A novel combination of silane-coated silica colloid with hybrid RNA extraction protocol and RNA enrichment for downstream applications of spermatozoal RNA.

Spermatozoa are specialised cells with low RNA content as compared to somatic cells. The suitable sperm RNA extraction and enrichment protocols for downstream applications are available for human, cattle, stallion and mouse but not for buffalo spermatozoa. Therefore, the present work was conducted to find out suitable colloidal solution for sperm purification and appropriate protocol for sperm RNA extraction and enrichment/amplification of RNA. For purification, we used PVP-coated silica colloidal solution (PVP-Si), silane-coated silica colloidal solution (Silane-Si) and iodixanol. Sperm recovery rate, total sperm motility and progressive sperm motility were significantly improved after separation by Silane-Si and iodixanol compared to PVA-Si method. The combined guanidinium thiocyanate-phenol-chloroform (GTPC) with silica matrix (SM)-based RNA extraction yielded more quantity of RNA in compared to individual method. The hybrid of SM and GTPC into a single protocol yielded 360-450 ng RNA from 30 million buffalo spermatozoa. For the first time, we adopted new way to enrich sperm RNA that increased the RNA concentration 4-5 times that was sufficient for downstream applications. The linear amplification of sperm RNA increased RNA concentration around 27-45 times. In summary, Silane-Si colloid for sperm separation, hybrid SM and GTPC protocol for sperm RNA extraction followed by enrichment or amplification of RNA was found suitable for high-throughput analyses of buffalo sperm RNA.

Terminal transferase in acute lymphoblastic leukemia in gibbons.

Terminal deoxynucleotidyl transferase, an enzyme which catalyzes the polymerization of deoxyribonucleoside triphosphates on a 3'-OH end of an initiator molecule in the absence of a template, has been suggested as a biological marker for human acute lymphoblastic leukemia. Examination of a cell line, 6G1, recently established from the peripheral blood of a gibbon ape with acute lymphoblastic leukemia showed the presence of terminal deoxynucleotidyl transferase. This enzyme after purification by successive column chromatography on DEAE-cellulose, phosphocellulose and hydroxyapatite, was found to have biochemical properties similar to those reported for terminal transferase from calf thymus and human leukemic cells. These studies suggest that terminal transferase can be used as a useful biological marker for acute lymphoblastic leukemia in both humans and subhuman primates.

Stimulation of luteinizing hormone release by gamma-aminobutyric acid (GABA) agonists: mediation by GABAA-type receptors and activation of chloride and voltage-sensitive calcium channels.

The mechanism by which gamma-aminobutyric acid (GABA) stimulates the release of LH was analyzed in cultured female rat pituitary cells. In 3-h incubations, GABA (1-100 microM) caused a dose-dependent increase in LH release, with the maximal response about 16% of that evoked by 10 nM GnRH. GABA action was independent of the GnRH receptor, since 1 microM GnRH antagonist [( N-acetyl-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10] GnRH), which completely inhibits GnRH action, did not affect the response to GABA. In studies on the effects of GABA receptor agonists and antagonists, 4,5,6,7-tetrahydoisoxazolo-[5,4-c]pyridin-3(2H)-one (THIP) and muscimol (GABAA agonists) gave similar response patterns, with the same maximal stimulation as GABA but much higher potencies. In contrast, the GABAB receptor agonist baclofen did not stimulate LH release. The GABAA receptor antagonist SR95531 caused dose-dependent inhibition of the LH-releasing effects of GABA and muscimol (10 microM), with complete blockade at 10 microM SR95531. T-Butylbicyclophosphorothionate, an inhibitor of the GABAA receptor-associated chloride channel, also dose-dependently reduced the releasing effect of 100 microM GABA. These results indicate that GABA action is mediated by the chloride channel-associated GABAA receptor. However, the other GABAA receptor antagonists, including bicuculline, picrotoxin, and strychnine, did not attenuate the LH-releasing effect of 100 microM GABA in concentrations up to 100 microM, suggesting that GABA action is mediated by nonclassical GABAA receptors. Incubation in the presence of nifedipine (1 microM) or in calcium-free medium inhibited the LH-releasing action of GABA, indicating that calcium influx through voltage-sensitive calcium channels (VSCC) is required for GABA-induced LH release. Such entry of Ca2+ would result from activation of VSCC by depolarization due to the increased Cl- conductance caused by GABAA receptor activation. In cell perfusion studies, the actions of GABA and muscimol were attenuated or abolished after repetitive stimulation, consistent with desensitization of the GABA receptors. These findings have demonstrated that the stimulation of LH release by GABA is independent of GnRH action, occurs via binding to nonclassical GABAA receptors, which rapidly desensitize, and is mediated by the activation of VSCC.

