BiP mRNA expression is upregulated by dehydration in vasopressin neurons in the hypothalamus in mice

The immunoglobulin heavy chain binding protein (BiP) is an endoplasmic reticulum (ER) chaperone that facilitates the proper folding of newly synthesized secretory and transmembrane proteins. Here we report that BiP mRNA was expressed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus in wild-type mice under basal conditions. Dual in situ hybridization in the SON and PVN demonstrated that BiP mRNA was expressed in almost all the neurons of arginine vasopressin (AVP), an antidiuretic hormone. BiP mRNA expression levels were increased in proportion to AVP mRNA expression in the SON and PVN under dehydration. These data suggest that BiP is involved in the homeostasis of ER function in the AVP neurons in the SON and PVN.     

Eki2 is upregulated specifically in the testis during mouse sex determination

We have identified a gene with gonad restricted expression throughout mouse development, which is orthologous to human EKI2 (ethanolamine kinase 2). Our studies showed that mouse Eki2 expression became upregulated in the male gonad during the period of sex determination. Expression was restricted to the Sertoli cells of the developing testis. Eki2 has sequence similarity to ethanolamine (73%) and choline kinases (54%).     

Lamination of the cerebral cortex is disturbed in Gli3 mutant mice

The layered organization of the cerebral cortex develops in an inside-out pattern, a process which is controlled by the secreted protein reelin. Here we report on cortical lamination in the Gli3 hypomorphic mouse mutant Xt^J/Pdn which lacks the cortical hem, a major source of reelin⁺ Cajal Retzius cells in the cerebral cortex. Unlike other previously described mouse mutants with hem defects, cortical lamination is disturbed in Xt^J/Pdn animals. Surprisingly, these layering defects occur in the presence of reelin⁺ cells which are probably derived from an expanded Dbx1⁺ progenitor pool in the mutant. However, while these reelin⁺ neurons and also Calretinin⁺ cells are initially evenly distributed over the cortical surface they form clusters later during development suggesting a novel role for Gli3 in maintaining the proper arrangement of these cells in the marginal zone. Moreover, the radial glial network is disturbed in the regions of these clusters. In addition, the differentiation of subplate cells is affected which serve as a framework for developing a properly laminated cortex.     

Effect of hypoxia on mitochondrial protein composition of cerebral cortex during aging

The effect of hypoxia on the protein composition of mitochondria from cerebral cortex of rats at 4, 12, and 24 months of age was investigated. The proteins were separated by electrophoresis on SDS polyacrilamide gels and the percent content was evaluated by measuring the optical density of the stained gels. The results demonstrate that hypoxic treatment causes a decrease in the amount of some proteins as follows: the 90 and the 16 kDa Mw proteins at 4 months; the 82 and the 79 kDa Mw proteins at 24 months; the 52-49, 35 and 20 kDa at all ages investigated; the 44 kDa protein at 4 and 12 months and the 28 kDa protein at 4 and 24 months of age. Our results show that hypoxic conditions affect mitochondrial protein composition to a greater extent than aging alone.Special issue dedicated to Dr. Santiago Grisolia     

Osteopontin expression during early cerebral ischemia-reperfusion in rats: enhanced expression in the right cortex is suppressed by acetaminophen

Osteopontin (OPN) is a pleiotropic protein implicated in various inflammatory responses including ischemia-reperfusion (I-R) injury. Two distinct forms of the protein have been identified: an extensively studied secreted form (sOPN) and a less-well-known intracellular form (iOPN). Studies have shown that increased OPN expression parallels the time course of macrophage infiltration into injured tissue, a late event in the development of cerebral infarcts. sOPN has been suggested to promote remodeling of the extracellular matrix in the brain; the function of iOPN may be to facilitate certain signal transduction processes. Here, we studied OPN expression in adult male Sprague-Dawley rats subjected to global forebrain I-R injury. We found iOPN in the cytoplasm of both cortices and the hippocampus, but unexpectedly only the right cortex exhibited a marked increase in the iOPN level after 45 min of reperfusion. Acetaminophen, a drug recently shown to decrease apoptotic incidence, caspase-9 activation, and mitochondrial dysfunction during global I-R, significantly inhibited the increase in iOPN protein in the right cortex, suggesting a role for iOPN in the response to I-R injury in the right cortex.     

