Characterization of the androgen-dependent 22Kdalton glycoprotein from rat ventral prostate

Oxidation by molecular oxygen converted the 22Kdalton glycoprotein from rat ventral prostate into a 34K species and this reaction could be reversed by thiol reducing reagent. Measurement of the level of the 22Kdalton glycoprotein in prostatic cytosol by the radial immunodiffusion technique showed that changes in the 22Kdalton glycoprotein concentration in response to androgen withdrawal and replacement were slow in comparison to androgen regulated levels of mRNA coding for the protein. (3) Charcoal absorption steroid binding assays of the 22Kdalton glycoprotein revealed that the protein did not bind testosterone, estradiol, progesterone or corticosterone. These results indicate that the 22Kdalton glycoprotein is metabolically stable, not steroid-binding, and exists as an oligomer through disulfide crosslinking.     

Characterization of photosystem II mutants of Chlamydomonas reinhardii lacking the psbA gene

Summary We have examined 78 chloroplast mutants of Chlamydomonas reinhardii lacking photosystem II activity. Most of them are unable to synthesize the 32 Kdalton protein. Analysis of 22 of these mutants reveals that they have deleted both copies of the psbA gene (which codes for the 32 Kdalton protein) in their chloroplast genome. Although these mutants are able to synthesize and to integrate the other photosystem II polypeptides in the thylakoid membranes, they are unable to assemble a stable functional photosystem II complex. The 32 Kprotein appears therefore to play an important role not only in photosystem II function, but also in stabilizing this complex.     

Modification of lipophilic proteins in Friend erythroleukemia cells during their differenciation

In mouse erythroleukemia cells (MELC) a lipophilic protein of apparent M. W. 9.5 kdaltons increases during differentiation. This increase is due either to an increase of biosynthesis or to a structural alteration impairing the capacity of the protein to form polymers of apparent high M.W. or favoring its extractability. The increase is related to differentiation and precedes hemoglobin synthesis by at least 1 day. It is not related cells (TFA-11) in which dimethylsulfoxide (DMSO) causes an increase in virus production. As it occurs in cells treated with 4 different inducers, and as the increase is less marked when antagonists of the inducers are also present, it is unlikely that the increase of the 9.5 Kdalton protein is due to an effect of the inducers unrelated to differentiation.     

Synaptosome-associated protein of 25 kilodaltons in oocytes and steroid-producing cells of rat and human ovary: molecular analysis and regulation by gonadotropins

The synaptosome-associated protein of 25 kDa (SNAP-25) is crucially involved in exocytosis in neurons. The aim of this study was to investigate whether it is present in the ovary. We found SNAP-25 to be expressed in nonneuronal cells of the rat and human ovary, namely in all oocytes and in steroidogenic cells, including granulosa cells (GC) of large antral follicles and luteal cells. Both isoforms, SNAP-25a and b, were found in the ovary. Oocytes obtained by laser capture microdissection were shown to express SNAP-25b, whereas SNAP-25a was found in rat GC and human luteinized GC. Immunohistochemical observations of strong SNAP-25 staining in GC of large growing antral follicles compared with absent or weak staining in small follicles suggested a role in folliculogenesis. To study a presumed regulation of SNAP-25, we used a rat GC line (GFSHR-17), which expresses FSH receptors, and luteinizing human GC, which express LH receptors. FSH elevated SNAP-25 mRNA and protein levels about fivefold within 24 h in GFSHR-17 cells. The cAMP analogue dibutyryl-cAMP (db-cAMP) mimicked this action of FSH. The effects of both db-cAMP and FSH were inhibited by the protein kinase A (PKA) inhibitor H89. In contrast, SNAP-25 protein and mRNA-levels were not altered by LH/hCG in luteinized human GC. Our results for the first time identify SNAP-25b in oocytes and SNAP-25a in steroidogenic cells of the mammalian ovary. SNAP-25a and b may be involved in different exocytotic processes in these cell types.     

