The novel untranslated first exon "exon 0N" of the rat estrogen receptor gene

The 5'-untranslated region (UTR) of the estrogen receptor (ER) mRNA in the rat liver was analyzed by the use of the 5'-rapid amplification of the cDNA ends (5'-RACE) method. The nucleotide sequence of one of the positive RACE clones (clone 9) revealed that the existence of the novel untranslated first exon (termed "exon N") being spliced onto the exon 1 of the rat ER mRNA. We further analyzed the distribution of the ER mRNA containing the "exon 0N" (ER mRNA (0N-1)) and the ER mRNA containing the previously reported exon 0 (ER mRNA (0-1)) in the rat brain and peripheral tissues. In contrast to the wide distribution of the ER mRNA (0-1), the distribution of the ER mRNA (0N-1) was almost limited in the peripheral tissues. These results indicate that the "exon 0N" is the novel untranslated first exon of the rat ER gene, and the tissue specific expression of the ER is regulated, at least in part, by differential promoter usage in the rat.     

The untranslated first exon ''exon 0S'' of the rat estrogen receptor (ER) gene

Occasionally, ipsilateral ischemia develops following the groin insertion of an intra-aortic balloon catheter. Various treatment options have evolved, and include replacing the catheter in the opposite groin, removing it completely, or performing a femorofemoral bypass to deliver blood flow below the catheter. Outlined in this paper is a simple method to restore blood flow to a threatened limb, during femoral artery exploration, in the presence of an intra-aortic balloon. This method is also appropriate for optimal positioning of the balloon catheter prior to femorofemoral bypass.     

Differential regulation of GABA(A) receptor gene expression by ethanol in the rat hippocampus versus cerebral cortex

Previous research has shown that chronic ethanol consumption dramatically alters GABA(A) receptor alpha1 and alpha4 subunit gene expression in the cerebral cortex and GABA(A) receptor alpha1 and alpha6 subunit gene expression in the cerebellum. However, it is not yet known if chronic ethanol consumption produces similar alterations in GABA(A) receptor gene expression in other brain regions. One brain region of interest is the hippocampus because it has recently been shown that a subset of GABA(A) receptors in the hippocampus is responsive to pharmacologically relevant concentrations of ethanol. Therefore, we directly compared the effects of chronic ethanol consumption on GABA(A) receptor subunit gene expression in the hippocampus and cerebral cortex. Furthermore, we investigated whether the duration of ethanol consumption (14 or 40 days) would influence regulation of GABA(A) receptor gene expression in these two brain regions. Chronic ethanol consumption produced a significant increase in the level of GABA(A) receptor alpha4 subunit peptide in the hippocampus following 40 days but not 14 days. The relative expression of hippocampal GABA(A) receptor alpha1, alpha2, alpha3, beta(2/3), or gamma2 was not altered by either period of chronic ethanol exposure. In marked contrast, chronic ethanol consumption for 40 days significantly increased the relative expression of cerebral cortical GABA(A) receptor alpha4 subunits and significantly decreased the relative expression of cerebral cortical GABA(A) receptor alpha1 subunits. This finding is consistent with previous results following 14 days of chronic ethanol consumption. Hence, chronic ethanol consumption alters GABA(A) receptor gene expression in the hippocampus but in a different manner from that in either the cerebral cortex or the cerebellum. Furthermore, these alterations are dependent on the duration of ethanol exposure.     

Expression and region-specific regulation of the oxytocin receptor gene in rat brain

