Effect of pregnant mare serum gonadotropin on the induction and degradation of FSH and LH receptors in the granulosa cell of the immature rat

The relative induction of FSH and LH receptors in the granulosa cells of immature rat ovary by pregnant mare serum gonadotropin (PMSG) has been studied. A single injection of PMSG (15 IU) brought about a 3- and 12-fold increase in FSH and LH receptor concentration,respectively, in the granulosa cells. Maximal concentration was reached by 72 h but the receptor levels showed a sharp decline during the next 24–48 h. The kinetic properties of the newly formed FSH receptors were indistinguishable from the pre-existing ones. The induced FSH receptors were functional as demonstrated by an increase in the in vitro responsiveness of the cells to exogenous FSH in terms of progesterone production. Treatment of immature rats with cyanoketone, an inhibitor of Δ5,3β-hydroxysteroid dehydrogenase, prior to PMSG injection effectively reduced the PMSG-stimulated increase in the serum estradiol, uterine weight and LH receptors but had no effect on the FSH receptor induction. The ability of PMSG to induce gonadotropin receptors can be arrested at any given time by injecting its antibody, thereby suggesting a continuous need for the hormonal inducer. Estrogen in the absence of the primary inducer was unable to maintain the induced LH and FSH receptor concentration. Inhibition of prostaglandin synthesis using indomethacin also had no effect on either the induction or degradation of gonadotropin receptors. Administration of PMSG antiserum, 48 h after PMSG injection, brought about a rapid decline in the induced receptors over the next 24 h, with a rate constant and \iota 1/2 of 0.078 h−1 and 8.9 h for FSH receptors and 0.086 h−1 and 8.0 h for the LH receptors, respectively.

Omega-Hydroxylation of acyclic monoterpene alcohols by rat lung microsomes

Rat lung microsomes were shown to �-hydroxylate acyclic monoterpene alcohols in the presence of NADPH and O2. NADH could neither support hydroxylation efficiently nor did it show synergistic effect. The hydroxylase activity was greater in microsomes prepared from β-naphthoflavone (BNF)-treated rats than from phenobarbital (PB)-treated or control microsomal preparations. Hydroxylation was specific to the C-8 position in geraniol and has a pH optimum of 7.8. The inhibition of the hydroxylase activity by SKF-525A, CO, N-ethylmaleimide, ellipticine, α-naphthoflavone, cyt. Image and p-CMB indicated the involvement of the cyt. P-450 system. However, NaN3 stimulated the hydroxylase activity to a significant level. Rat kidney microsomes were also capable of �-hydroxylating geraniol although the activity was lower than that observed with lungs.

Metabolism of 1,8-Cineole in Rat: Its Effects on Liver and Lung Microsomal Cytochrome P-450 Systems

The monoterpene cyclic ether, cineole (l,8-cineole, I) also known as eucalyptol, is a component of many essential oils and is widely distributed in nature. It is extensively used in pharmaceutical preparations for external application and also as a nasal spray. It was reported earlier that cineole when administered to sheep may be largely oxidized in the system (Scheline 1978). However the mode of metabolism of cineole is not known. Hence the present study was undertaken to investigate the metabolic fate of this ubiquitous terpenoid following its administration to rats by gastric intubation.

Adriamycin treatment increases 3-hydroxy-3-methylglutaryl CoA reductase and isoprene biosynthesis in the rat liver

Treatment of rats with Adriamycin caused an increase in the incorporation into hepatic cholesterol of [1-14C] acetate, but not of [2-14C] mevalonate. The step affected was found to be 3-hydroxy-3-methylglutaryl CoA reductase whose activity in the liver microsomes increased in Adriamycin-treated animals, but was inhibited when the drug was added in the assay medium. Also, the concentration of ubiquinone in the liver and of cholesterol in the plasma increased.

A universal test to determine the integrity of RNA, and its suitability for reverse transcription, in animal tissue laboratory specimens

Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.

Development of a small-plant bioassay to assess banana grown from tissue culture for consistent infection by Fusarium oxysporum f. sp. cubense

Two reliable small-plant bioassays were developed using tissue-cultured banana, resulting in consistent symptom expression and infection by Fusarium oxysporum f. sp. cubense (Foc). One bioassay was based on providing a constant watertable within a closed pot and the second used free-draining pots. Culture medium for spore generation influenced infectivity of Foc. Inoculation of potted banana by drenching potting mix with a conidial suspension, consisting mostly of microconidia, few macroconidia and no chlamydospores, generated from one-quarter-strength potato dextrose agar + streptomycin sulfate, resulted in inconsistent infection. When a conidial suspension that consisted of all three spore types, microconidia, macroconidia and chlamydospores, prepared from spores generated on carnation leaf agar was used, all plants became infected, indicating that the spore type present in conidial suspensions may contribute to inconsistency of infection. Inconsistency of infection was not due to loss of virulence of the pathogen in culture. Millet grain precolonised by Foc as a source of inoculum resulted in consistent infection between replicate plants. Sorghum was not a suitable grain for preparation of inoculum as it was observed to discolour roots and has the potential to stunt root growth, possibly due to the release of phytotoxins. For the modified closed-pot system, a pasteurised potting mix consisting of equal parts of bedding sand, perlite and vermiculite plus 1 g/L Triabon slow release fertiliser was suitable for plant growth and promoted capillary movement of water through the potting mix profile. A suitable potting mix for the free-draining pot system was also developed.

