Effect of cysteamine on glutathione level and developmental capacity of bovine oocyte matured in vitro

The present study was carried out to evaluate if the addition of cysteamine to the culture medium during in vitro maturation of bovine oocytes increased the glutathione (GSH) levels in the mature oocytes, and if these changes may promote an improvement on in vitro development to the blastocyst stage. Follicular oocytes from slaughterhouse ovaries were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 (control), 25, 50, or 100 μM of cysteamine for 24 hr. After in vitro maturation the oocytes were fertilized and cultured for 8 days. The percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.01) for oocytes matured in medium containing 100 μM of cysteamine than for those matured in control medium. Moreover, the intracellular GSH levels were increased (P < 0.05) in oocytes matured with 100 μM of cysteamine with respect to control. No differences were observed in maturation and cleavage rates, and in the mean cell numbers per blastocyst among treatments (P > 0.05). These results indicate that the addition of thiol compounds such as cysteamine to maturation medium increases the efficiency of in vitro blastocyst production from immature bovine oocytes. The higher levels of GSH in oocytes matured in the presence of cysteamine suggest that the beneficial effects of cysteamine on in vitro maturation and subsequent development after in vitro fertilization are mediated by GSH. © 1995 wiley-Liss, Inc.     

Troponin subunits contribute to altered myosin ATPase activity in diabetic cardiomyopathy

Our group has documented that myocardial performance is impaired in the hearts of chronically diabetic rats and rabbits. Abnormalities in the contractile proteins and regulatory proteins may be responsible for the mechanical defects in the streptozotocin (STZ)-diabetic hearts. Previously, the major focus of our research on contractile proteins in abnormal states has concentrated on myosin ATPase and its isoenzymes. Our present study is based on the overall hypothesis that regulatory proteins, in addition to contractile protein, myosin contribute to altered cardiac contractile performance in the rat model of diabetic cardiomyopathy. The purpose of our research was to define the role of cardiac regulatory proteins (troponin-tropomyosin) in the regulation of actomyosin system in diabetic cardiomyopathy.For baseline data, myofibrillar ATPase studies were conducted in the myofibrils from control and diabetic rats. To focus on the regulatory proteins (troponin and tropomyosin), individual proteins of the cardiac system were reconstituted under controlled conditions. By this approach, myosin plus actin and troponin-tropomyosin from the normal and diabetic animals could be studied enzymatically. The proteins were isolated from the cardiac muscle of control and STZ-diabetic (4 weeks) rats. Sodium dodecyl sulfate gel electrophoretic patterns demonstrate differences in the cardiac TnT and TnI regions of diabetic animals suggesting the different amounts of TnT and/or TnI or possibly different cardiac isozymes in the regulatory protein complex. Myofibrils probed with a monoclonal antibody TnI-1 (specific for adult cardiac TnI) show a downregulation of cardiac TnI in diabetics when compared to its controls. Enzymatic data confirm a diminished calcium sensitivity in the regulation of the cardiac actomyosin system when regulatory protein(s) complex was recombined from diabetic hearts. Actomyosin ATPase activity in the hearts of diabetic animals was partially reversed when myosin from diabetic rats was regulated with the regulatory protein complex isolated from control hearts. To our knowledge, this is the first study which demonstrates that the regulatory proteins from normal hearts can upregulate cardiac myosin isolated from a pathologic rat model of diabetes. This diminished calcium sensitivity along with shifts in cardiac myosin heavy chain (V1V3) may be partially responsible for the impaired cardiac function in the hearts of chronic diabetic rats. (Mol Cell Biochem151: 165–172, 1995)     

Responses to T cell receptor/CD3 and interleukin-2 receptor stimulation are altered in T cells from B cell non-hodgkin's lymphomas

T cells infiltrating (T-TIL) B cell non-Hodgkin's lymphomas (NHL) are thought to represent a local host response to the tumor. However, tumor progression in the presence of this T cell infiltrate suggests that the T-TIL may be functionally impaired. To address this issue we determined whether response to stimulation of T-TIL from 25 patients with NHL through the T cell receptor (TCR/CD3) and the interleukin-2 (IL-2) receptor (IL-2R) was intact, since activation of these receptors is important for proliferation and cytokine production. Our results demonstrate defects in response to stimulation via TCR/CD3 and the IL-2R in T-TIL cells from patients with NHL that were not observed with T cells from the peripheral blood. T-TIL showed minimal proliferation to anti-CD3 and only modest proliferation to IL-2 alone or when combined with anti-CD3. Moreover, cytokine production in T-TIL was impaired since stimulation through the TCR/CD3 complex did not induce mRNA for interferon (IFN), IL-2, IL-4 or IL-10. The functional unresponsiveness of these cells may be linked to altered signalling through the TCR/CD3 since an abnormal tyrosine phosphorylation pattern was detected in T-TIL after stimulation with anti-CD3.     