The neuropeptide oxytocin (OT) exerts its various neurotransmitter functions via specific OT receptors (OTRs) that have been localized to distinct brain regions, including the ventromedial hypothalamus, the bed nucleus of stria terminalis, the amygdala, the subiculum, the hippocampus, and the olfactory nuclei. In the present study, we have characterized OTR gene expression by Northern blot and by semiquantitative RT-PCR in these brain regions and studied its regulation in response to estrogen (E2), progesterone, and the antiestrogen tamoxifen. We find that all regions analyzed express two messenger RNA (mRNA) bands (6.7 and 4.8 kb) that hybridize to a rat OTR complementary DNA probe and that correspond in size to two of the three OTR mRNA bands expressed in rat uterus. Analysis by RT-PCR, with two different primer pairs, did not reveal any structural differences between the coding regions of uterine and brain OTR mRNA. E2 treatment and gestation led to an 8-fold and a 6.5-fold increase in OTR mRNA levels, respectively. Progesterone was without effect, if administered alone, and did not influence the E2-induced rise in OTR mRNA. The E2 effect was restricted to E2-sensitive regions, such as the hypothalamus, and was not observed in the subiculum or the olfactory nuclei. Tamoxifen had a dual effect: on the one hand, it acted as a partial agonist in raising OTR mRNA levels in the hypothalamus of ovariectomized animals; on the other hand, it suppressed the E2-induced OTR mRNA rise in E2-sensitive brain regions. Although the present data do not exclude the possible existence of OTR subtype(s) in brain, they show that the uterine-type OTR gene is expressed in all major OTR-containing brain regions. Moreover, they show that region-specific regulation of OTR gene expression underlies the previously observed region-specific steroid regulation of central OT binding sites.     

Differential assembly of coexpressed glutamate receptor subunits in neurons of rat cerebral cortex

In the rat, subunits of the glutamate receptor family fall into three pharmacologically distinct groups: alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid preferring receptors (Glu R1-4), kainate preferring receptors (Glu R5-7, KA 1, KA 2), and N-methyl-D-aspartate preferring receptors (NMDA R1, NMDA R2A-2D). In the present study, we demonstrate immunocytochemically that the majority of neurons in rat cerebral cortex coexpress members of all three groups of glutamate receptor subunits, Glu R2/3, Glu R5/6/7, and NMDA R1. Using immunoaffinity purified or immunoprecipitated alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, kainate and N-methyl-D-aspartate receptors, we show that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors containing Glu R1-4, kainate receptors containing Glu R6, Glu R7, and KA 2 and N-methyl-D-aspartate receptors containing NMDA R1 each form distinct protein complexes that do not share subunits. Our data indicate that a mechanism exists which allows for the specific assembly of selected glutamate receptor subunits into functionally and structurally distinct heteromeric receptors.     

Expression of the thrombin receptor in developing bone and associated tissues

Thrombin, a serine protease with a central role in thrombosis and hemostasis, is also a specific agonist for a variety of cellular responses in osteoblasts and stimulates bone resorption in organ culture. Cultured osteoblast-like cells express the proteolytically activated thrombin receptor, but the significance of this finding in vivo remains unknown. Immunohistochemistry was used to investigate the normal tissue distribution of the proteolytically activated thrombin receptor in developing rat bones and associated tissues. In hind limbs, the receptor was first observed on embryonic day 16 and became more abundant within the limb as gestation progressed. Thrombin receptor staining was detected on osteoblasts, macrophages, muscle cells, and endothelial cells, but not osteoclasts. Similarly, osteoblasts in developing calvariae stained positively for the thrombin receptor. The pattern of receptor expression by primary osteoblast cultures and freshly isolated macrophages and osteoclasts corresponded to that observed in vivo. The observed pattern of thrombin receptor expression in bone cells supports the hypothesis that cell-mediated thrombin-induced bone resorption is mediated by osteoblasts.     

Presence of cross-reacting thymus and brain antigens in the cerebral cortex

Rabbit antisera against antigens of mouse, rabbit, guinea pig and human whole brain cross-reacted in the cytotoxic test with the lymphocytes of the thymus, lymph nodes and the spleen of these animal species. Mouse thymocytes were most sensitive to the antibrain sera (cytotoxic index -- 63--100 per cent); cells from other mouse lymphoid organs and lymphocytes of rabbit, guinea pig and man were more resistant. Bone marrow lymphocytes were not damaged by any of these sera. The antigens which induced the cytotoxic properties of the sera were found only in the human cerebral cortex, but not in the white matter of the brain stem.     

