NOS3 gene variants and male infertility: Association of 4a/4b with oligoasthenozoospermia.

Results of recent studies confirmed that oxidative stress negatively affects sperm motility and causes sperm DNA damage. Produced by nitric oxide synthase 3 (NOS3), nitric oxide is considered to be one of the important mediators of oxidative stress in testis tissue. The aim of this study was to assess the possible association of three genetic variants (rs2070744, rs1799983 and intron variant 4a/4b) in NOS3 gene and infertility occurrence in two groups of infertile men (idiopathic azoospermia and oligoasthenozoospermia) and fertile controls. Genotypes for the single-nucleotide genetic variants rs1799983 and rs2070744 were determined by PCR-RFLP, while genotyping of intron 4 variant 4a/4b was performed by gel electrophoresis of PCR products. Statistical analysis was performed by SNPStats software. No significant association between the three genetic variants of the NOS3 gene and infertility risk was determined comparing allele and genotype frequencies among group of patients diagnosed with azoospermia and the control group. Nevertheless, there was a significant positive association between 4a/4b and infertility in the group of males diagnosed with oligoasthenozoospermia, under overdominant genetic model. Our findings suggest that tandem repeat variant within intron 4 of the NOS3 gene is associated with an increased risk of infertility in men diagnosed with idiopathic oligoasthenozoospermia.

Induced resistance in the human non small cell lung carcinoma (NCI-H460) cell line in vitro by anticancer drugs.

Exposure of human non-small cell lung cancer cells (NCI-H460) to gradually increasing concentrations of doxorubicin resulted in the appearance of a new cell line (NCI-H460/R) that was resistant to doxorubicin (96.2-fold) and cross-resistant to etoposide, paclitaxel, vinblastine and epirubicin. Slight cross-resistance to two MDR-unrelated drugs 8-Cl-cAMP and sulfinosine was observed. Flow cytometry analysis showed that the accumulation of doxorubicin in the resistant cells was 88.4% lower than in the parental cells. Also, verapamil significantly decreased the efflux rate in NCI-H460 and NCI-H460/R cells, whereas curcumin inhibited the efflux in NCI-H460 cells only. Gene expression data confirmed the induction of mdr1 (P-gp), as judged by the observed 15-fold increase in its mRNA concentration in doxorubicin-resistant NCI-H460/R cells. In contrast, mrp1 and lrp expression was unaffected by the doxorubicin resistance. Further work should develop a rationale for a novel treatment of NSCLC with appropriate modulators of resistance aimed at improving the outcome of the acquired drug resistance.

Differential effects of amphetamine and phencyclidine on the expression of growth-associated protein GAP-43.

The purpose of the present study was to test changes in the expression of growth-associated protein (GAP-43) after chronic treatment with two different psychotomimetic drugs: amphetamine and phencyclidine. Rats were treated chronically for 7 days (twice daily) with 5 mg/kg of amphetamine and phencyclidine and sacrificed after 2, 5 or 7 days of treatment, and following 7, 14 or 21 days of recovery after full treatment (7 days). Separate groups of rats were treated on the same regiment with haloperidol, and control group was treated with vehicle. To determine the effects of different psychotomimetic drugs on the expression of GAP-43 we have used Northern blotting and quantitative in situ hybridization. Treatment with amphetamine induced decrease of GAP-43 mRNA expression, that was detected also during recovery period, up to 14 days after the last day of 7 days treatments. On the contrary, PCP induced increase of GAP-43 mRNA expression, that was detectable from the first days of treatment until 21 days after the last day of treatment. Treatment with haloperidol did not produce significant changes in GAP-43 mRNA expression. It can be suggested that GAP-43 upregulation upon phencyclidine treatment occurs as a result of functional activation of pathways able to participate in remodeling, while amphetamine showed neurotoxic effect, decreasing expression of GAP-43 mRNA.

Glucocorticoid receptors in ageing rats.

The role of the glucocorticoid receptor (GR) in senescence was studied in rats of increasing age. Statistically significant changes in the number of GRs from rat liver were detected, whereas the affinity for the ligand triamcinolone acetonide (TA) did not change with increasing age, and was in the range of 1-2 nM. In all cases the number of receptors was lower in rats treated with hormone in vivo relative to untreated animals. In addition, we have found changes in GR activation, as measured by the binding to DNA cellulose in the mentioned age groups. Furthermore, expression of the glucocorticoid hormone (GH)-inducible gene, tyrosine amino transferase (TAT) also showed age-related alterations. We conclude that receptor function shows oscillatory changes during ageing. In addition, response to GH generally declines towards the older age. This specific periodicity in functional characteristics of the GR may reconcile conflicting results about the receptor number and properties during the ageing process, and marks particular age at which individual organism shows the highest or the lowest sensitivity to the actions of GH.

