Occurrence of histone H1° protein in rat alveolar macrophages

Histone H1 of rat alveolar macrophages, neutrophilic granulocytes and monocytes extracted with 5% (v/v) perchloric acid was studied in order to see whether a protein similar to histone H1° of rat liver exists in these specialized cells. The biochemical methods used involved SDS and acid-urea polyacrylamide gel electrophoresis, gel filtration on BioGel P100 and raising antisera against chromatographically purified rat liver H1° and histone H1. The antiserum was applied for further characterization of the presumptive H1° fraction using ELISA and Western blot analysis.The results from our studies showed that histone H1° protein is present in rat alveolar macrophages, monocytes and neutrophilic granulocytes, but its quantity in neutrophilic granulocytes is very much less than macrophages and monocytes.     

Lipoxygenation in rat brain?

It has been previously claimed that rodent brain possesses lipoxygenase activity, based upon the structure of products which were formed from arachidonic acid and the inhibition of this activity by "lipoxygenase inhibitors." Our studies confirm that various positional isomers of hydroxyeicosatetraenoic acids (HETE) are formed (e.g., 15-, 12-, 11-, 9-, 8- and 5-HETE) by brain homogenate and that their production is inhibited by certain lipoxygenase inhibitors, such as nordihydroguaiaretic acid (NDGA) but not by cyclooxygenase or cytochrome P-450 inhibitors. However, stereochemical analysis indicated racemic distributions of these products suggesting that they were not formed by a lipoxygenase enzyme but rather by a peroxidative process. It should also be noted that the presence of 12(S)-lipoxygenase activity could be demonstrated by stereochemical analysis only when the brain was not perfused properly, indicating this activity was due to blood cell contamination. It is known that many lipoxygenase inhibitors are also capable of inhibiting peroxidative reactions apparently due to their free radical scavenging properties. For these reasons, it is essential that the stereochemical purity of purported lipoxygenase products be determined and that previous claims of lipoxygenase activity in mammalian brain be reexamined.     

Effects of combined administration ofl-tryptophan and tricyclic antidepressants on α2- and β-adrenoceptors and monoamine levels in rat brain

In order to test whether co-administration of a serotonin precursor with antidepressant drugs could potentiate the effects of the antidepressants on monoamines or adrenoceptors in rat brain,l-tryptophan (20 mg/kg) was administered to rats daily for 7 or 15 days, either alone or in combination with desipramine (10 mg/ kg) or amitriptyline (10 mg/kg). After treatment withl-tryptophan for 7 days, increases were observed in rat hypothalamic and frontal cortex 5-hydroxy-3-indoleacetic acid levels as well as in hypothalamic dopamine and nucleus accumbens 3,4-dihydroxyphenylacetic acid levels. After 15 days, hippocampal -adrenoceptor density was found to be decreased. There was no evidence of potentiation of desipramine or amitriptyline action whenl-tryptophan was administered in combination with the antidepressants. On the contrary, the antidepressants appeared to interact withl-tryptophan to reduce its effects.     

Regional distribution of α-neo-endorphin in rat brain and pituitary

α-Neo-endorphin was isolated as the first form of “big” Leu-enkephalin and its complete amino acid sequence has recently been established. Using an antiserum raised against synthetic α-neo-endorphin, a highly sentitive and specific radioimmunoassay was developed. The antiserum practically possesses no cross-reactivity to Leu-enkephalin, dynorphin[1–13] and PH-8P, and very little to β-neo-endorphin. Distribution of α-neo-endorphin has been determined in rat brain and pituitary by the use of the highly specific antiserum. The highest concentration was observed at posterior lobe of pituitary. Furthermore, immunoreactive α-neo-endorphin was characterized by gel-filtration and high performance liquid chromatography, and shown to be identical with authentic α-neo-endorphin.     

Transplated neuronal precursors migrate and differentiate in the developing mouse brain

The subventricular zone(SVZ),lining the lateral ventricle in forebrain,retains a population of neuronal precursors with the ability of proliferation in adult mammals.To test the potential of neuronal precursors in adult mice,we transplanted adult SVZ cells labeled with fluorescent dye PKH26 into the lateral ventricle of the mouse brain in different development stages.The preliminary results indicated that the grafted cells were able to survive and migrate into multiople regions of the recipient brain,including SVZ, the third ventricle,thalamus,superior colliculus,inferior colliculus,cerebellum and olfactory bulb etc;and the amount of survival cells in different brain regions was correlated with the development stage of the recipient brain.Immunohistochemical studies showed that most of the grafted cells migrating into the specific target could express neuronal or astrocytic marker.Our results revealed that the neuronal precursors in adult SVZ still retained immortality and ability of proliferation,which is likely to be induced by some environmental factors.     