The distribution of neurotrophin receptor TrkC-like immunoreactive fibers and varicosities in the rhesus monkey brain

The distribution of immunoreactivity for the neurotrophin receptor tyrosine kinase TrkC was examined in the brain of the adult rhesus monkey. TrkC-like immunoreactivity was widespread and consisted primarily of varicose fibers. The most dense populations of fibers were in the basal forebrain (in the cholinergic cell groups Ch1, Ch2 and Ch4), in the raphé complex throughout its rostrocaudal extent, and in the locus coeruleus. Other fibers were present in the thalamus, hypothalamus, central gray matter of the midbrain, dorsal midline of the brainstem and the cerebral cortex. The only neuronal cell bodies with consistent labeling were located in the lateral hypothalamus. Purkinje cells in the cerebellum showed variable labeling. Specific labeling of varicosities and cell bodies was abolished by omission of the primary antiserum or by preabsorption with the TrkC peptide antigen. We conclude that TrkC-like immunoreactivity can be detected in a wide variety of subcortical locations in the adult rhesus monkey. Labeling was particularly prominent in the vicinity of the major cholinergic, serotonergic and adrenergic nuclei, known from other studies to be vulnerable in the ageing brain. This suggests that the ligand for TrkC, neurotrophin-3, may persist as a survival factor for critical neurons into adulthood.     

The capacity of central and peripheral catecholaminergic neurons to innervate the pineal organ and cerebral cortex of the rat: in vitro immunohistochemical observations

The locus coeruleus (LC) or superior cervical ganglion (SCG) of neonatal rats were co-cultured either with the pineal organ or cerebral cortex (CX) to investigate the innervating capacity of central and peripheral catecholamine neurons under these experimental conditions. After 2 weeks of co-culturing, cultures were fixed for tyrosine hydroxylase (TH) immunohistochemistry to examine the distribution of catecholamine neurons and their fibers. Glial fibrillary acidic protein and fibronectin immunohistochemistry was performed to determine the cell types proliferating around the explants. In LC/CX co-cultures, numerous astrocytes spread between the two explants, and TH-immunoreactive neurites were generally seen to invade CX explants. In contrast, neurite extension from LC to pineal explants occurred only when a glial cell sheet grew between the two explants, and when the pineal explants were not surrounded by a tight fibronectin-positive cell layer. Neurites of the SCG usually invaded both CX and pineal explants, regardless of the existence of glial or non-glial cell layer. These results indicate that central and peripheral catecholamine neurites have the potential of invading both the cortex and pineal, although they are distributed only in particular regions of the intact brain. The distribution of LC neurites, however, seems to be profoundly affected by the cell types spreading around the explants; glial cells appear to support LC neurite extension, whereas non-glial cells appear to inhibit it.     

Effect of guanine nucleotides and temperature on calcitonin gene-related peptide receptor binding sites in brain and peripheral tissues

Recent data have suggested the existence of at least two major classes of calcitonin gene-related peptide (CGRP) receptors in brain and peripheral tissues [Henke et al., Brain Res., 410 (1987) 404-408; Dennis et al., J. Pharmacol. Exp. Ther., 251 (1989) 718-725; ibid, 254 (1990) 123-128; Quirion et al., Ann. NY Acad. Sci., 657 (1992) 88-105]. However, little is currently known in the structure characteristics of CGRP receptors as cloning as yet to be reported. In the present study, the sensitivity of [125I]humanCGRP alpha binding to guanine nucleotides and temperature was investigated in guinea pig atria (prototypical CGRP1 tissue) guinea pig vas deferens (prototypical CGRP2 tissue) and in the rat brain and cerebellum (mixed assay). Binding isotherms of [125I]hCGRP alpha in those four tissue preparations were curvilinear and best fitted to a two-site model under most assay conditions. The high affinity binding component was highly temperature-sensitive and accounted, under experimental conditions, for up to 18% of the total population of receptors. Moreover, these high affinity sites were also highly sensitive to guanine nucleotides (Gpp(NH)p, 100 microM) in all preparations although to a different extend depending upon assay temperatures. Taken together, this suggests that the different CGRP receptor subtypes present in these tissue all belong to a G-protein coupled receptor family.     