Surface properties of crosslinked erythrocytes as studied by counter-current distribution in aqueous polymer two-phase systems

The bifunctional imidoester dimethyl suberimidate hydrochloride can stabilize rat red blood cells (RBCs) by membrane protein crosslinking, and in that way they can be used as carrier systems for exogenous substances. Counter-current distribution fractionation in charge-sensitive dextran-polyethyleneglycol two-phase systems has been used to detect slight changes in surface charge in stabilized cells. A decrease in the surface charge of crosslinked RBCs and an apparent masking of the age-related cell surface properties have been found to result from the protein crosslinking. Digitonin treatment used to permeabilize crosslinked RBCs produces a significant decrease of the cell surface charge while the age-related surface properties do not seem to be modified by the treatment.     

Tissue and subcellular distributions of an inhibitory GDP/GTP exchange protein (GDI) for smg p25A by use of its antibody

We have recently purified from bovine brain cytosol to near homogeneity a GDP/GTP exchange protein for smg p25A, named smg p25A GDI, that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A. In the present study, we made an antiserum against smg p25A GDI and studied its tissue distribution in rat and its subcellular distribution in rat cerebrum by use of this antiserum. smg p25A GDI was found in secretory cells with both regulated and constitutive secretion types. Since smg p25A was previously found in only secretory cells with a regulated secretion type, this result suggests that small GTP-binding proteins different from smg p25A but recognized by smg p25A GDI are present in secretory cells with a constitutive secretion type, and that smg p25A GDI is involved in both regulated and constitutive secretory processes. In subcellular fractionation analysis of rat cerebrum, smg p25A GDI was mostly found in the cytosol fraction of neuron body and synaptosome. In synaptosome, it was mainly found in the synaptic cytosol.     

Arsenate induces stress proteins in cultured rat myoblasts

The induction of stress proteins was examined in rat myoblast cultures by two-dimensional gel electrophoresis. Data obtained by this analysis led to the following observations. (a) Arsenate, which behaves as a phosphate analogue in cellular phosphate-transfer reactions, stresses cultured rat cells and induces the synthesis of a unique set of proteins. (b) Most of the proteins synthesized after the addition of arsenate are identical to proteins synthesized in rat myoblasts in response to heat shock or arsenite stress. (c) However, both arsenic salts induce the synthesis of two unique proteins not induced by heat shock. (d) Five 25-30-kdalton stress proteins of rat cells do not contain methionine residues. (e) A majority of the proteins synthesized in stressed myogenic cells are also induced by stress in other rat cells such as hepatoma cells, pituitary tumor cells, and fibroblasts. The 25-30-kdalton stress-related proteins identified in myogenic cells, on the other hand, are induced in fibroblasts but not hepatoma or pituitary cells.     

Origin of 25-hydroxyvitamin D3 binding protein from tissue cytosol preparations.

The 6 S, cytosolic 25-hydroxyvitamin D₃ binding protein found in several rat tissues reacts with an antibody directed to the serum 25-hydroxyvitamin D₃ transport protein. The 6 S “cytosolic” protein is not found in carefully washed intestinal mucosal cells isolated from chicks and rats, but can be made to appear by adding serum to the cytosol itself or to the cells prior to homogenization. On the other hand, the rat intestinal 3.2 S cytosol binding protein for 1,25-dihydroxyvitamin D₃ does not react with the antibody to the serum transport protein. Thus the 6 S, 25-hydroxyvitamin D₃ binding protein does not appear to be a physiologically significant substance, but rather the result of the serum 25-hydroxyvitamin D₃ transport protein interacting with a cytosolic protein in vitro.     

Influence of exercise on cardiac and skeletal muscle myofibrillar proteins

The purpose of this study was to examine the Ca²⁺-Mg²⁺ myofibrillar ATPase and protein composition of cardiac and skeletal muscle following strenuous activity to voluntary exhaustion. Sprague-Dawley rats (200 g) were assigned to a control and exercised group, with the run group completing 25 m·min^–1 and 8% grade for 1 hour. Following activity, the myocardial Ca²⁺–Mg²⁺ myofibrillar ATPase activity -pCa relationship had undergone a rightward shift in the curve. Electrophoretic analysis revealed a change in the pattern of cardiac myofibrillar protein bands, particularly in the 38–42 Kdalton region. Enzymatic analysis of myofibrillar proteins from plantaris muscle, revealed no change in Ca²⁺ regulation following exercise. Electronmicrographic and electrophoretic analysis revealed extensively disrupted sarcomeric structure and a change in the ratio of several plantaris myofibrillar proteins. No difference was observed for myosin: Actin: tropomyosin ratios; however a dramatic reduction in 58 and 95 Kdalton proteins were evident. The results indicate that prolonged running is associated with similar responses in cardiac and skeletal muscle myofibrillar protein compositions. The abnormalities in myofibrillar ultrastructure may implicate force transmission failure as a factor in exercised-induced muscle damage and/or fatigue.     