Benzafibrate induces acyl-CoA oxidase mRNA levels and fatty acid peroxisomal β-oxidation in rat white adipose tissue

Rats treated with bezafibrate, a PPAR activator, gain less body weight and increase daily food intake. Previously, we have related these changes to a shift of thermogenesis from brown adipose tissue to white adipose tissue attributable to bezafibrate, which induces uncoupling proteins (UCP), UCP-1 and UCP-3, in rat white adipocytes. Nevertheless, UCP induction was weak, implying additional mechanisms in the change of energy homeostasis produced by bezafibrate. Here we show that bezafibrate, in addition to inducing UCPs, modifies energy homeostasis by directly inducing aco gene expression and peroxisomal fatty acid -oxidation in white adipose tissue. Further, bezafibrate significantly reduced plasma triglyceride and leptin concentrations, without modifying the levels of PPAR or ob gene in white adipose tissue. These results indicate that bezafibrate reduces the amount of fatty acids available for triglyceride synthesis in white adipose tissue.     

α1,3 Fucosyltransferase, α-L-fucosidase, α-D-galactosidase, β-D-galactosidase, and Lex glycoconjugates in developing rat brain

Fucosyltransferases (FTs) and various glycosidases that are involved in the biosynthesis or degradation of SSEA-1 (Lex) antigens and their precursors in the CNS are developmentally regulated. In forebrain and cerebellum with lactosamine (LacNAc) as acceptor the FT activity was maximal at P15–P22, but with the glycolipid substrate paragloboside (nLc4) the maximal activity in cerebellum was obtained at P10–P15. The FT activity, with these substrates, was insensitive to N-ethylmaleimide (NEM) and the glycolipid product had an α1,3 linkage (Fuc to GlcNAc) suggesting similarities of the investigated enzyme to the cloned human and rat FT IV. However, the observation of different patterns of FT activity in isoelectrofocused fractions (pH 3.5–10) with different types of acceptors, and the differential expression of Lex containing glycolipids and glycoproteins during development strongly suggest the presence of more than one type of FT during development. Data on developmental expression of the hydrolytic enzymes, α-L-fucosidase, β-D-galactosidase and α-D-galactosidase, which can potentially hydrolyse SSEA-1 or its precursors, support the notion that SSEA-1 expression is the result of a dynamic balance between the activity of transferases and hydrolases. © 1998 Rapid Science Ltd     

Kinetics of [3H]prazosin and [3H]dihydroalprenolol binding to α1- and β-adrenoreceptors in rat brain cortex membranes

The binding of nonselective α₁- and β-adrenoreceptor antagonists [³H]prazosin and [³H]dihydroalprenolol ([³H]DHA) to rat cerebral cortex synaptosomal membranes has been studied. It is found that ligand-receptor interactions of α₁-adrenoreceptors fit into a single receptor pool model, which assumes the binding of two ligand molecules to one receptor molecule. The parameters of [³H]prazosin binding to α₁-adrenoreceptors are as follows: K _d = 2.58 ± 0.20 nM; B _m = 2.95 ± 1.12 fmol/mg protein; Hill coefficient, n = 2. For β-adrenoreceptors, ligand-receptor interactions fit into a model assuming the presence of two receptor pools in the same effector system and binding of two ligand molecules to one receptor molecule. The corresponding parameters of the [³H]DHA binding to β-adrenoreceptors are as follows: K _d1 = 0.74 ± 0.09 nM; K _d2 = 7.63 ± 0.70 nM; B _m1 = 25 ± 2 fmol/mg, B _m2 = 48 ± 2 fmol/mg, n ₁ = 2; n ₂ = 2. We suggest that in rat cerebral cortex membranes α-and β-adrenoreceptors exist as dimers.     

Enzymatic alterations in developing rat brain cells exposed to a low-intensity 16.5 GHz microwave radiation

The present study deals with the effects of chronic exposure of low-level microwave radiation on developing rat brain. Starting at 35 days of age, male rats were exposed to 2?h/day for another 35 days to a 16.5-GHz microwave radiation field. After the exposure period, the rats were sacrificed, and brain tissues dissected out and used for biochemical assay. Results showed that exposure to a 16.5-GHz radiation caused significant changes in the activity of protein kinase C as compared to the control group. Furthermore, electron microscopic study revealed an increase in glial cell population. These results confirm that brain cell membrane is an interactive site for electromagnetic field causing an inflammation and possibly tumor promotion.     