Estrogens and non-estrogenic ovarian influences combine to promote the recruitment and decrease the turnover of new neurons in the adult female canary brain

The higher vocal center (HVC) of the songbird forebrain exhibits persistent neurogenesis in adulthood, particularly in a region of the mediocaudal neostriatum that is associated with a subventricular layer of estrogen receptive cells. We asked whether estrogens might influence adult neurogenesis, by assessing the effect of ovariectomy on HVC neuronal production in the adult female canary. Fifteen 1-year-old females were separated into groups of ovariectomized, estradiol-replaced ovariectomized, and gonadally intact birds. To label dividing cells and their progency, the birds were given [³H]thymidine for 8 days, killed 32 days later, and their brains autoradiographed. A significant rise was noted in the number of HVC neurons per section in estradiol-treated birds relative to the untreated control birds. The number of [³H]-thymidine-labeled HVC neurons was also higher in the estrogen-treated birds; however, the neuronal labeling index (LI) did not vary as a function of estradiol replacement, as the total number of HVC neurons rose in parallel with the added new neurons. In contrast, the neuronal LI did rise as a result of ovariectomy, and this ovariectomy-associated increase in the LI was not reversed by estradiol. Among non-neuronal cell types, the endothelial LI was higher in estrogen-treated birds than in their untreated counterparts, suggesting estrogen-associated angiogenesis. Radioimmunoassay confirmed that serum estradiol was reduced in the castrated birds. Since estrogen appeared to promote the survival of [³H]thymidine⁺ neurons, we next sought to determine whether estrogen acted directly on the newly generated neurons, or rather indirectly through an intermediary cell population. To this end, we asked whether the new neurons or their precursors expressed estrogen receptor immunoreactivity (ER-IR). Five adult male canaries were given [³H]thymidine for periods ranging from 2 to 28 days, killed at varying times up to 3 weeks therafter, then probed for ER-IR and autoradiographed. [³H]thymidine⁺ cells displayed no detectable ER-IR within their first 4 weeks of postmitotic life. Rather, during migration from the ventricular zone (VZ), the new neurons traversed a layer of mitotically quiescent, ER⁺ subventricular cells. Double labeling for ER-IR and cell-type selective antigens confirmed that these ER⁺ cells were neurons. These results indicate that the early survival of new neurons in the adult songbird HVC is promoted by estrogen, and may be mediated by the estrogen-stimulated paracrine release of neurotrophic agents by ER-IR subventricular neurons. Our data suggest that estrogen's promotion of neuronal survival may operate concurrently with an estrogen-independent ovarian suppression of neuronal mitogenesis.     

Apolipoprotein(a): A Puzzling Evolutionary Story

Human apolipoprotein(a), a risk factor for heart disease, has over 80% sequence identity to plasminogen. Plasminogen contains five distinct kringle domains plus a catalytic protease subunit. Human apo(a) consists of multiple copies (the number varies in individuals) of a domain resembling kringle 4, a single copy of a domain resembling kringle 5, and a protease-like domain. The recently cloned hedgehog version of apolipoprotein(a), which contains 31 nearly identical copies of plasminogen kringle 3 and lacks a protease domain, has prompted us to investigate the evolutionary history of the apolipoprotein (a) gene in mammals. Our analysis supports the nonfunctionality of the human apolipoprotein(a) protease domain, and a single (or multiple) duplication of plasminogen gene before mammal radiation, which originated apolipoprotein(a) in mammals. Received: 26 February 1996 / Accepted: 6 August 1996     

Antioxidant Enzymes in Phytoparasitic Nematodes

Presence of different antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and ascorbate, p-phenilendiamine-pyrocathecol (PPD-PC), o-dianisidine, and guaiacol isoperoxidases, was shown in the phytoparasific nematode species Meloidogyne incognita, M. hapla, Globodera rostochiensis, G. pallida, Heterodera schachtii, H. carotae, and Xiphinema index. The activity of the enzymes tested differed among the life stages examined. SOD was present in cysts but was not detected in Meloidogyne egg masses. Catalase activity of Meloidogyne females was higher than that of preparasitic stages and cyst-nematode females. For the first time, ascorbate peroxidase was found to occur commonly in phytoparasitic nematodes, with the highest activity in the invading life-stages. In all the life stages examined, the antioxidant enzyme activities of M. hapla were markedly higher than those of M. incognita. Glutathione peroxidase was not found in the species examined.     

