active component of vitamin E, is elevated in liver tumor cells, contributing to their greater resistance towards lipid peroxidation compared to cells from normal tissues. Also, in regenerating rat liver the level of vitamin E has been found to fluctuate in phase with the rate of cell division. In order to study the biokinetcis and mechanisms of the distribution of vitamin E in organs and within tissues of animals, deuterated forms of -tocopherol have been synthesized and their uptake into blood and tissues has been measured by gas chromatography-mass spectrometry. Measurement of the competitive uptake from a mixture of the RRR-and SRR--tocopherol stereoisomers labelled with different amounts of deuterium shows that the liver exerts a strong preference for secretion of the natural (RRR) stereoisomer into the plasma. It is suggested that a tocopherol-binding protein plays a key role in this process.     

Impact of sar and agr on methicillin resistance in Staphylococcus aureus

Abstract The global regulators agr and sar control expression of cell wall and extracellular proteins. Inactivation of either sar and/or agr in a typical heterogeneously methicillin-resistant Staphylococcus aureus resulted in a small but reproducible decrease in the number of cells in the subpopulation expressing high methicillin resistance. The amount of low affinity penicillin-binding protein PBP2', the prerequisite for methicillin resistance, was apparently not affected, however, a reduction in PBP1 and PBP3 production was observed, suggesting that these resident PBPs of the cells might be involved somehow together with PBP2' in high level methicillin resistance.     

Immunohistochemical characteristics of the constituents of senile plaques and amyloid angiopathy in aged cynomolgus monkeys

Abstract: In this study, we immunohistochemically examined the several constituents of senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in aged cynomolgus monkeys. Apolipoprotein E (apoE) deposited in all mature plaques and CAA, and in half of the diffuse plaques. Alpha-1-antichymotripsin (αACT) deposited in half of the mature plaques and in one third of the CAA. Amyloid precursor protein (APP), ubiquitin (Ub), and microtubule-associated protein-2 (MAP-2) accumulated in the swollen neurites of mature plaques. Glial fibrillary acidic protein (GFAP) was detected in the astrocytes and their processes surrounding the mature plaques. Tau was detected in neither the SPs nor CAA. Therefore, mature plaques involved extracellular Aβ, apoE, and αACT, and also astrocytes and swollen neurites. However, diffuse plaques involved only extracellular Aβ and apoE. Since these features, except for tau, were consistent with those in humans, this animal model will be useful for studying the pathogenesis of cerebral amyloid deposition.     

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.