Sequence of a cDNA encoding bovine apolipoprotein H.

The nucleotide sequence of the ApoH cDNA encoding the bovine apolipoprotein H (ApoH) has been determined. The deduced protein, which contains a 19-amino-acid (aa) signal peptide and the 326-aa mature ApoH, shows 89% and 86% homology with human and rat ApoH, respectively.

L-carnitine uptake into primary rat cortical cultures: interaction with GABA.

The ability of the primary rat cortical cells to take up L-carnitine increased with the age of the cultures and plateaued at around day 11 up to 25 days in vitro (DIV) when a slight decline was evident and by 32 DIV there was a major decrease in L-carnitine uptake. The uptake of L-carnitine displayed complex components. Elimination of mitochondrial energy supply by NaCN (1 mM), rotenone (1.25 microM) and DNP (50 microM), caused a small but significant decrease in the uptake (21, 11 and 16%, respectively). The uptake was highly dependent on the Na gradient, since ouabain (0.5 mM) and Na free buffer (replaced by 250 mM sucrose), reduced uptake by 54 and 63%, respectively. There was competition of L-carnitine uptake by molecules resembling its structure, e.g. gamma-aminobutyric acid (GABA), acetyl-L-carnitine (ALC), D-carnitine, L-aminocarnitine and L-choline, with GABA being the most potent inhibitor (57% at 50 microM) and L-choline not being significantly active. The Na-dependent uptake of L-carnitine was saturable with a high Km (692 microM) and Vmax (839 pmol/min/mg). This Na-dependent component was not further additive with the GABA (500 microM) or the DNP (50 microM) inhibitable component, suggesting that it represented the same phenomenon, probably the Na gradient dependent transport of L-carnitine. The results indicate that the uptake of L-carnitine occurs by Na-dependent saturable process as well as non-saturable, Na-independent processes. At least the former uptake mechanism is potently inhibited by GABA.

Actions of acetyl-L-carnitine on the hypothalamo-pituitary-gonadal system in female rats.

Acetyl-L-carnitine (ALC) is known to affect several aspects of neuronal activity. To evaluate the neuroendocrine actions of this compound, several endocrinological parameters were followed in ALC-treated and control animals during recovery from dark-induced anestrus. In treated animals, serum luteinizing hormone (LH) and prolactin levels were higher than those of controls during the proestrous and estrous phases of the cycle, and serum estradiol levels were higher during estrus. No significant changes were observed in serum levels of follicle-stimulating hormone and progesterone. Uterine weight was increased in ALC-treated rats during proestrus and estrus, but not in diestrus. The basal release of gonadotropin-releasing hormone (GnRH) from perifused hypothalamic slices of ALC-treated animals was elevated at proestrus and diestrus, and GnRH release elicited by high K+ was higher during all three phases of the cycle. The basal release of LH from perifused pituitaries of treated animals was elevated in diestrus, and the LH response to GnRH was higher in estrus and diestrus I. Depolarization with K+ caused increased LH secretion during proestrus and estrus in treated animals. In contrast to these effects of ALC treatment in vivo, no direct effects of ALC were observed during short- or long-term treatment of cultured pituitary cells. These results indicate that ALC treatment influences hypothalamo-pituitary function in a cycle stage-dependent manner, and increases the secretory activity of gonadotrophs and lactotrophs. Since no effects of ALC on basal and agonist-induced secretory responses of gonadotrophs were observed in vitro, it is probable that its effects on gonadotropin release are related to enhancement of GnRH neuronal function in the hypothalamus.

The action of acetyl-L-carnitine on the neurotoxicity evoked by amyloid fragments and peroxide on primary rat cortical neurones.

