Intra-Nasally Administered Oligopeptide Lunasin Acts as a Possible Anti-Psychotic Agent in Mice Models.

Background and Objectives: Previously we have shown that synthetic lunasin, a 43 amino acid residue-containing peptide, after its central (intracisternal) administration in mice demonstrated antagonism against dopaminergic drug behavioural effects, indicating a putative antipsychotic/anti-schizophrenic profile of lunasin. The aims of the present studies were: to test whether lunasin would show an influence on the dopaminergic system after intranasal administration, and to examine the effect(s) of lunasin on serotonin and glutamatergic systems, which could play an essential role in antipsychotic action. Materials and Methods: Lunasin was administered intra-nasally at doses 0.1 and 1 nmol/mouse in ICR mice (n = 7-8) and tested in an open field on hyperlocomotion caused by amphetamine; serotonin 5-HT 2A/2C receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)- 2-aminopropane (DOI); and glutamate NMDA receptor antagonist phencyclidine. Following behavioural testing, the contents of neurotransmitters and their metabolites in brain hemispheres (n = 6-8) were assessed by ultra-high-performance liquid chromatography-time of flight mas-spectrometry (UHPLC-TOF-MS) method. Also, lunasin binding to serotonin receptors was assessed. Results: Lunasin intra-nasally fully normalized hyper-locomotion and brain monoamine levels in amphetamine- and DOI-treated mice brains. Phencyclidine behavioural effects were not influenced. In vitro receptor binding data demonstrated a low affinity of lunasin (at µM concentrations) compared with DOI (nM concentrations) for the 5-HT2A and 5-HT2C receptors. Conclusions: These results demonstrated, for the first time, that the intranasal administration of oligopeptide lunasin normalized mice behaviour and brain monoamine levels in experimental psychosis mice models. Its neuro-regulatory effects indicated a usefulness of this peptide molecule for the design of novel psychotropic agents.

Itching in a trichophytin contact dermatitis mouse model and the antipruritic effect of antifungal agents.

BACKGROUND: Tinea is an infectious disease by dermatophytes, of which Trichophyton species accounts for the overwhelming majority of case. Tinea often causes itching with inflammation. In terms of pruritus by fungal infection, however, tinea has not been investigated sufficiently to date. AIM: To evaluate itch caused by Trichophyton infection and the effect of antifungal agents on the infection, by measuring scratch behaviour and profiles of inflammatory cytokines and chemokines. METHODS: We used a previously established mouse model of contact hypersensitivity induced by trichophytin, a crude extract from Trichophyton mentagrophytes. Scratching behaviour was recorded using a counting device that measured an electric current induced in a coil by movement of magnets that had been inserted into the hind paws of each animal. We investigated expression of various genes in lesional skin of mice and in normal human epidermal keratinocytes. We also investigated the antipruritic effects of the corticosteroid dexamethasone (DEX) and three antifungal agents: ketoconazole (KCZ), terbinafine (TBF) and liranaftate (LNF). RESULTS: Biphasic peaks of scratching were observed at 1 h and at 6-7 h during an observation period of 14 h after trichophytin induction. For lesional skin, RNA was extracted 24 h after trichophytin challenge, and increased expression was seen in the genes for interleukin (IL)-17A, interferon-γ, tumour necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-2 and Dectin-1, whereas there was no obvious change in the genes for IL-31 and prostaglandin (PG)E2. Furthermore, KCZ inhibited histidine decarboxylase (HDC) expression in vitro and in vivo, and inhibited scratching in the very early phase. LNF inhibited expression of thymic stromal lymphopoietin (TSLP) and IL-8 in vitro, and TSLP, TNF-α, IL-1α and MIP2 in vivo, and also scratching in the early phase. TBF did not induce any significant alterations in either gene expression or scratching. DEX suppressed expression of all the chemical mediators except HDC in vitro and in vivo, and inhibited scratching. CONCLUSION: Antifungals can inhibit itching induced by fungal infection through different mechanisms.

Sprouty2 is involved in the control of osteoblast proliferation and differentiation through the FGF and BMP signaling pathways.

Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) play essential roles in bone formation and osteoblast activity through the extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad pathways. Sprouty family members are intracellular inhibitors of the FGF signaling pathway, and four orthologs of Sprouty have been identified in mammals. In vivo analyses have revealed that Sprouty2 is associated with bone formation. However, the mechanism by which the Sprouty family controls bone formation has not been clarified. In this study, we investigated the involvement of Sprouty2 in osteoblast proliferation and differentiation. We examined Sprouty2 expression in MC3T3-E1 cells, and found that high levels of Sprouty2 expression were induced by basic FGF stimulation. Overexpression of Sprouty2 in MC3T3-E1 cells resulted in suppressed proliferation compared with control cells. Sprouty2 negatively regulated the phosphorylation of ERK1/2 after basic FGF stimulation, and of Smad1/5/8 after BMP stimulation. Furthermore, Sprouty2 suppressed the expression of osterix, alkaline phosphatase, and osteocalcin mRNA, which are markers of osteoblast differentiation. Additionally, Sprouty2 inhibited osteoblast matrix mineralization. These results suggest that Sprouty2 is involved in the control of osteoblast proliferation and differentiation by downregulating the FGF-ERK1/2 and BMP-Smad pathways, and suppresses the induction of markers of osteoblast differentiation.

Protein restriction does not affect body temperature pattern in female mice.