Expression of Cytochrome P-450 and Albumin Genes in Rat Liver: Effect of Xenobioticst

Thioacetamide, a hepatocarcinogen and an inhibitor of heme synthesis, blocks the phenobarbitone- mediated increase in the transcription of cytochrome P-450b+e messenger RNA in rat liver. This property is also shared by CoCl, and 3-amino-l,2,4-triazole, two other inhibitors of heme synthesis. Thus, it appears feasible that heme may serve as a positive regulator of cytochrome P-450b+e gene transcription. Thioacetamide enhances albumin messenger RNA concentration, whereas phenobarbitone decreases the same. However, these changes in albumin messenger RNA concentration are not accompanied by corresponding changes in the transcription rates. Therefore, drug-mediated changes in albumin messenger RNA concentration are due to posttranscriptional regulation. The property of thioacetamide to enhance the albumin messenger RNA concentration is not shared by CoC1, and 3-amino- 1,2,4-triazole. Therefore, heme does not appear to be a regulatory molecule mediating the reciprocal changes brought about in the concentrations of cytochrome P-450b+e and albumin messenger RNAs.

Spatial patterns of sub-tidal seagrasses and their tissue nutrients in the Torres Strait, northern Australia: Implications for management

The distribution and nutritional profiles of sub-tidal seagrasses from the Torres Strait were surveyed and mapped across an area of 31,000 km2. Benthic sediment composition, water depth, seagrass species type and nutrients were sampled at 168 points selected in a stratified representative pattern. Eleven species of seagrass were present at 56 (33.3%) of the sample points. Halophila spinulosa, Halophila ovalis, Cymodocea serrulata and Syringodium isoetifolium were the most common species and these were nutrient profiled. Sub-tidal seagrass distribution (and associated seagrass nutrient concentrations) was generally confined to northern-central and south-western regions of the survey area (rations than the below-ground component (roots and rhizomes), which possessed greater starch concentrations. S. isoetifolium had the highest total nitrogen concentrations (1.40±0.05% DW). However, it also had higher fibre concentrations (38.2±0.68% DW) relative to the other four species. H. ovalis possessed the highest starch concentrations (2.76±0.12% DW) and highest digestibility (83.24±0.66% DW) as well as the lowest fibre (27.2±0.66% DW). The high relative abundance (found at 55% of the sites that had seagrass) and nutrient quality characteristics of H. ovalis make it an important source of energy to marine herbivores that forage sub-tidally in the Torres Strait. There were two regions in Torres Strait (north-central and south-western) where sub-tidal seagrass meadows were prevalent and of relatively higher nutritional value. This spatial and nutritional information can be used by local agencies to manage and to protect the ecological, economic and cultural values of the sub-tidal seagrass ecosystems and associated fisheries of the Torres Strait.

Prolonged hyperinsulinemia affects metabolic signal transduction markers in a tissue specific manner

Insulin dysregulation is common in horses although the mechanisms of metabolic dysfunction are poorly understood. We hypothesized that insulin signaling in striated (cardiac and skeletal) muscle and lamellae may be mediated through different receptors as a result of receptor content, and that transcriptional regulation of downstream signal transduction and glucose transport may also differ between tissues sites during hyperinsulinemia. Archived samples from horses treated with a prolonged insulin infusion or a balanced electrolyte solution were used. All treated horses developed marked hyperinsulinemia and clinical laminitis. Protein expression was compared across tissues for the insulin receptor and insulin-like growth factor 1 receptor (IGF-1R) by immunoblotting. Gene expression of metabolic insulin-signaling markers (insulin receptor substrate 1, Akt2, and glycogen synthase kinase 3 beta [GSK-3β]) and glucose transport (basal glucose transporter 1 and insulin-sensitive glucose transporter 4) was evaluated using real-time reverse transcription polymerase chain reaction. Lamellar tissue contained significantly more IGF-1R protein than skeletal muscle, indicating the potential significance of IGF-1R signaling for this tissue. Gene expression of the selected markers of insulin signaling and glucose transport in skeletal muscle and lamellar tissues was unaffected by prolonged hyperinsulinemia. In contrast, the significant upregulation of Akt2, GSK-3β, GLUT1, and GLUT4 gene expression in cardiac tissue suggested that the prolonged hyperinsulinemia induced an increase in insulin sensitivity and a transcriptional activation of glucose transport. Responses to insulin are tissue-specific, and extrapolation of data across tissue sites is inappropriate.

In vitro propagation of Eucalyptus grandis L. by tissue culture

Multiple shoots were induced from nodal segments of five year old trees of Eucalyptus grandis L. on solid medium containing Murashige and Skoog's (MS) Basal medium supplemented with additional thiamine, BAP and NAA. Rooting could be achieved from shoot culture on half strength MS salts or white's medium supplemented with low auxins like IAA, IBA and NAA.