大鼠力竭运动诱导的氧化应激损伤对红细胞变形性的影响

目的:探讨一次性力竭运动诱导的氧化应激反应对大鼠红细胞的抗氧化能力和细胞变形性的影响。方法:大鼠分为3组(n=10):对照组(Control)、适度运动组(MRE)和力竭运动组(ERE)。力竭运动组大鼠运动的前20 min保持5%的坡度和20 m/min的速度,20 min后调整为15%的坡度和25 m/min的速度,直至运动力竭。适度运动组大鼠在5%的坡度和20 m/min的速度下跑40 min。检测各组大鼠红细胞的抗氧化能力,并对氧化应激反应诱导的红细胞膜蛋白巯基水平、膜脂质过氧化水平和膜蛋白SDS-Page电泳条带变化进行了分析。通过激光衍射法对不同运动组大鼠红细胞变形性进行了检测。结果:力竭运动条件下大鼠红细胞受到严重的氧化应激损伤,红细胞内抗氧化能力下降。导致膜脂质过氧化损伤和膜蛋白巯基交联为主的蛋白聚簇化,形成高分子聚合物(HMW)。力竭组大鼠红细胞变形性(0.314±0.013 at 3 Pa and 0.534±0.009 at 30 Pa)显著低于对照组(0.41±0.01 at 3 Pa and 0.571±0.008 at 30 Pa;P0.05 and P0.01,respectively)和适度运动组。结论:力竭运动诱导的氧化损伤导致了红细胞变形能力(EI)的显著下降,使红细胞在微循环的转运受到限制,导致组织缺血缺氧进而引起休克、死亡等运动性疾病。     

HIV-1 Tat-mediated protein transduction of Cu,Zn-superoxide dismutase into pancreatic beta cells in vitro and in vivo

Reactive oxygen species (ROS) are considered an important mediator in pancreatic beta cell destruction, thereby triggering the development of insulin-dependent diabetes mellitus. In the present study, we investigated the HIV-1 Tat protein transduction domain-mediated transduction of Cu,Zn-superoxide dismutase (SOD), which supplies SOD activity exogenously in pancreatic beta cells under oxidative stress. Tat-SOD fusion protein was successfully delivered into insulin-producing RINm5F cells and rat islet cells. The intracellular dismutation activities of SOD were found to increase in line with the amount of protein delivered into the cells. ROS, nitric oxide-induced cell death, lipid peroxidation, and the DNA fragmentation of insulin-producing cells were found to be significantly reduced when the cells were pretreated with Tat-SOD. Next, we examined the in vivo transduction of Tat-SOD into streptozotocin-induced diabetic mice. A single intraperitoneal injection of Tat-SOD resulted in the delivery of this biologically active enzyme to the pancreas. Moreover, increased radical scavenging activity in the pancreas was induced by multiple injections of Tat-SOD, and this enhanced the tolerance of pancreatic beta cells to oxidative stress. These results suggest that the transduction of Tat-SOD offers a new strategy for protecting pancreatic beta cells from destruction by relieving oxidative stress in ROS-implicated diabetes.     

The presence and localization of heat shock protein 70 in rat mast cells

Heat shock protein 70 (Hsp70) is considered not only as a cytosolic stress protein, but also as an extracellular molecule with immunomodulatory and signaling functions that play a role in adaptation to stress on cellular and systemic levels. The active involvement of mast cells in adaptation to stress may be associated with the presence of Hsp70 in secretory granules. Using immunoelectron microscopy, we showed that Hsp70 localized in secretory granules of rat pericardial and peritoneal mast cells. Localization of Hsp70 in rat perinoneal mast cells isolated by centrifugation on Percoll was confirmed by immunoblotting. The proposed involvement of mast cells in production of extracellular Hsp70 and possible functions of Hsp70 inside the mast cells granules are discussed.     