Skeletal muscle and bone: effect of sex steroids and aging

Both estrogen and testosterone are present in males and females. Both hormones contribute to the well being of skeletal muscle and bone in men and women, and there is evidence that the loss of sex hormones is associated with the age-related decline in bone and skeletal muscle mass. Hormonal supplementation of older adults to restore estrogen and testosterone levels to those of young men and women is not without penalty.     

Apolipoprotein A-I gene expression is upregulated by polychlorinated biphenyls in rat liver

Xenobiotics such as polychlorinated biphenyls (PCB) increase serum cholesterol level (especially high density lipoprotein cholesterol) and apolipoprotein A-I (apo A-I) level in rats. The effect of PCB on serum apo A-I and hepatic apo A-I gene expression and the relationship between apo A-I and drug-metabolizing enzymes in rats were investigated. Serum levels of cholesterol and apo A-I were increased by dietary addition of PCB in a dose-dependent manner (0-500 mg/kg diet). Hepatic apo A-I mRNA level was also elevated by PCB in a similar fashion. Serum level of cholesterol gradually increased during feeding period of PCB (200 mg/kg diet, 105 days) and reached a two-fold higher level in PCB group than in controls. The levels of serum apo A-I and hepatic apo A-I mRNA linearly elevated during feeding period of PCB and were increased 3- or 4-fold, respectively, compared to controls. Although acute administration (16 hr) of PCB, 3-methylcholanthrene, and phenobarbital induced cytochrome P-450 gene expression in the liver, hepatic apo A-I gene expression was not increased by these xenobiotics. These results indicated that the serum levels of cholesterol and apo A-I had positive correlation with hepatic level of apo A-I mRNA in rats fed PCB, and that hepatic apo A-I gene expression was dependent upon intake of PCB but was not directly related to the induction of drug-metabolizing enzymes. This study demonstrated that xenobiotic-induced hyper-alpha-cholesterolemia would be caused by the increased apo A-I gene expression and cholesterol synthesis in the liver, coordinately.     

Role of sex steroids in development of anxiety in female mice

Level of sex steroid hormones being changed throughout an estrous cycle influences physiological and behavioral features of female subjects. To test how estrogen and progesterone affect the anxiety level in mice the ovariectomy (OVX) followed by hormone treatment was carried out. After 1 week of recovery period estradiol benzonate (20 micrograms, s/c) was injected once a day during 7 consequent days. By the 7th day in addition to EB injection progesterone (500 micrograms, s/c) was also injected. Four hours later the mice were tested in elevated plus-maze to measure the anxiety level. Control animals were treated with sesame oil only. Behavioral data obtained demonstrate that the hormonal treatment altered anxiety state in experimental animals. In plusmaze paradigm, it has been demonstrated that progesterone-treated mice revealed the lowest level of open arm activity. In contrast, these mice showed the highest grooming activity as compared to other experimental groups. Immunohistochemical data on progesterone receptor (PR), immunoreactivity in brain have shown that the manipulation with different hormonal treatments modified the number of PR-ir cells in many brain areas. Our data suggest that sex steroid hormones play an important role in induction of anxiety level, as measured by elevated plus-maze, and this action might be partially mediated through the classical steroid receptors.     

Human cytotrophoblast expression of the von Hippel-Lindau protein is downregulated during uterine invasion in situ and upregulated by hypoxia in vitro

The von Hippel-Lindau tumor-suppressor protein (pVHL) regulates the stability of HIF1 alpha and HIF2 alpha and thus is pivotal in cellular responses to changes in oxygen tension. Paradoxically, human cytotrophoblasts proliferate under hypoxic conditions comparable to those measured in the early gestation placenta (2% O(2)), but differentiate into tumorlike invasive cells under well-oxygenated conditions such as those found in the uterus. We sought to explain this phenomenon in terms of pVHL expression. In situ, pVHL immunolocalized to villous cytotrophoblast stem cells, and expression was enhanced at sites of cell column initiation; in both of these relatively hypoxic locations, cytoplasmic staining for HIF2 alpha was also detected. As cytotrophoblasts attached to and invaded the uterus, which results in their increased exposure to oxygen, pVHL staining was abruptly downregulated concordant with localization of HIF2 alpha to the nucleus. In vitro, hypoxia (2% O(2)) upregulated cytotrophoblast pVHL expression together with HIF2 alpha, which localized to the cytoplasm; culture under well-oxygenated conditions greatly reduced levels of both molecules. These results, together with the placental defects previously observed in VHL(-/-) mice, suggest that pVHL is a component of the mechanism that transduces local differences in oxygen tension at the maternal-fetal interface to changes in the biological behavior of cytotrophoblasts. Furthermore, these data provide the first example of oxygen-dependent changes in pVHL abundance.     