Biosynthesis of cytoplasmic subunits of cytochrome C oxidase in rat liver

The biosynthesis of the cytoplasmic subunits of cytochrome oxidase from rat liver has been studied in vitro by translating liver poly (A)-containing RNA in the wheat germ cell-free system and immunoprecipitating the products with anti-cytochrome oxidase antibody. Analysis of the labelled immunoprecipitate on SDS-gels does not reveal the presence of a polyprotein precursor. On the other hand discrete products which are either slightly bigger or closely similar to the mature subunits present in purified cytochrome oxidase have been detected.

Structural and functional characterization of dendritic arbors and GABAergic synaptic inputs on interneurons and principal cells in the rat basolateral amygdala

The basolateral amygdala (BLA) is a complex brain region associated with processing emotional states, such as fear, anxiety, and stress. Some aspects of these emotional states are driven by the network activity of synaptic connections, derived from both local circuitry and projections to the BLA from other regions. Although the synaptic physiology and general morphological characteristics are known for many individual cell types within the BLA, the combination of morphological, electrophysiological, and distribution of neurochemical GABAergic synapses in a three-dimensional neuronal arbor has not been reported for single neurons from this region. The aim of this study was to assess differences in morphological characteristics of BLA principal cells and interneurons, quantify the distribution of GABAergic neurochemical synapses within the entire neuronal arbor of each cell type, and determine whether GABAergic synaptic density correlates with electrophysiological recordings of inhibitory postsynaptic currents. We show that BLA principal neurons form complex dendritic arborizations, with proximal dendrites having fewer spines but higher densities of neurochemical GABAergic synapses compared with distal dendrites. Furthermore, we found that BLA interneurons exhibited reduced dendritic arbor lengths and spine densities but had significantly higher densities of putative GABAergic synapses compared with principal cells, which was correlated with an increased frequency of spontaneous inhibitory postsynaptic currents. The quantification of GABAergic connectivity, in combination with morphological and electrophysiological measurements of the BLA cell types, is the first step toward a greater understanding of how fear and stress lead to changes in morphology, local connectivity, and/or synaptic reorganization of the BLA.

Localization of testis-variant histones in rat testis chromatin

Nucleosome core particles and oligonucleosomes were isolated by digesting rat testis nuclei with micrococcal nuclease to 20% acid-solubility, followed by fractionation of the digest on a Bio-Gel A-5m column. The core particles thus isolated were characterized on the basis of their DNA length of 151 +/- 5 base-pairs and sedimentation coefficient of 11.4S. Analysis of the acid-soluble proteins of the core particles indicated that histones TH2B and X2 are constituents of the core particles, in addition to the somatic histones H2A, H2B, H3 and H4. The acid-soluble proteins of the oligonucleosomes comprised all the histones, including both the somatic (H1, H2A, H2B, H3, H4 and X2) and the testis-specific ones (TH1 and TH2B). It was also observed that histones TH1 and H1 are absent from the core particles and were readily extracted from the chromatin by 0.6 M-NaCl, which indicated that both of them are bound to the linker DNA.

Influence of starvation and clofibrate administration on oxidative phosphorylation by rat liver mitochondria

Whole cells, homogenates and mitochondrial obtained from the livers of albino rats which were starved for 6 days or more showed a 50% decrease in oxidative activity. The decrease could be corrected by the addition of cytochrome c in vitro. The phosphorylative activity of mitochondria remained unaffected. The decrease in oxidative rate was not observed when starving animals were given the anti-hypercholesterolaemic drug clofibrate. The total cellular concentration of cytochrome c was not affected by starvation. However, the concentration of the pigment in hepatic mitochondria isolated from starving animals was less than half that in normal mitochondria. Clofibrate-treated animals did not show a decreased concentration of cytochrome c in hepatic mitochondria. Mitochondria isolated from starving animals, though deficient in cytochrome c, did not show any decrease in succinate dehydrogenase activity or in the rate of substrate-dependent reduction of potassium ferricyanide or attendant phosphorylation. In coupled mitochondria, ferricyanide may not accept electrons from the cytochrome c in the respiratory chain. Starvation decreases the concentration of high-affinity binding sites for cytochrome c on the mitochondrial membrane. The dissociation constant increases in magnitude.

Purification of clofibrate-induced carnitine acetyltransferase from rat liver mitochondria

The enzyme carnitine acetyltransferase (acetyl-CoA:carnitine O-acetyltransferase, EC 2.3.1.7) has been purified to homogeneity from hepatic mitochondria of clofibrate-fed rats. It is a protein of molecular weight 56 000 composed of two non-identical subunits of molecular weight 34 000 and 25 000. The enzyme is inhibited by palmityl-CoA as well as acetyl carnitine. The inhibition by fatty acyl-CoA is competitive with respect to both the substrates, carnitine and acetyl-CoA. The inhibition by acetylcarnitine is reversed by carnitine but not by acetyl-CoA.