Regulation of cytosol 5'-nucleotidase by adenylate energy charge

In the physiological range of the adenylate energy charge in liver (0.7-0.9), th rate of AMP-hydrolysis catalysed by rat liver cytosol 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) increased sharply with decreasing energy charge. In addition, a decrease in the concentration of Pi caused marked acceleration of the AMP-hydrolysing activity over the physiological range of adenylate energy charge. These responses seem to serve to protect the cells against a metabolic stress which could result from sudden utilization of ATP by removal of AMP. The AMP-hydrolysing activity of this enzyme decreased sharply as the size of the adenine nucleotide pool decreased in the physiological range. This effect may be a self-limiting response to prevent excess depletion of the pool. IMP-hydrolysing activity of this enzyme increased with increasing adenylate energy charge. But no marked response to its variation within the physiological range was observed. On the basis of the data obtained in this study, the IMP-hydrolysing activity of the cytosol 5'-nucleotidase in rat liver cells seems to be comparable to that of AMP deaminase reaction, but the AMP-hydrolysing activity was estimated to be less than 10% of AMP deaminase reaction at energy charge value of about 0.7. This strongly suggests that the AMP leads to IMP leads to inosine pathway is more significant that the AMP leads to adenosine leads to inosine pathway in rat liver.     

Changes in cell surface charge and transmembrane potential accompanying neoplastic transformation of rat kidney cells

Free flow electrophoresis measurements have been used to determine the surface charge density of normal rat kidney (NRK) cells and a clone of NRK, designated as 6m2, that exhibit a transformed phenotype at 33 degrees C and a non-transformed phenotype at 39 degrees C. A clone of 6m2, designated 54-5A4, which is transformed at both 33 degrees C and 39 degrees C was also studied. A surface charge density of -1.42 microC/cm2 was obtained for the NRK and non-transformed 6m2 cells at 39 degrees C, whereas at 33 degrees C values of -1.85 and -1.78 microC/cm2 were determined for the transformed 6m2 and 54-5A4 cells, respectively. It was found that 72% of the increased charge that appeared on the transformed 6m2 cells compared with the non-transformed 6m2 cells was RNAase sensitive. The time-dependent decrease in surface charge that accompanied the shift of the 6m2 cells from their transformed to non-transformed state was found to mirror the increase in transmembrane potential previously reported using a fluorescent dye technique, and was also comparable to the reported temporal changes in their morphology and virally-coded protein content.     

Distribution,phosphorylation, and activities of Hsp25 in heat-stressed H9c2 myoblasts: a functional link to cytoprotection

The behavior of the endogenous heat shock protein 25 (Hsp25) in heat-stressed rat H9c2 myoblasts was studied. After mild or severe heating, this protein became less extractable with Triton X-100 and displayed characteristic immunofluorescence patterns, namely (1) granules in the nucleus, and (2) association with F-actin bundles in the cytoplasm. The intranuclear granulation of Hsp25 and its association with F-actin were sensitive to drugs affecting Hsp25 phosphorylation (cantharidin, sodium orthovanadate, SB203580, SB202190). Isoform analysis of Hsp25 translocated to the nucleus-free cytoskeletal fraction revealed only mono- and biphosphorylated Hsp25 and no unphosphorylated Hsp25. Transfected luciferase with initial localization in the nucleosol became colocalized with the Hsp25-containing granules after a heat shock treatment that denatured the enzyme in the cells. The association of Hsp25 with actin filaments after a mild heat stress conferred protection from subsequent F-actin-damaging treatments with cytochalasins (D and B) or severe heat stress. We hypothesize that (1) the binding of heat-denatured nucleosolic proteins to the Hsp25 contained in specific granular structures may serve for the subsequent chaperoning or degradation of the bound proteins, and (2) the actin cytoskeleton is stabilized by the direct targeting of phosphorylated Hsp25 to microfilament bundles.     