The amyloid beta-peptides have been implicated in the excitotoxic mechanism of neuronal injury in the pathogenesis of Alzheimer's disease. In this paper we examine the effect of different amyloid fragments (beta A1-40, A1-28, and A25-35), as well as potential neuroprotective compounds on rat cortical neuron viability. Exposure of neurones to beta A25-35 or A1-40 at concentrations as low as 1 microgram/ml inhibited, significantly, the MTT response and this level of inhibition was similar after 24-h or three-day exposure. Furthermore, the level of inhibition was not affected by the presence or absence of 5% horse serum in the medium. Preexposure (10 min) of neurones to ALC at concentrations of 0.1, 1, 5, and 10 mM attenuated the inhibition of the MTT response caused by beta A25-35 (50 micrograms/ml) in serum free medium for 24 h. The treatment of cells with vitamin E (100 microM), catalase (4 mg/ml), NGF (0.1 and 10 ng/ml), or cycloheximide (0.1 microgram/ml) significantly restored the MTT response that was inhibited by beta A25-35. The mechanism for the protective actions of these compounds against beta A25-35 toxicity is not clear but may involve free radical scavenger action and preservation of energy production, although other mechanisms, especially for ALC, such as a direct effect on A-beta interaction with charged anionic phospholipids and/or stabilizing action on membranes, are also possible.

Peptidases involved in the catabolism of neurotensin: inhibitor studies using superfused rat hypothalamic slices.

In order to identify which peptidases are involved in the catabolism of neurotensin in the CNS, [3H-Tyr3,11]-neurotensin was superfused over rat hypothalamic slices in the presence and absence of peptidase inhibitors. The degree of degradation of the peptide was determined by reverse phase HPLC separation of 3H-labelled neurotensin from 3H-labelled products. Very little degrading activity was released from the slice into the medium during the superfusion. In the absence of inhibitors, 20 to 50% of 3H-neurotensin was degraded giving mainly 3H-Tyr along with other unidentified 3H-labelled products. Inhibitors of endopeptidase 24.11 (phosphoramidon) and proline endopeptidase (antibody) had no effect on the degradation. Captopril, an inhibitor of angiotensin converting enzyme, had a small inhibitory effect. In contrast, dynorphin(1-13), an inhibitor of a soluble, thiol dependent metallopeptidase which hydrolyses neurotensin at Arg8-Arg9, gave greater than 80% inhibition of 3H-neurotensin degradation in the slice preparation. 1,10-Phenanthroline, an inhibitor of metallopeptidases, was also an effective inhibitor. The dynorphin sequence responsible for the inhibition contains the Arg6-Arg7 bond. Other peptides (bradykinin and angiotensin) which are substrates of the soluble metallopeptidase also inhibited neurotensin breakdown by the slice. This evidence suggests that this thiol dependent metalloendopeptidase is the major neurotensin catabolizing enzyme in hypothalamic slices.

Cerebral alteration in calmodulin levels associated with the induction of physical dependence upon ethanol in rats.

Calmodulin levels were measured in different areas of brain in rats rendered physically dependent and after single doses (6 g/kg) of ethanol. After single doses of ethanol no changes in the calmodulin levels were found in the cortex, but those in the hippocampus and caudate nuclei were increased while those in the cerebellum were reduced. In the dependent intoxicated (prodromal) rats, calmodulin levels were elevated in all these regions except the cerebellum. In rats undergoing ethanol withdrawal syndrome, the calmodulin levels decreased in all regions of the brain except caudate nuclei.

Alteration in calcium-binding activity in synaptosomal membranes from rat brains in association with physical dependence upon ethanol.

The effects of ethanol treatment on calcium-binding activity in synaptosomal membrane fraction from rat brains were studied. The synaptosomal membrane fraction from the hippocampus, the cortex and the cerebellum from control, single dose (6 g/kg), dependent intoxicated (prodromal) and dependent withdrawing (ethanol withdrawal syndrome) rats were used. Two different methods were used for determining the calcium activity in these membrane preparations: the calcium chelator fluorescence probe, chlortetracycline (CTC), was used to measure Ca2+ binding sites in the membranes, and radioactive calcium (45Ca) was used to measure the calcium binding to the synaptosomal membranes. Both methods provided similar conclusions; the calcium activity was higher during the dependent intoxicated phase of the ethanol withdrawal period. The synaptosomal membranes from the hippocampus showed more drastic changes in calcium-binding activity than the cortex and the cerebellum. These results suggest that ethanol dependence is associated with changes in calcium-binding activity in certain areas of the rat brain.

