The cell cycle gene SKP1 is regulated by light in postnatal rat brain

During the early postnatal phase of high neuronal plasticity, an altered visual input leads to great modifications of visual cortex organization [Y. Frégnac, M. Imbert, Development of neuronal selectivity in primary visual cortex of cat, Physiol. Rev., 64 (1984) 375-434; D.H. Hubel, T.N. Wiesel, S. LeVay, Plasticity of ocular dominance columns in monkey striate cortex, Philos. Trans. R. Soc. London, Ser. B, 278 (1977) 377-409.]. We used refined differential screening of an organized cDNA library to identify the genes that may participate in this plasticity. We isolated a candidate plasticity gene encoding for a 163 aa protein that is closely related to the human and yeast Skp1p, a key factor in cell cycle progression [C. Ba?, K. Hofman, L. Ma, M. Goebl, J.W. Harper, S.J. Elledge, SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box, Cell, 86 (1996) 263-274; C. Connelly, P. Hieter, Budding yeast SKP1 encodes an evolutionary conserved kinetochore protein required for cell cycle progression, Cell, 86 (1996) 275-285; H. Zhang, R. Kobayashi, K. Galaktionov, D. Beach, p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase, Cell, 82 (1995) 915-925.]. Northern blot analysis showed that the expression of SKP1 (Skp1p gene) dramatically decreased after 2 h of light stimulation in the visual cortex of young dark-reared rats. This down regulation lasted at least 72 h. It was specific for the critical period as we did not observe any significant regulation of SKP1 mRNA by light in adult dark-reared rat brain. The down regulation was observed in the superior colliculus but also in the frontal cortex and in the hippocampus. The fact that this down regulation was not restricted to the visual system, suggested that it could be produced by dark rearing-induced hormonal changes. The significance of SKP1 expression in the brain and its regulation are discussed.     

Estrogen receptor expression in bovine and rat retinas

PURPOSE: To investigate the expression and distribution of estrogen receptor protein and mRNA in bovine and rat retinas. METHODS: Western blot analysis with an antiestrogen receptor monoclonal antibody (mAb) was used to detect estrogen receptor protein in the bovine retina. Immunohistochemistry with an antiestrogen receptor mAb and in situ hybridization with an oligodeoxynucleotide sequence coding for estrogen receptor were applied to study the cellular distribution of estrogen receptor protein and its mRNA in male bovine retina and rat retina of both sexes. RESULTS: Estrogen receptor protein was detected in bovine retina by Western blot analysis. Immunohistochemical staining with the antiestrogen receptor mAb was widespread throughout the neural retina. Specific staining showed extensive distribution localizing in the nerve fiber layer, the ganglion cell layer, the inner nuclear layer, and the outer plexiform layer. Retinal pigment epithelium and choroid were also stained with the antiestrogen receptor mAb. By in situ hybridization, the expression of estrogen receptor mRNA was predominantly observed in ganglion cell layer, the inner nuclear layer, and outer portion of the outer nuclear layer. No significant difference was found between male and female rats in the immunostaining of retinas with the antiestrogen receptor mAb. CONCLUSIONS: This study provides the first evidence for the presence of estrogen receptor in bovine and rat retinas. Expression of estrogen receptor throughout the retina suggests that estrogen may have important functions in the retina.     

Transient c-fos gene expression in cerebellar development and functional stimulation