Calcium-binding protein regucalcin mRNA expression in the kidney cortex is suppressed by saline ingestion in rats

The effect of adrenalectomy (ADX) or saline ingestion, which is a hypertensive factor, on the expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open-reading frame). Regucalcin mRNA was expressed in the kidney cortex but not the medulla. Rats were adrenalectomized, and 48 h later they were sacrificed. ADX caused a reduction of regucalcin mRNA levels in the kidney cortex, suggesting that adrenal glands participate in the regulation of the mRNA expression. This reduction was not restored by the subcutaneous administration of dexamethasone with an effective dose (1 mg/kg body weight), which can stimulate kidney regucalcin mRNA expression. Regucalcin mRNA levels in the kidney cortex of rats were markedly suppressed by the ingestion of saline for 7 days. The ADX-induced decrease of renal cortex regucalcin mRNA levels was not appreciably restored by saline ingestion. Moreover, regucalcin mRNA levels in the kidney cortex of spontaneous hypertensive rats (SHR) were clearly decreased as compared with that of control (Wistar-Kyoto) rats. Meanwhile, calcium content in the kidney cortex was not significantly decreased by ADX or saline ingestion. The present study suggests that the expression of regucalcin mRNA in the kidney cortex of rats is suppressed by saline administration.     

Transthyretin is up-regulated by sex hormones in mice liver

The chaperone function of alpha-crystallin is significantly affected in diabetes. Increased formation of advanced glycation end products (AGEs) is the likely cause. This study was aimed to investigate the effect of AGE crosslinks on the chaperone activity of alpha-crystallin and to show the effect of an AGEs crosslink breaker, phenacyl-4,5-dimethylthiazolium bromide (DMPTB). Recombinant alphaA-crystallin was prepared by expressing it in Escherichia coli and purified by size exclusion chromatography. Glycation of alphaA-crystallin was performed with 1-100 mM glucose-6-phosphate (G6P) as the glycating agent for a period of 1-15 days. To break AGE crosslinks, pre-glycated alphaA-crystallin was treated with 0.1-20 mM DMPTB for 3 days. Excess G6P and DMPTB were removed by gel filtration before performing additional experiments. AGEs and crosslinked proteins were estimated by measuring non-tryptophan fluorescence and by SDS-PAGE. Chaperone activity was determined with alcohol dehydrogenase as the target protein. With increasing duration of glycation and G6P concentration, chaperone activity of alpha-crystallin decreased. When pre-glycated alphaA-crystallin was treated with 5-20 mM DMPTB, a DMPTB concentration-dependent recovery of chaperone activity was seen. Lower concentrations, 0.1, 0.5, and 1.0 mM, of DMPTB also showed significant recovery of the chaperone activity. SDS-PAGE analysis after DMPTB treatment showed 40% decrease in crosslinked proteins and fluorescence scan indicated 30% decrease in AGEs. DMPTB is expected to regain alpha-crystallin chaperone activity and provide structural stability to other eye lens proteins that are in aggregation mode which emphasizes the clinical importance of the present finding.     