Stimulation of gonadotropin-releasing hormone secretion by acetyl-L-carnitine in hypothalamic neurons and GT1 neuronal cells.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion from perifused hypothalamic cells and GT1-1 neuronal cells was significantly increased after culture in medium containing 100 microM acetyl-L-carnitine (ALC). This action of ALC was largely due to an increase in the spike amplitude of GnRH release. In addition, the receptor-mediated release of GnRH by N-methyl-D-aspartic acid and endothelin was significantly increased in perifused cells cultured in ALC-enriched medium. Stimulatory effects of ALC on basal, high K(+)- and agonist-induced GnRH release were also observed during long-term culture of primary hypothalamic neurons. Similar effects of ALC were evident in cultured GT1-1 cells and were accompanied by a significant increase in cell number. These observations in normal and transformed GnRH neurons demonstrate that ALC promotes the growth and secretory activity of neuropeptide-producing cells of the hypothalamus.

Enrichment of terminal deoxynucleotidyl transferase activity by cell separation.

Terminal deoxynucleotidyl transferase is a unique DNA polymerase that can carry out DNA synthesis on an initiator molecule in the absence of a template. The usefulness of this enzyme as a biological marker for following patients during treatment and remission has been suggested. The potential usefulness of this enzyme in predicting the onset of relapse before any morphological indications has been demonstrated in chronic myelogenous leukemia patients in blast phase of the disease. In order to be able to detect low levels of TdT activity especially during remission phase, we have used cell separation techniques which can enrich cell populations containing TdT activity. A number of cell separation techniques have been developed to separate different cell types. We have used the techniques of unit gravity sedimentation and free flow electrophoresis to achieve enrichment of TdT positive cell populations. Our results show that up to 20 fold enrichment of TdT activity in normal human bone marrow can be accomplished by using cell separation techniques. With the use of free flow electrophoresis, we have achieved enrichment of TdT positive cell populations from normal human bone marrow, cells from patients with acute lymphoblastic leukemia and chronic myelogenous leukemia in blast phase of the disease. No TdT positive cells were detected in patients with acute myelogenous leukemia. These cell separation techniques should prove to be useful in early detection of relapse in patients in remission.

Calcium regulation of magnesium dependent phosphorylation of human erythrocyte ghost spectrin.

Phosphorylation of human erythrocyte ghost membrane proteins was found to be affected by micromolar calcium concentrations. Increasing Ca2+ concentration to 0.2 microM decreased spectrin (band 2) phosphorylation to 30 +/- 6% of control (to which no calcium was added). Decreasing calcium concentration by adding EGTA (0.2mM) to the standard membrane preparation increased spectrin phosphorylation to 575% control. This effect of Ca2+ was more pronounced at higher temperature. At 0 degree C, Ca2+ (0.05mM) had no effect on protein phosphorylation. Sodium fluoride like EGTA caused a four to five fold increase in phosphorylation. Pyrophosphate, a phosphoprotein phosphatase inhibator, had no effect. Once spectrin was phosphorylated in the presence of [gamma-32P]ATP the addition of Ca2+ or EGTA did not decrease or increase its phosphorylation. It is suggested that calcium regulates spectrin phosphorylation either by decreasing kinase activity or by decreasing substrate availability.

Biphasic protein kinase C translocation in PC12 cells in response to short-term and long-term ethanol exposure.

Short-term and long-term effects of ethanol on protein kinase C (PKC) activity and PKC translocation from cytosol to membrane were examined in PC12 cells, a clonal cell line of neural crest origin. Treatment of PC12 cells with ethanol (30-100 mM) for 2 hr had no effect on PKC activity and PKC translocation. When PC12 cells were treated with 100 mM ethanol for 18, 44 and 74 hr, there was a biphasic effect on PKC translocation. At 18 and 44 hr ethanol treatment, PKC translocation was significantly (P < 0.001) increased, at 74 hr ethanol treatment, there was a significant decrease (P < 0.05). Less than 100 mM of ethanol had no effect on PKC activity and PKC translocation. Cyclic AMP and cyclic GMP-dependent protein kinase had no effect on PKC translocation. These findings indicate that biphasic PKC translocation from cytosol to membrane forms the basis of acute and chronic effects of ethanol on neurotransmission.