The effect of ischemic preconditioning and superoxide dismutase (SOD) on endothelial glycocalyx and endothelium-dependent vasodilation in the postischemic isolated guinea-pig hearts was examined. Seven groups of hearts were used: group 1 underwent sham aerobic perfusion; group 2 was subjected to 40 min global ischemia without reperfusion; group 3, 40 min ischemia followed by 40 min reperfusion; group 4 was preconditioned with three cycles of 5 min global ischemia followed by 5 min of reperfusion (IPC), prior to 40 min ischemia; group 5 was subjected to IPC prior to standard ischemia/ reperfusion; group 6 underwent standard ischemia/reperfusion and SOD infusion (150 U/ml) was begun 5 min before 40 min ischemia and continued during the initial 5 min of the reperfusion period; group 7 was subjected to 80 min aerobic perfusion with NO-synthase inhibitor, L-NAME, to produce a model of endothelial dysfunction independent from the ischemia/reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and endothelium-independent vascular function, respectively. Reduction in coronary flow caused by NO-synthase inhibitor, L-NAME, served as a measure of a basal endothelium-dependent vasodilator tone. After completion of each experimental protocol, the hearts were stained with ruthenium red or lanthanum chloride for electron microscopy evaluation of the endothelial glycocalyx. While ischemia led only to a slightly flocculent appearance of the glycocalyx, in ischemia/reperfused hearts the glycocalyx was disrupted, suggesting that it is the reperfusion injury which leads to the glycocalyx injury. Moreover, the coronary flow responses to ACh and L-NAME were impaired, while the responses to SNP were unchanged in the ischemia/reperfused hearts. The disruption of the glycocalyx and the deterioration of ACh and L-NAME responses was prevented by IPC. In addition, the alterations in the glycocalyx and the impairment of ACh responses were prevented by SOD. The glycocalyx appeared to be not changed in the hearts subjected to 80 min aerobic perfusion with L-NAME. In conclusion: (1) the impairment of the endothelium-dependent coronary vasodilation is paralleled by the endothelial glycocalyx disruption in the postischemic guinea-pig hearts; (2) both these changes are prevented by SOD, suggesting the role of free radicals in the mechanism of their development; (3) both changes are prevented by IPC. We hypothesize, therefore, that alterations in the glycocalyx contribute to the mechanism of the endothelial dysfunction in the postischemic hearts.     

Cytokine gene expression in intestine of rat during the postnatal developmental period: increased IL-1 expression at weaning

In the present study we have investigate whether cytokines are constitutively and differently expressed in intestine during the differentiative processes that take place at weaning. We have analyzed the expression of IL-1 beta, IL-2, IL-4 and IFN gamma by polymerase chain reaction in Peyer's patches (PP) and in intestine deprived of PP (I-PP) of rats from 16 to 30 days of age. The results showed a constitutive and marked expression of the cytokines already before weaning, with the exception of IL-2 in PP and IFN gamma in I-PP. IL-beta was the only cytokine to show a different expression at various ages with an initial increase at 19 days and a further elevation at 21 days when intestinal epithelium passes through major differentiative stages, suggesting an involvement of this cytokine in intestinal development. We have also tested whether treatment of rats with the immunosuppressor cyclosporin A (CsA) could affect intestinal differentiation. The results showed that only some markers of differentiation were affected (proliferation of staminal crypt cells and length of crypts). This was probably due to a direct effect rather than an immunomediated effect of CsA, since treatment of three intestinal cell lines (Caco-2, HT-29, FRIC) with CsA indicated that this drug can exert a cytostatic activity on intestinal cells.     

Quipazine and light have similar effects on c-fos induction in the rat suprachiasmatic nucleus

The effects of the serotonin agonist, quipazine, on the induction of c-fos in the suprachiasmatic nucleus of the rat was examined at different times of the 24 h cycle. Quipazine administered at night induced Fos production in a dose dependent manner (1, 3, 10, 30 mumol/kg) in the ventrolateral portion of the suprachiasmatic nucleus at ZT18. Administration of the highest dose at other times resulted in c-fos induction at ZT15 but not at other times of the day or subjective day examined (CT6 and ZT12). When compared to the effects of light pulses (2 lux/1 min), quipazine only caused c-fos induction at times when light caused induction. Our results support a role of serotonergic pathways in the transmission or modulation of photic information from the retina to the suprachiasmatic nucleus of the rat.     