Expression of βA3/A1-crystallin in the developing and adult rat eye

Crystallins are very abundant structural proteins of the lens and are also expressed in other tissues. We have previously reported a spontaneous mutation in the rat βA3/A1-crystallin gene, termed Nuc1, which has a novel, complex, ocular phenotype. The current study was undertaken to compare the expression pattern of this gene during eye development in wild type and Nuc1 rats by in situ hybridization (ISH) and immunohistochemistry (IHC). βA3/A1-crystallin expression was first detected in the eyes of both wild type and Nuc1 rats at embryonic (E) day 12.5 in the posterior portion of the lens vesicle, and remained limited to the lens fibers throughout fetal life. After birth, βA3/A1-crystallin expression was also detected in the neural retina (specifically in the astrocytes and ganglion cells) and in the retinal pigmented epithelium (RPE). This suggested that βA3/A1-crystallin is not only a structural protein of the lens, but has cellular function(s) in other ocular tissues. In summary, expression of βA3/A1-crystallin is controlled differentially in various eye tissues with lens being the site of greatest expression. Similar staining patterns, detected by ISH and IHC, in wild type and Nuc1 animals suggest that functional differences in the protein, rather than changes in mRNA/protein level of expression, likely account for developmental abnormalities in Nuc1.     

β-d-glucosidase in fractions from rat brain

-Glucosidase activity has been determined in homogenate and in centrifugation fractions of 7-day-old and adult rat brain; maximum activity was found at pH 4 and pH 5. Of the adult brain, more than 50% of the activity was concentrated in the 800-g sediment fraction (P₁), while in the brain of 7-day-old rat about 20% was found in the corresponding fraction. The activity maximum in all fractions after a 2% Triton X-100 treatment occurs at pH 5. Addition of Triton to adult brain homogenate enhances the activity, but this stimulation is less than the sum of the activities observed at pH 4 and pH 5 in the absence of Triton. Triton addition to brain homogenate of 7-day-old rat results in a fall in activity at pH 4 and in a maximum at pH 5. In rat brain homogenate subjected to sonication, a loss of activity is observed at pH 4, scarcely at pH 5; the activity loss is completely abolished and turned into an increase under the influence of Triton. This increase equals the level obtained when Triton is added to an untreated brain homogenate. Sonication of rat brain homogenate leads to changes in the distribution pattern; about 25% of the activity of the adult brain is found in the P₁ fraction compared to 50% in the corresponding fraction of the untreated brain. Fractionation of a sonicated brain homogenate from adult rat reveals that at pH 4 most activity (52%) is concentrated in the 20,000-g pellet (P₂), 23% in supernatant fluid (S₂); at pH 5 the opposite is observed: most activity (49%) is found in the 20,000-g supernatant (S₂) and 23% in the 20,000-g pellet (P₂). In the presence of Triton the activity of the sonicated brain homogenate of adult rat increases; this stimulation roughly equals the sum of the corresponding activities measured at pH 4 and pH 5 in the absence of Triton.     

The B°AT1 amino acid transporter from rat kidney reconstituted in liposomes: kinetics and inactivation by methylmercury

The neutral amino acid transporter B°-like from rat kidney, previously reconstituted in liposomes, was identified as B°AT1 by a specific antibody. Collectrin was present in the brush-border extract but not in functionally active proteoliposomes, indicating that it was not required for the transport function. Neutral amino acids behaved as competitive inhibitors of the glutamine transport mediated by B°AT1 with half saturation constants ranging from 0.13 to 4.74mM. The intraliposomal half saturation constant for glutamine was 2.0mM. By a bisubstrate kinetic analysis of the glutamine-Na(+) cotransport, a random simultaneous mechanism was found. Methylmercury and HgCl(2) inhibited the transporter; the inhibition was reversed by dithioerythritol, Cys and, at a lower extent, N-acetylcysteine but not by S-carboxymethylcysteine. The IC(50) of the transporter for methylmercury and HgCl(2) was 1.88 and 1.75μM, respectively. The reagents behaved as non-competitive inhibitors toward both glutamine and Na(+) and no protection by glutamine or Na(+) was found for the two inhibitors.     

Pharmacological characterization of [³H]CHIBA-3007 binding to glycine transporter 1 in the rat brain