Calcium-induced proteolysis of spectrin and band 3 protein in rat erythrocyte membranes.

Calcium-dependent protease activity capable of degrading a number of endogenous proteins was found in rat red blood cell membranes. This protease activity, like that found in human red blood cells, was activated by low concentrations of calcium, but in the rat red blood cells, unlike the human red blood cells, calcium-activated protease activity was membrane-bound. A number of endogenous membrane-bound proteins were degraded after the addition of calcium to the membranes. These included spectrin bands 1 and 2 as well as bands 3, 2.1, and 2.2. No calcium-induced aggregation (transglutaminase activity) was noted in the rat red blood cell membranes.

Changes in erythrocyte ghost protein phosphorylation associated with physical dependence upon ethanol.

The effects of ethanol on phosphorylation in the erythrocyte ghost membranes was studied. Four groups of rats were used: controls; prodromal phase; overt withdrawal syndrome; and treated with a single dose (6g/kg) of ethanol. Spectrin (Band 2) phosphorylation was enhanced during prodromal phase (200 +/- 12%) and during overt ethanol withdrawal syndrome (150 +/- 18%), while a single dose of ethanol produced no significant change. Addition of micromolar calcium chloride solution to the phosphorylating medium produced pronounced decrease in spectrin phosphorylation in the ghost membranes isolated from controls and those treated with a single dose of ethanol, while this effect of calcium was less pronounced in the ghost membranes isolated from rats in the prodromal phase and in the withdrawal syndrome. This suggests that due to prolonged ethanol treatment membrane phosphorylating properties were less sensitive to the change in calcium concentration. The membrane polypeptide composition was unaffected upon ethanol treatment.

Enrichment of cell populations containing terminal deoxynucleotidyl transferase activity by free flow electrophoresis.

Terminal deoxynucleotidyl transferase (TdT) is an enzyme that has been utilized as a biochemical marker for following certain leukemia patients during treatment and remission. The potential usefulness of this enzyme in predicting the onset of relapse before any morphological indications has been demonstrated in chronic myelogenous leukemia patients in blast phase of the disease. In order to be able to detect low levels of TdT activity, especially during remission phase, we have used the technique of free flow electrophoresis to enrich cell populations containing TdT. With this technique we have achieved up to 20-fold enrichment of TdT-positive cell populations from normal human bone marrow, cells from patients with acute lymphoblastic leukemia and chronic myelogenous leukemia in blast phase of the disease. TdT positive cells were not detected in patients with acute myelogenous leukemia and B cell lines. This technique should prove to be useful in early detection of relapse in patients in remission and for enrichment of cell populations containing other biochemical markers.

Efflux of putative transmitters from superfused rat brain slices induced by low chloride ion concentrations.

Slices of rat cerebral cortex, preloaded with [14C]gamma-aminobutyric acid (GABA) and either [3H]5-hydroxytryptamine (5-HT) or [3H]noradrenaline, were superfused with media in which varying concentrations of Cl- had been replaced with other monovalent anions. Rapid reduction of [Cl-], by superfusion with media containing instead the impermeant anions propionate, isethionate, gluconate, or methyl sulphate, caused increases in the efflux of tritiated biogenic amines, but the increase in that of [14C]-GABA was not significant. The increased efflux of [3H]5-HT evoked by superfusion with low Cl- levels when propionate was the replacement anion, was transient and was linearly related to the log[Cl-]-1. It was not affected by removal of Ca2+ or by addition of 10 mM Mg2+ and was delayed but not abolished by tetrodotoxin. The low Cl(-)-evoked efflux of [3H]5-HT was not affected by pretreatment with neuronal reuptake blockers but was inhibited by picrotoxin, strychnine, and 4-acetamido-4-isothiocyanostilbene-2,2-disulphonic acid and was enhanced by glycine. Muscimol and GABA were without effect. These observations are taken to indicate that the efflux of biogenic amines is brought about by terminal depolarisation due to outward movement of Cl- in low chloride-containing media. They are of relevance to other physiological and pharmacological studies in which anion concentrations are manipulated and suggest that the anion-evoked release phenomenon may provide a model for the analysis of Cl(-)-dependent mechanisms in nerve terminals.