Prolonged expression of c-fos suppresses cell cycle entry of dormant hematopoietic stem cells

The proto-oncogene c-fos was transiently upregulated in primitive hematopoietic stem (Lin-Sca-1(+)) cells stimulated with stem cell factor, interleukin-3 (IL-3), and IL-6. To investigate a role of the c-fos in hematopoietic stem cells, we used bone marrow (BM) cells from transgenic mice carrying the c-fos gene under the control of the interferon-alpha/beta-inducible Mx-promoter (Mx-c-fos), and fetal liver cells from c-fos-deficient mice. Prolonged expression of the c-fos in Lin-Sca-1(+) BM cells inhibited factor-dependent colony formation and hematopoiesis on a stromal cell layer by keeping them at G0/G1 phase of the cell cycle. These Lin-Sca-1(+) BM cells on a stromal layer entered into the cell cycle whenever exogenous c-fos was downregulated. However, ectopic c-fos did not perturb colony formation by Lin-Sca-1(+) BM cells after they entered the cell cycle. Furthermore, endogenous c-fos is not essential to cell cycle progression of hematopoietic stem cells because the factor-dependent and the stroma-dependent hematopoiesis by Lin-Sca-1(+) fetal liver cells from c-fos-deficient mice was not impaired. These results suggest that the c-fos induced in primitive hematopoietic stem cells negatively controls cell cycle progression and maintains them in a dormant state.     

cAMP-mediated c-fos expression in pressure-overloaded acceleration of protein synthesis in adult rat heart

This study was carried out to investigate the amplification of HER-2/neu oncogene in 66 patients with primary breast cancer and 90 samples from benign breast disease (BBD). The amplification of HER-2/neu oncogene in the DNA of paraffin-embedded specimens was determined by differential PCR. Nineteen out of 66 (28.8%) breast cancer patients showed amplification of the gene. No gene amplification was found in benign breast disease. There was no significant correlation of HER-2/neu amplification with, age, menopausal status, the number of positive nodes, tumor size, estrogen receptor, however, amplification of HER-2/neu gene was strongly correlated with nodal status (p = 0.0049). In node positive patients, the incidence of HER-2/neu amplification was high (43%). These findings indicate that the amplification of HER-2/neu gene may be of pathogenetic significance in breast cancer and may have a poor prognosis in node positive breast cancer patients while no gene amplification in benign breast disease suggests that HER-2/neu amplification is a late molecular alteration event in the pathogenesis of breast cancer.     

Roles of glutamate receptor in c-fos gene expression and epilepsy susceptibility in rats

We use NMDA to induce expression of c-fos mRNA as a marker to observe the activity of NMDA receptor in neurons during development, and compare the activity of NMDA receptor between audiogenic epilepsy -prone (P77PMC) and audiogenic epilepsy resistant rats brain. In primary culture of rats cerebral cortical neurons NMDA induced c-fos mRNA expression exhibits dose and time-dependent changes, which can be prevented by antagonists. During the development of neurons, the NMDA -induced c-fos mRNA expression reaches a maximum at day 24. NMDA-induced c-fos mRNA expression of P77PMC rats is higher than that of controls during 6 to 24 days in vitro with significant difference (P < 0.05) at day 18. To present changes in c-fos mRNA expression induced by NMDA in cultured P77PMC rat cortical neurons may be one of the factors related to susceptibility of epilepsy in P77PMC rats.     

Increased intraluminal pressure induces DNA synthesis and c-fos expression in perfused rat aorta