Odorant receptor expression in the mouse cerebral cortex

Mammalian odorant receptors have been known to be involved not only in odorant detection but also in neuronal development of olfactory sensory neurons. We have examined a possibility of odorant receptor expression in nonolfactory neurons in the mouse. Mouse odorant receptors (M71, C6, and OR3), two of which were already shown to be functionally activated by odorants in heterologous systems, were detected by polymerase chain reactions (PCRs) from the cerebral cortex but not from other brain tissues. Degenerate PCR further suggested that other odorant receptors were also expressed in the mouse cerebral cortex. One of these receptors showed high sequence-match with a putative chick odorant receptor OR7 transiently expressed in the notochord during development. In situ hybridization detected signals for M71 and C6 receptors in the layer II cortical pyramidal neurons located in the occipital pole. In the M71-IRES-tauLacZ mouse, in which M71 expression was genetically marked with tauLacZ, X-gal staining signals were mostly localized in the layer II neurons in the occipital pole, being consistent with the in situ hybridization result. Fluorescent immunohistochemistry using anti-beta-galactosidase antibody further detected the tauLacZ signals in the same cells. X-gal staining began at P3, peaked at P8, and continued to adults, although signals gradually decreased. These data showed that at least a few odorant receptors are expressed not only in olfactory sensory neurons but also in pyramidal neurons in the cerebral cortex, possibly playing an important role either in chemical detection of exogenous or endogenous ligands or in a developmental process such as axon guidance and target recognition.     

APP mRNA splicing is upregulated in the brain of biglycan transgenic mice

Many of the risk factors for cerebrovascular disease and atherosclerosis also increase the risk of Alzheimer's disease, characterized by the cerebral deposition of beta-amyloid plaques resulting from the abnormal processing of the transmembrane amyloid precursor protein (APP). The initiating event of cholesterol-induced atherosclerosis is the retention and accumulation of atherogenic apolipoprotein B (apoB) together with low-density lipoproteins in the vascular intima. Biglycan, a member of the small leucine-rich protein family, was suspected of contributing to this process. The individual and combined overexpressions of biglycan and apoB-100 were therefore examined on the cortical APP mRNA levels of transgenic mice by means of semiquantitative PCR. As compared with the control littermates, transgenic biglycan mice had significantly increased cortical APP695 (122%) and APP770 (157%) mRNA levels, while the double transgenic (apoB(+/-)xbiglycan(+/-)) mice did not exhibit any changes. These results provide the first experimental evidence that the atherogenic risk factor biglycan alters APP splicing and may participate in the pathogenesis of both Alzheimer and vascular dementias.     

Differential expression patterns of gangliosides in the ischemic cerebral cortex produced by middle cerebral artery occlusion

Neuronal damage subsequent to transient cerebral ischemia is a multifactorial process involving several overlapping mechanisms. Gangliosides, sialic acid-conjugated glycosphingolipids, reduce the severity of acute brain damage in vitro. However their in vivo effects on the cerebral cortex damaged by ischemic infarct are unknown. To assess the possible protective role of gangliosides we examined their expression in the cerebral cortex damaged by ischemic infarct in the rat. Ischemia was induced by middle cerebral artery (MCA) occlusion, and the resulting damage was observed by staining with 2, 3, 5-triphenylterazolium chloride (TTC). High-performance thin-layer chromatography (HPTLC) showed that gangliosides GM3 and GM1 increased in the damaged cerebral cortex, and immunofluorescence microscopy also revealed a significant change in expression of GM1. In addition, in situ hybridization demonstrated an increase in the mRNA for ganglioside GM3 synthase. These results suggest that gangliosides GM1 and GM3 may be synthesized in vivo to protect the cerebral cortex from ischemic damage.     

Unusual estrogen-binding liver protein in rats and the metabolism of sex steroids

A study was made of the effect of a highly purified preparation of an unusual estrogen-binding protein (UEBP) of rat liver on 3H-estradiol (3H-E2) and 3H-testosterone (3H-T) metabolism by female rat liver homogenates. The UEBP decreased the rate of metabolic conversions of 3H-E2 and 3H-T in a dose-dependent and specific manner. The effectiveness of the inhibitory action of the UEBP declined with increase of metabolic activities of homogenate enzymes. The effects of the UEBP were reduced fully or partly by the ligands specifically binding to the UEBP, estrone (E1), and estriol (E3) respectively. The latter fact was used to demonstrate the effectiveness of the endogenous male rat liver homogenate UEBP in the control of 3H-E2 metabolism. The UEBP did not exhibit any oxidoreductase activity on the use of 3H-E1, 3H-E2, 3H-E2, 3H-T and 3H-androstenedione with NAD+, NADH, NADP+, and NADPH as cofactors. These data present the first direct experimental evidence of the regulatory function of the UEBP in the time course of changes in sex steroids.