Inhibition of L-carnitine uptake into primary rat cortical cell cultures by GABA and GABA uptake blockers.

L-carnitine plays a central role in mitochondrial function and is found to be differentially distributed in the brain. We have shown before that the uptake of L-carnitine into cultured rat cortical neurones was temperature-dependent, as well as potently inhibited by factors affecting the sodium gradient as well as by molecules resembling its structure, e.g. D-carnitine, acetyl-L-carnitine and gamma-aminobutyric acid (GABA). GABA was the most potent inhibitor of L-carnitine uptake. In the present study we have found that specific GABA uptake blockers, nipecotic acid, cis-4-hydroxynipecotic (HNA), guvacine, 2,4-diaminobutyric acid (DABA) and NO 711 inhibit L-carnitine uptake even more potently than GABA. However, apart from NO 711, they caused about the same maximal inhibition, 67.4% at 50 microM for guvacine, compared to 60.5% by GABA. NO 711 was extremely potent and blocked 80.5% of the L-carnitine uptake. In contrast, the GABAA receptor agonists, isonipecotic acid and isoguvacine, or the antagonist bicuculline, at similar concentrations (50 microM), did not significantly inhibit the uptake of the L-carnitine. However, bicuculline at relatively high concentration (500 microM) was inhibitory (38%). The GABAB receptor agonist, baclofen, or antagonist, phaclofen, were ineffective, although 5-aminovaleric acid did significantly inhibit uptake at both 50 and 500 microM, causing 22 and 48% inhibition respectively. Like bicuculline, it was not as effective as GABA or the specific GABA uptake blockers. The results indicate that the uptake of L-carnitine by rat cortical neurones occurs in part by a process that can be potently inhibited by GABA and GABA uptake blockers.

Selective regulation of beta 2-adrenergic receptor gene expression by interleukin-1 in cultured human lung tumor cells.

The regulation of beta 1- and beta 2-adrenergic receptors (beta 1AR and beta 2AR) and receptor gene expression by interleukin-1 alpha (IL-1 alpha) was studied in cultured A549 human lung adenocarcinoma cells. The density and affinity of beta 1 AR and beta 2 AR were analyzed by computerized curve fitting of 125I-pindolol binding and its displacement by subtype selective antagonists. Steady state levels of receptor mRNAs were quantified by DNA excess solution hybridization assays. A549 cells in preconfluent cultures had fewer beta 1AR than beta 2AR (beta 1: 1.9 +/- 0.3 vs beta 2: 4.0 +/- 0.5 fmol/mg protein, means +/- SE), but lost most of their beta 2 AR upon reaching confluency (beta 1: 2.7 +/- 0.4, beta 2: 0.8 +/- 0.3 fmol/mg). Incubation of preconfluent cells for 24 hr with 20 pM of human recombinant IL-1 alpha did not modify the density of either of the beta AR subtypes. Similar incubations of confluent cells increased the density of beta 2 AR from 0.8 +/- 0.3 to 4.2 +/- 0.9 fmol/mg, while the density of beta 1 AR and the antagonist affinities of both receptors remained unaltered. The IL-1 alpha-induced increase in beta 2 AR density in confluent cells was antagonized in a concentration-dependent manner by a recombinant protein antagonist of type I IL-1 receptors (IC50: 0.2 nM). The IL-1 alpha-induced increase in beta 2AR density was preceded by an increase in the steady state level of beta 2AR mRNA, while levels of beta 1AR mRNA remained unchanged. IL-1 alpha increased the stability as well as the rate of transcription of beta 2AR mRNA. These findings demonstrate for the first time that activation of type I IL-1 receptors in A549 cells leads to a cell density-dependent, selective upregulation of beta 2AR, and that the mechanism of this effect involves increased formation and stability of the beta 2AR message.