The metabolic syndrome is a well-known risk for the development of cardiovascular disease. In the present study the possible importance of an altered visceral adipocyte beta-adrenoceptor function in this syndrome was investigated. In 65 subjects of both sexes undergoing elective surgery for non-malignant disorders, the metabolic syndrome phenotype and the lipolytic sensitivity for various beta-adrenoceptor subtype agonists in omental adipocytes were determined. The study group represented a wide range of abdominal adipose tissue distribution (waist-to-hip ratios 0.76-1.13), but was otherwise apparently healthy. The subjects were divided into three subgroups according to their waist-to-hip (WHR) ratios: 1) WHR < 0.92; 2) WHR 0.92-1.04; 3) WHR > 1.04. The subgroups demonstrated significant differences regarding body mass index, sagittal diameter, systolic and diastolic blood pressures, plasma concentrations of glucose, insulin, triglycerides and HDL-cholesterol (p = 0.005-0.0001). Furthermore, in omental adipocytes beta 3-adrenoceptor sensitivity, but not beta 1-and beta 2-adrenoceptor sensitivities, differed significantly between the WHR subgroups (p = 0.0001). beta 3-adrenoceptor sensitivity was also related to the other components of the metabolic syndrome, although a strong covariation between WHR and beta 3-adrenoceptor sensitivity vs blood pressure and the metabolic parameters was found. The present data provide evidence of a relationship between upperbody obesity and its associated metabolic complications and also, an increased visceral fat beta 3-adrenoceptor sensitivity. We suggest that the latter finding results in an augmented release of non-esterified fatty acids from the visceral fat depot to the portal venous system. This may in turn contribute to the development of the metabolic syndrome.     

The effect of adrenalectomy on stress-induced c-fos mRNA expression in the rat brain

Hydrophilic-interaction liquid chromatography (HILIC) has recently been introduced as a highly efficient chromatographic technique for the separation of a wide range of solutes. The present work was performed with the aim of evaluating the potential utility of HILIC for the separation of postranslationally acetylated histones. The protein fractionations were generally achieved by using a weak cation-exchange column and an increasing sodium perchlorate gradient system in the presence of acetonitrile (70%, v/v) at pH 3.0. In combination with reversed-phase high-performance liquid chromatography (RP-HPLC) we have successfully separated various H2A variants and posttranslationally acetylated forms of H2A variants and H4 proteins in very pure form. An unambiguous assignment of the histone fractions obtained was performed using high-performance capillary and acid-urea-Triton gel electrophoresis. Our results demonstrate that for the analysis and isolation of modified core histone variants HILIC provides a new and important alternative to traditional separation techniques and will be useful in studying the biological function of histone acetylation.     

ALG gene expression and cell cycle progression

The evolutionarily conserved ALG genes function in the dolichol pathway in the synthesis of the lipid-linked oligosaccharide precursor for protein N-glycosylation. Increasing evidence suggests a role for these genes in the cell cycle. In Saccharomyces cerevisiae, coordinate regulation of the ALG genes makes up the primary genomic response to growth stimulation; several features of the ALG genes' expression resemble mammalian early growth response genes. However, only the first gene in the pathway, ALG7, is downregulated in response to an antimitogenic signal that leads to cell cycle arrest and differentiation, suggesting that selective inhibition of the first gene may be sufficient to regulate the dolichol pathway for the withdrawal from the cell cycle. The availability of mutants in the early essential ALG genes has established functional relationships between these genes' expression and G1/S transition, budding, progression through G2 and withdrawal from the cell cycle. Analysis of the regulation of ALG7 has provided insights into how this gene's expression is controlled at the molecular level. Recent studies have also begun to reveal how ALG7 expression is linked to cell cycle arrest in response to antimitogenic cues and have identified G1 cyclins as some of its downstream targets. Since the functions of the ALG genes appear to be as conserved among eukaryotes as the cell cycle machinery, it is likely that these genes play a similar role in mammalian cell proliferation and differentiation.     

Norepinephrine induces expression of c-fos mRNA through the alpha-adrenoceptor in rat aortic rings

We examined whether norepinephrine at pharmacologically relevant doses induces increased expression of c-fos mRNA in rat aortic rings. c-fos mRNA was expressed at norepinephrine concentrations known to cause minimum and maximum contraction of rat aorta in vitro. At the concentration known to cause maximum contraction, norepinephrine produced a marked and sustained increase of c-fos mRNA expression. Induction of c-fos was blocked completely by the alpha 1-adrenergic antagonist prazosin, partially by the alpha 2-adrenergic antagonist yohimbine, and not at all by the beta-adrenergic antagonist propranolol. A prazosin inhibition curve showed that 1 nmol/L prazosin inhibited 10 micromol/L norepinephrine induced c-fos expression by 40%. At the pharmacologic dose known to cause maximum contraction, norepinephrine induces c-fos mRNA expression through the alpha-adrenoceptor in rat aortic rings.     