25-Hydroxylation of 1 alpha-hydroxyvitamin D-3 in rat and human liver

1 alpha-Hydroxyvitamin D-3 25-hydroxylase activity was measured in subcellular fractions of rat and human liver. The formation of 1,25-dihydroxyvitamin D-3 was determined by high pressure liquid chromatography. In rat liver 1 alpha-hydroxyvitamin D-3 25-hydroxylase activities were found in the purified nuclei, the heavy mitochondrial fraction and the microsomal fraction. The enrichment of 25-hydroxylase activity was highest in the heavy mitochondrial fraction. With this fraction a minimum amount (about 0.5 mg) of protein was required before formation of 1,25-dihydroxyvitamin D-3 could be detected. Above this amount the reaction was linear with amount of protein up to at least 2 mg/ml. The reaction was also linear with time up to 60 min. An apparent Km value of 2 X 10(-5) M was found. The mitochondrial 25-hydroxylase was stimulated by addition of cytosolic protein or bovine serum albumin. The degree of stimulation was dependent on the amount of mitochondrial protein present in the incubation mixture. Maximal stimulation was seen with 0.2 mg/ml of either protein in the presence of 0.5 mg mitochondrial protein. The stimulating effect remained after heating the protein for 5 min at 100 degrees C. The cytosolic protein did not stimulate a reconstituted mitochondrial 1 alpha-hydroxyvitamin D-3 25-hydroxylase. The mitochondrial vitamin D-3 25-hydroxylase was inhibited both by cytosolic protein and by bovine serum albumin. Human liver revealed only one 1 alpha-hydroxyvitamin D-3 25-hydroxylase activity located to the heavy mitochondrial fraction. The results are in agreement with previous studies on the localization of vitamin D-3 25-hydroxylase in rat and human liver. The difference in localization of the 25-hydroxylase between rat and human liver implies that studies on the regulation of the microsomal 25-hydroxylase in rat liver may not be relevant to the situation in human liver.     

Reduction in S100 protein beta subunit mRNA in C6 rat glioma cells following treatment with anti-microtubular drugs

S100 protein is a calcium-binding protein found in vertebrate nervous tissue. Synthesis of S100 protein in the rat glioma cell line, C6, is inhibited by the addition of anti-microtubular drugs. We have cloned a cDNA for the beta subunit of S100 protein from rat brain in a lambda gt 11 expression vector and used this cDNA to measure the amounts of S100 beta subunit mRNA in C6 cells after treatment with anti-microtubular drugs. Levels of alpha-tubulin and beta-actin mRNAs were also measured. All measurements were performed using RNA-RNA hybridization techniques at high stringency with rat mRNA-specific probes. After 24 h of treatment, the S100 beta subunit mRNA was reduced to levels of 25% by colchicine and 32% by vinblastine when compared to untreated controls. In contrast, the levels of tubulin and actin mRNAs were only slightly changed by these treatments. These studies demonstrate that disruption of the microtubular cytoskeleton causes a specific reduction in the level of S100 protein mRNA in C6 cells.     

The role of beta2-glycoprotein I in liposome-hepatocyte interaction

Adsorption of serum proteins to the liposomal surface plays a critical role in liposome clearance from the blood. The aim of this study was to investigate the role of liposome-adsorbed serum proteins in the interaction of liposomes with hepatocytes. We analyzed the serum proteins adsorbing to the surface of differently composed small unilamellar liposomes during incubation with human or rat serum, and found that one protein, with a molecular weight of around 55 kDa, adsorbed in a large amount to negatively charged liposomes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). The binding was dependent on the liposomal charge density. The approximately 55-kDa protein was identified as beta2-glycoprotein I (beta2GPI) by Western blotting. Despite the high affinity of beta2GPI for strongly negatively charged liposomes, in vitro uptake and binding experiments with isolated rat hepatocytes, Kupffer cells or liver endothelial cells, and with HepG2 cells showed no enhancing effect of this protein on the association of negatively charged liposomes with any of these cells. On the contrary, an inhibitory effect was observed. We conclude that despite abundant adsorption to negatively charged liposomes, beta2GP1 inhibits, rather than enhances, liposome uptake by liver cells.