Optimization of in situ hybridization histochemistry for localization of GAP-43 mRNA in fetal brain during human development.

In situ hybridization histochemistry has proven to be an important tool for cellular and molecular studies in neurobiology. The method basically involves the anatomic localization of labeled RNA or DNA molecules that hybridize with complementary target RNA or DNA sequence in the cell. In regard to gene expression, in situ hybridization allows the study of specific mRNA level and distribution between various cell types, or of comparative levels of mRNA throughout development. Major advantages of in situ hybridization are: (a) specific mRNAs can be detected in heterogeneous cell populations where only one of multiple sets of cells present may be transcribing the mRNA of interest; (b) it allows the maximal use of rare tissues that may be in short supply (clinical biopsies, embryos, cultured cells); (c) the location of the mRNA can be identified on conventional microscopic sections without the necessity of electron microscopy; and (d) identification of specific gene without metabolic incorporation of radiolabeled precursor into the endogenous protein in the cells. Because of these advantages, we decided to utilize this approach for specific studies involving the expression and distribution of GAP-43 (growth-associated protein 43) mRNA in early human development. The method we employed for in situ hybridization is basically a modification of the many original protocols developed by others and can be easily applied to many studies of cellular gene expression in the human nervous system.

Enhanced serum response element binding activity correlates with down-regulation of c-fos mRNA expression in the rat brain following repeated cortical lesions.

Repeated lesions of rat cerebral cortex result in transient peaks in the level of the c-fos transcript, but after the second lesion, this peak is substantially diminished. Using this lesion paradigm, we have analyzed the participation of the c-fos promoter elements SRE and DSE in the regulation of c-fos transcription. Following a single lesion, SRE/DSE binding activity peaked at 2 h, subsequent to the maximal levels of c-fos mRNA and parallel to the peak of c-Fos protein. After a second lesion (reinduction), 4 h following the initial lesion, SRE/DSE binding activity peaked after only 30 min and was significantly higher than following the first lesion. Once again, this peak occurred after the peak of c-fos mRNA expression and parallel with the second peak of c-Fos protein expression. These results suggested that the SRE and DSE promoter elements participated in the induction and down-regulation of c-fos transcription in vivo and suggested the possible involvement of Fos protein in its own regulation. The ability of Fos/Fra proteins to participate in a transcriptional complex was confirmed in gel-shift experiments with an AP-1 element, and the biphasic trend of binding activity was observed. Supershift experiments were performed to directly determine whether Fos protein was participating in SRE and/or DSE transcriptional complexes. No alterations in the position or intensity of the shifted band were observed using Fos/Fra antiserum suggesting that Fos/Fra proteins could be involved in c-fos down-regulation through mechanisms other than direct participation in the SRE/DSE transcription complex.

GAP-43 mRNA expression in early development of human nervous system.

The temporal and spatial distribution of GAP-43 mRNA in early human development, from 6 to 23 gestational weeks (g.w.), was examined by in situ hybridization histochemistry. GAP-43 mRNA was expressed as early as 6 g.w. in all regions of developing nervous system, the spinal cord, brainstem, cerebellum, diencephalic and telencephalic regions. Although the pronounced level of expression persisted during the entire examined period, the intensity of expression varied along the spatial axis over time. Analysis at the cellular level revealed that early on in development (6 g.w.) GAP-43 mRNA was expressed in the entire neuroblast population. With the onset of differentiation, at 13-23 g.w., GAP-43 mRNA expression had switched to the neurons that are in the process outgrowth. The highest level of GAP-43 mRNA expression was localized in the regions consisting of differentiating neurons, such as the cortical plate and intermediate zone of the telencephalic wall, and several delineated subcortical and thalamic nuclei. The spatial and temporal pattern of GAP-43 mRNA expression obtained suggests a possible dual role of GAP-43 in the development of the human nervous system: in the embryonic brain it could be involved in fundamental processes underlying cell proliferation; in the fetal brain its expression is specifically correlated with differentiation and the outgrowth of axons.

Immunocytochemical localization of growth-associated protein GAP-43 in early human development.