Effects of the novelty or familiarity of visual stimuli on the expression of the immediate early gene c-fos in rat brain

To investigate substrates of recognition memory, the cellular expression of Fos protein in rat brain has been studied after groups of rats were either shown sets of novel or highly familiar objects, or were exposed to the same pattern of illumination without objects being shown. Counts of stained nuclei were made in eight brain regions, where information about novel or familiar visual stimuli is likely to be processed or stored. The counts were relatively high in occipital visual association cortex and area TE of temporal cortex, intermediate in perirhinal cortex, entorhinal cortex, anterior cingulate cortex and the diagonal band of Broca, and low in the hippocampal formation and mediodorsal nucleus of the thalamus. The number of Fos-stained cells was significantly higher for the rats shown novel objects than for those shown familiar objects in perirhinal cortex, area TE, occipital cortex and anterior cingulate cortex. Arguments are advanced that these differences in counts indicate areas involved in the processing and/or storage of information about the novelty or familiarity of visual stimuli, information important to recognition memory.     

Cocaine-induced expression of striatal c-fos in the rat is inhibited by NMDA receptor antagonists

To assess the possible involvement of NMDA receptors in mediating the expression of striatal c-fos by cocaine injection, we investigated the effects of the noncompetitive NMDA receptor antagonists, ketamine and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), as well as the competitive NMDA receptor antagonist, 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), in the perikarya of cocaine-treated rat brains. As previously shown by our group, administration of 20 mg/kg cocaine (IP) resulted in the immunocytochemical expression of the protooncogene in numerous cells of the caudate putamen (dorsal/sensorimotor striatum). A ketamine mixture anesthetic (2 mg/kg), however, administered 30 min prior to cocaine exposure completely blocked such genomic expression. Pretreatment with MK-801 (1 mg/kg) or CPP (5 mg/kg) also interfered, albeit to a lesser extent, with the expression of c-fos by cocaine in awake animals. These results indicate that cocaine induction of cellular c-fos in the caudate putamen is mediated at least in part by NMDA-sensitive receptors.     

Cochlear ablation alters acoustically induced c-fos mRNA expression in the adult rat auditory brainstem

Expression of c-fos mRNA was studied in the adult rat brain following cochlear ablations by using in situ hybridization. In normal animals, expression was produced by acoustic stimulation and was found to be tonotopically distributed in many auditory nuclei. Following unilateral cochlear ablation, acoustically driven expression was eliminated or decreased in areas normally activated by the ablated ear, e.g., the ipsilateral dorsal and ventral cochlear nuclei, dorsal periolivary nuclei, and lateral nucleus of the trapezoid body and the contralateral medial and ventral nuclei of the trapezoid body, lateral lemniscal nuclei, and inferior colliculus. These deficits did not recover, even after long survivals up to 6 months. Results also indicated that neurons in the dorsal cochlear nucleus could be activated by contralateral stimulation in the absence of ipsilateral cochlear input and that the influence of the contralateral ear was tonotopically organized. Results also indicated that c-fos expression rose rapidly and persisted for up to 6 months in neurons in the rostral part of the contralateral medial nucleus of the trapezoid body following a cochlear ablation, even in the absence of acoustic stimulation. This response may reflect a release of constitutive excitatory inputs normally suppressed by missing afferent input or changes in homeostatic gene expression related to sensory deprivation. Instances of transient, surgery-dependent increases in c-fos mRNA expression in the absence of acoustic stimulation were observed in the superficial dorsal cochlear nucleus and the cochlear nerve root on the ablated side.