Glycine transporter-1 (GlyT-1) in glial cells regulates extracellular levels of glycine, which acts as an obligatory co-agonist at the N-methyl-D-aspartate (NMDA) receptors in the brain. In the present study, we developed a novel radioligand, [³H]3-chloro-N-((S)-((R)-1-methylpiperidin-2-yl)(thiophen- 3-yl)methyl)-4- (trifluoromethyl)picolinamide ([³H]CHIBA-3007), for studying GlyT-1 in the brain. The presence of a single saturable high-affinity binding component for [³H]CHIBA-3007 binding to the rat brain membranes was detected. Scatchard analysis revealed an apparent equilibrium dissociation constant (K_d) of 1.61±0.16 nM and a maximal number of binding sites (B_max) of 692.8±22.8 fmol/mg protein (mean ± SEM, n = 3). The specific binding of [³H]CHIBA-3007 was inhibited by a number of GlyT-1 inhibitors, such as CHIBA-3007, desmethyl-CHIBA-3007, CHIBA-3008, SSR504734, NFPS/ALX5407, LY2365109 and {"type":"entrez-protein","attrs":{"text":"Org24598","term_id":"1179171570","term_text":"ORG24598"}}Org24598, consistent with the pharmacological profiles of GlyT-1 inhibitors. Interestingly, the potency of eight GlyT-1 inhibitors (CHIBA-3007, desmethyl-CHIBA-3007, NFPS/ALX5407, LY2365109, {"type":"entrez-protein","attrs":{"text":"Org24598","term_id":"1179171570","term_text":"ORG24598"}}Org24598, SSR504734, sarcosine, and glycine) for blocking in vitro specific binding of [³H]CHIBA-3007 was significantly correlated with the potency of these inhibitors for inhibiting [¹⁴C]glycine uptake in the rat brain membranes. In contrast, the GlyT-2 inhibitor ALX1393 exhibited very weak for [³H]CHIBA-3007 binding. Furthermore, the regional distribution of [³H]CHIBA-3007 binding in the rat brain was similar to the previously reported distribution of GlyT-1. The present findings suggest that [³H]CHIBA-3007 would be a useful new radioligand for studying GlyT-1 in the brain.     

αMSH-like peptides in rat brain: Identification and changes in level during development

MSH-like peptides were extracted from rat brains and separated by high pressure liquid chromatography. Using C and N terminally directed antibodies, and a bioassay for melanotropic activity, two major melanotropic peptides were detected. One peptide was identified as αMSH and the other as des-acetyl αMSH, a form which has not been previously reported in brain. Analysis of the level of αMSH-like peptides in brain and pituitary gland during development, showed steady increases of pituitary αMSH from day 18 fetuses to adults, whereas in brain, significant increases were not observed until one day post partum. This difference in the time of onset of αMSH production in the two tissues suggests the presence of biosynthetically independant pools of αMSH-like peptides in pituitary and brain.     

Cistanches Herba aqueous extract affecting serum BGP and TRAP and bone marrow Smad1 mRNA, Smad5 mRNA, TGF-β1 mRNA and TIEG1 mRNA expression levels in osteoporosis disease

We studied molecular mechanism of Cistanches Herba aqueous extract (CHAE) in ovariectomized (OVX) rats, as an experimental model of postmenopausal osteoporosis. Female rats were either sham-operated or bilaterally OVX; and at 60 days postoperatively. The OVX group (n = 8) received an ovariectomy and treatment with normal saline for 90 days commencing from 20th post ovariectomy day. The ovariectomized +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy and were treated with Cistanches Herba aqueous extract of 100 mg/kg body weight daily for 90 days commencing from 22nd post ovariectomy day. The ovariectomy +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy, and were treated with the of 200 mg/kg body weight daily for 90 days commencing from 20th post ovariectomy day. Serum BGP and TRAP, E2, FSH and LH level, bone marrow Smad1, Smad5, TGF-β1 and TIEG1 mRNA expression levels were examined. Results showed that serum BGP and TRAP, FSH and LH levels were significantly increased, whereas E2, Smad1, Smad5, TGF-β1 and TIEG1 mRNA and proteins expression levels were significantly decreased in OVX rats compared to sham rats. 90 days of CHAE treatment could significantly decrease serum BGP and TRAP, FSH and LH levels, and increase E2, Smad1, Smad5, TGF-β1 and TIEG1 mRNA and proteins expression levels in OVX rats. It can be concluded that CHAE play its protective effect against OVX-induced bone degeneration partly by regulating some bone metabolism related genes, e.g. Smad1, Smad5, TGF-β1 and TIEG1.     

The occurrence and properties of histone H1° in quiescent rabbit tissue

• 1.1. The issue of the existence and the properties of histone H1° in quiescent rabbit tissues was approached by electrophoretic, Chromatographic and immunochemical techniques.
• 2.2. The results demonstrated that the rabbit tissues did contain a typical histone H1°, its properties being comparable to those of H1° from all other sources studied thus far.

     

Effects of ligands of α<Superscript>2</Superscript>-adrenoceptors on mRNA level of apoptotic proteins in developing rat brain

The effects of antagonist of α²-adrenoceptors yohimbine and their agonist clonidine on Bax and Bcl-X _L mRNA levels in neonatal rat brain were studied. Yohimbine decreased Bax mRNA level in the cerebellum, increased the ratio between Bcl-X _L and Bax mRNA levels in the cerebellum, cortex, and hippocampus, and increased Bcl-X _L mRNA level in the cortex and hippocampus of 6-day-old-rat pups 24 h after injection. Administration of clonidine 20 min after yohimbine administration abolished its effect on Bcl-X_L mRNA level in the hippocampus. The data obtained indicate that the blockade of α²-adrenoceptors induces antiapoptotic changes in the developing rat brain, some of which can be abolished after coadministration with the agonist of these receptors.