Redescription and distribution of Boeckella gibbosa (Brehm, 1935) (Copepoda,Calanoida, Centropagidae)

Thirty-five species of the family Lecanidae are examined from Tripura state in North-Eastern India. Of these, Lecane levistyla and L. scutata are interesting cold-water forms; L. batillifer is an Australasian element; L. acanthinula and L. sinuata are Oriental endemics and L. braumi, L. lateralis and L. simonneae are palaeotropical species. The lecanid fauna also includes the pantropical L. thienemanni and L. sola while the remainder are cosmopolitan and tropicopolitan elements. This revised version was published online in August 2006 with corrections to the Cover Date.     

A Procedure for Human Pregnancy Zone Protein (and Human α2-Macroglobulin) Purification Using Hydrophobic Interaction Chromatography on Phenyl–Sepharose CL-4B Column

In the present work we describe a procedure for the purification of human pregnancy zone protein (PZP) from pooled late pregnancy plasma by using hydrophobic interaction chromatography (HIC) on a phenyl–Sepharose column. The HIC step allowed the complete isolation of haptoglobins and the partial separation of human α₂-macroglobulin (α₂-M) from a protein fraction containing PZP previously obtained by a DEAE-Sephacel chromatography. Pure and native PZP, with a recovery of nearly 25% and biological activity of protease-binding, was obtained by two definitive final steps consisting of zinc-chelate and size-filtration chromatographies. Moreover, we further present an alternative procedure for the purification of α₂-M from the same pregnancy plasma, based on the differential elution of PZP and α₂-M from the HIC. This purification step gave rise to a highly purified product with a recovery of 10%. This differential elution could be explained by differences in surface hydrophobicity observed between both proteins. In addition, considering the different hydrophobic properties exhibited by native PZP and PZP–protease complexes, HIC on phenyl–Sepharose column could also be used for separating both conformational states of PZP.     

Urinary markers of nephrotoxicity following administration of 2 bromoethanamine hydrobromide a comparison with hexachlorobutadiene

2 bromoethanamine hydrobromide (BEA) has been widely considered to be a target selective nephrotoxin that causes necrosis of the medulla in 24-48 h, but recent reports suggest that early cortical injury is also associated with this lesion. In order to assess the cortical effects of BEA (100 mg kg^-1 bw single ip injection), several urinary markers of renal injury were evaluated over a 7 day period in male Wistar Albino rats. Hexachlorobutadiene (HCBD 150 mg kg^-1 bw in peanut oil ip), a renal toxin which targets selectively for the proximal tubule, was used as a comparison. After BEA treatment, urinary levels of alanine aminopeptidase, gamma-glutamyl-transpeptidase, alkaline phosphatase and glucose increased transiently. Each of the proximal tubule marker enzymes peaked earlier following HCBD treatment and elevation of alanine aminopeptidase and gamma glutamyl transpeptidase was sustained for longer periods than for BEA. Following BEA treatment, lactate dehydrogenase rose prominently on day 1 followed by a return to control values on day 2 and a further rise on day 3 and remained high until the end of the study. BEA also increased the urinary excretion of total protein and albumin. After HCBD treatment, lactate dehydrogenase showed a transient elevation and glucose levels were slightly increased. Based on the present observations the changes induced by BEA administration on urinary markers of renal injury are different from those observed following HCBD treatment. These findings suggest that BEA toxicity also involves other parts of the kidney besides the papilla.     

Differential gene expression in apoptotic 32Dcl3 cells: induction of metallothionein

Growth factor deprivation induced cell death of the hematopoietic cell line 32Dcl3 is widely used as a model system to study apoptotic signalling pathways. Here we show that the onset of cell death after IL-3 withdrawal can be strongly delayed by either cycloheximide or actinomycin D, indicating that de novo protein synthesis is required. Subtractive cDNA library hybridization was used to identify genes upregulated in apoptotic 32Dcl3 cells. Here we present data showing metallothionein-I (MT-I) mRNA transiently upregulated by a factor of three- to 20-fold. Increased levels of total MT-I+II protein after IL-3 withdrawal were demonstrated. An induction of MT-I RNA as well as of MT-I+II total protein was also observed in serum deprived NIH3T3 fibroblasts. Testing the effect of different inducers of apoptosis on 32Dcl3 cells we found that only IL-3 withdrawal and ethanol treatment led to an upregulation of MT-I mRNA level. Since MTs are believed to play a role in the metabolism of zinc, we tested the effect of zinc on induced cell death. When 32Dcl3 cells are treated with zinc (50-300 M) in the absence of IL-3, loss of viability as well as degradation of the cellular DNA were delayed, indicating that zinc represses apoptosis. On the other hand zinc pre-treatment induced MT expression and accelerated the onset of apoptosis. Our data, therefore, suggest that MT exerts a proapoptotic function.