Diminution of preprosomatostatin-mRNA in cerebral cortex of the aged rat

The aim of the present study was to examine the effect of normal aging on somatostatin neurotransmission. Somatostatin gene expression was analysed in several regions of the cerebral cortex and hippocampus in 3, 7 and 21 month-old Sprague-Dawley rats using quantitative in situ hybridization with a 48mer oligodeoxynucleotide antisense probe. Furthermore the distribution of 125I-Tyr11 somatostatin receptor binding sites was studied using quantitative receptor autoradiography. The results demonstrated a significant reduction of preprosomatostatin-mRNA in the frontal cortex of the aged (21 month) group compared with the young (3 month) and the middle-aged (7 month) groups. The receptor binding densities of the aged (21 month) group tended to be lower, compared to the other groups although no significant region-specific changes were evident. These results indicate neurochemical changes in somatostatin-containing neurons in the frontal cortex during aging.     

The orthostatic resistance of the cerebral and peripheral circulations in patients with contusive brain lesions

Rat pancreatic islets were immunoisolated within alginate capsules with additional polyethyleneimine-protamine-heparin highly biocompatible membrane. Perifusion study in vitro demonstrated satisfactory similarities between the insulin release profiles of encapsulated and free islets. Concordant xenotransplantation of microencapsulated rat islets significantly prolonged mean time of restored normoglycemia (46 +/- 15 days) in streptozotocin-diabetic BALB/c mice recipients comparing to uncoated grafts (7 +/- 2 days).     

Daily variation of CNS gene expression in nocturnal vs. diurnal rodents and in the developing rat brain

To identify risk factors associated with an increased risk for ipsilateral breast tumor recurrence following breast-conserving surgery, a cohort of 759 women with T1-T2 tumors were studied. The majority of the patients (88%) had received postoperative radiation therapy to the breast. Median follow-up time was 10 (range: 6-17) years. There was a 1-1.5% yearly increase in ipsilateral breast tumor recurrences. For women < 50 ys the cumulative recurrence rate at 10 years was 18% and for women > or = 50 ys, 9%. Node positive women had a cumulative breast recurrence rate of 25% versus 10% for node negative women. Ten years postoperatively, irradiated patients had a cumulative recurrence rate of 11% versus 26% when no irradiation was given. The beneficial effect of radiotherapy was substantial during the first four postoperative years. The relative risk for an ipsilateral breast tumor recurrence during this period was 4.5 times higher than for non-irradiated patients. However, the protective effect of radiotherapy decreased with time. After ten years the relative risk of ipsilateral breast tumor recurrence was the same among irradiated and non-irradiated patients although the number of events during this period was low. Univariate analysis showed that seven factors were significantly associated with an increased risk of ipsilateral breast tumor recurrence, namely age < 50 ys, increasing tumor size, uncertain microscopic margins, axillary lymph node metastases, no postoperative tamoxifen treatment, premenopausal status, and no postoperative radiotherapy. Three factors remained independently significant after multivariate analysis: age < 50 ys, no postoperative radiation therapy, and positive lymph nodes. In conclusion, radiotherapy reduced the breast recurrence rate, but the effect decreased with time. Node-negative women > or = 50 were a low risk-group for ipsilateral breast tumor recurrence, with a cumulative risk at 10 years of 9% without radiation therapy and 5% with breast irradiation.     

Aromatase immunoreactivity in axon terminals of the vertebrate brain. An immunocytochemical study on quail, rat, monkey and human tissues

Correct detection of premature contractions and incompetent uterine cervix is still a challenging obstetrical problem since these factors remain a major cause of perinatal loss. Ultrasonography offers additional important data for the prediction of these pathologies which have common sonographic patterns; shortening of the cervical length, funneling of the membranes and dilatation of the endocervical canal. The first section of this review highlights sonography of normal cervical anatomy, while the second section focuses on recent advances in sonographic detection of premature contractions and incompetent cervix. It is believed that due to its accuracy and reproducibility, this noninvasive technique should become more integrated into this aspect of antenatal care.