Fibers labelled with antibody to the growth associated protein (GAP-43) were observed as early as 4 gestational weeks (g.w.) in the nervous system of human embryos. At 6 g.w. these fibers could be traced throughout the brainstem and the diencephalon. None of the immunolabeled fibers entered the telencephalic wall at that point, but 2 weeks later at 8 g.w., GAP-43 positive fibers were observed below the newly formed cortical plate of the cerebral cortex. GAP-43 positive fiber bundles had the same distribution as those previously labeled with tyrosine hydroxylase antibodies at the same age. These results strongly suggest that this growth associated protein is localized in the early growing dopaminergic fibers.

Desensitization of c-fos mRNA expression in rat brain following cortical lesions.

The effect of cortical lesions on the c-fos mRNA expression in adult rat brain was studied using in situ hybridization and Northern blot analysis. It was observed that the single cortical lesion evoked a rapid increase in the content of c-fos mRNA in whole brain. However, when rats received a second and third cortical injury 4 and 8 h after the first one, there was reduced elevation of c-fos mRNA after the second (60%) and third (75%) injuries in comparison with those seen after a single cortical lesion. These results represent the desensitization period during which the induction of c-fos mRNA by repeated cortical lesions was decreased. On the contrary, when animals were treated with a fourth cortical lesion 24 h after the first injury, with recovery for 16 h after the third one, the levels of c-fos mRNA were again elevated to a degree almost comparable to that seen after the single injury alone. The anatomical distribution of c-fos mRNA-expressing cells after cortical lesions revealed a large increase of hybridization to a heterogeneous population of hippocampal neurons including small cells of stratum granulosum in the dentate gyrus and larger cells within hippocampal stratum pyramidale. A dramatic labeling of neurons with c-fos riboprobe in frontoparietal and piriform cortex was also observed but with predominantly localized hybridization to c-fos mRNA on the damaged side of the cortex following the first and second cortical injury. However, hippocampal distribution of c-fos mRNA-containing cells was bilateral as a result of polysynaptic potentials evoked by cortical lesions.

Temporal and spatial preferences of c-fos mRNA expression in the rat brain following cortical lesion.

The expression of the proto-oncogene c-fos is increased in neuronal cells by a number of stimuli and the usefulness of this gene as a marker of neuronal activity has been demonstrated. The temporal and spatial expression of c-fos mRNA following the induction of a unilateral cortical lesion have been investigated in the rat brain by Northern blot analysis and in situ hybridization histochemistry. It was observed that the lesion evoked a rapid increase (20-fold) in the content of c-fos mRNA in the ipsilateral cortex, whereas in the contralateral cortex c-fos mRNA expression was more modest (7-fold). In the whole hippocampus a large and very rapid increase (17-fold) of c-fos mRNA expression was detected. The effect of a cortical lesion on Ca2+ uptake and membrane potential was also investigated. Using synaptosomes as a model system, we have provided evidence that Ca2+ entry via membrane depolarization increases in coordination with c-fos gene expression in neuronal cells. The principal conclusions from this study are that cortical lesions induce transient expression of the c-fos gene in specific neuronal cells of the rat brain.

Synapsins: mosaics of shared and individual domains in a family of synaptic vesicle phosphoproteins.

Synapsins are neuronal phosphoproteins that coat synaptic vesicles, bind to the cytoskeleton, and are believed to function in the regulation of neurotransmitter release. Molecular cloning reveals that the synapsins comprise a family of four homologous proteins whose messenger RNA's are generated by differential splicing of transcripts from two genes. Each synapsin is a mosaic composed of homologous amino-terminal domains common to all synapsins and different combinations of distinct carboxyl-terminal domains. Immunocytochemical studies demonstrate that all four synapsins are widely distributed in nerve terminals, but that their relative amounts vary among different kinds of synapses. The structural diversity and differential distribution of the four synapsins suggest common and different roles of each in the integration of distinct signal transduction pathways that modulate neurotransmitter release in various types of neurons.

Effects of cortisol treatment on protein synthesis in septum and hippocampus of rat brain.

The effects of cortisol on protein synthesis in distinct brain regions were studied. Hormone administration produced increases in tubulin and actin content in the soluble fraction isolated from septum and hippocampus. Therefore, the poly(A)RNAs were isolated from the same brain regions and their translational capacity was tested in a heterologous cell-free system. Poly(A)RNAs from cortisol-treated animals, as compared to control animals, had a stimulatory effect on 35S-methionine incorporation into tubulin and actin. Thus, our results suggest that cortisol treatment increases the amount of mRNAs coding for tubulin and actin. More importantly, this study indicates that the molecular mechanism of glucocorticoid action in the brain involves modulation of protein synthesis.