Daily torpor is a physiological adaptation in mammals and birds characterized by a controlled reduction of metabolic rate and body temperature during the resting phase of circadian rhythms. In laboratory mice, daily torpor is induced by dietary caloric restriction. However, it is not known which nutrients are related to daily torpor expression. To determine whether dietary protein is a key factor in inducing daily torpor in mice, we fed mice a protein-restricted (PR) diet that included only one-quarter of the amount of protein but the same caloric level as a control (C) diet. We assigned six non-pregnant female ICR mice to each group and recorded their body weights and core body temperatures for 4 weeks. Body weights in the C group increased, but those in the PR group remained steady or decreased. Mice in both groups did not show daily torpor, but most mice in a food-restricted group (n=6) supplied with 80% of the calories given to the C group exhibited decreased body weights and frequently displayed daily torpor. This suggests that protein restriction is not a trigger of daily torpor; torpid animals can conserve their internal energy, but torpor may not play a significant role in conserving internal protein. Thus, opportunistic daily torpor in mice may function in energy conservation rather than protein saving.

Manipulation of dopamine metabolism contributes to attenuating innate high locomotor activity in ICR mice.

Attention-deficit hyperactivity disorder (ADHD) is defined as attention deficiency, restlessness and distraction. The main characteristics of ADHD are hyperactivity, impulsiveness and carelessness. There is a possibility that these abnormal behaviors, in particular hyperactivity, are derived from abnormal dopamine (DA) neurotransmission. To elucidate the mechanism of high locomotor activity, the relationship between innate activity levels and brain monoamines and amino acids was investigated in this study. Differences in locomotor activity between ICR, C57BL/6J and CBA/N mice were determined using the open field test. Among the three strains, ICR mice showed the greatest amount of locomotor activity. The level of striatal and cerebellar DA was lower in ICR mice than in C57BL/6J mice, while the level of L-tyrosine (L-Tyr), a DA precursor, was higher in ICR mice. These results suggest that the metabolic conversion of L-Tyr to DA is lower in ICR mice than it is in C57BL/6J mice. Next, the effects of intraperitoneal injection of (6R)-5, 6, 7, 8-tetrahydro-l-biopterin dihydrochloride (BH4) (a co-enzyme for tyrosine hydroxylase) and L-3,4-dihydroxyphenylalanine (L-DOPA) on DA metabolism and behavior in ICR mice were investigated. The DA level in the brain was increased by BH4 administration, but the increased DA did not influence behavior. However, L-DOPA administration drastically lowered locomotor activity and increased DA concentration in several parts of the brain. The reduced locomotor activity may have been a consequence of the overproduction of DA. In conclusion, the high level of locomotor activity in ICR mice may be explained by a strain-specific DA metabolism.

Preventive effect of rosiglitazone on liver injury in a mouse model of decompression sickness.

BACKGROUND AND AIMS: Severe decompression sickness (DCS) is a multi-organ injury. This study investigated the preventive effects of rosiglitazone on liver injury following rapid decompression in mice and examined the underlying mechanisms. METHODS: Mice were randomly divided into four groups: a control group, vehicle group, and rosiglitazone (5 and 10 mg·kg⁻¹) groups, the latter three being exposed to a pressure of 911 kPa. Haematoxylin and eosin staining, plasma levels of alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase and blood cell counts were used to evaluate liver injury at 30 min after rapid decompression. The expression of endothelial and inducible nitric oxide synthase (iNOS) and its phosphorylation were measured to uncover the underlying molecular mechanisms. RESULTS: A significant increase in plasma ALT, red blood cells and platelets, and a decrease in neutrophils were observed in the vehicle group. Furthermore, the expression of iNOS, E-selectin and the total level of NO in hepatic tissue, and soluble E-selectin in the plasma were significantly elevated in the vehicle group. Rosiglitazone pre-treatment prevented the increases in ALT (and AST), soluble E-selectin concentration, red blood cells and platelet counts. Moreover, rosiglitazone reduced over-expression of iNOS and the NO level, prevented the fall in neutrophil count and promoted the phosphorylation of iNOS in the liver. CONCLUSIONS: Pre-treatment with rosiglitazone ameliorated liver injury from severe DCS. This preventive effect may be partly mediated by stimulating endothelial NO production, improving endothelial function and limiting inflammatory processes.

Laboratory domestication changed the expression patterns of oxytocin and vasopressin in brains of rats and mice.

The process of domestication is recognized to exert significant effects on the social behaviors of various animal species, including defensive and cognitive behaviors that are closely linked to the expression of oxytocin (OT) and vasopressin (AVP) in selected areas of the brain. However, it is still unclear whether the behavioral changes observed under domestication have resulted in differences in the neurochemical systems that regulate them. In this study, we compared the differences in distribution patterns and regional quantities of OT and/or AVP staining in the forebrains of wild and laboratory strains of rats and mice. Our results indicated that, in the anterior hypothalamus (AH), laboratory strains showed significantly higher densities of OT-ir (immunoreactive) and AVP-ir cells than wild strains, while no significant difference in the densities of those cells in the lateral hypothalamus (LH) was detected between wild and laboratory strains. Laboratory strains showed higher densities of OT-ir and AVP-ir cells than wild strains in the medial preoptic area (MPOA), and differed in almost every MPOA subnucleus. Our results suggest that domestication significantly alters the expression of OT and AVP in related brain areas of laboratory rats and mice, an observation that could explain the identified changes in behavioral patterns.

[The differences in absorption and metabolism of bisphenol A between rats and mice].

OBJECTIVE: To investigate the mechanism of the different levels of serum bisphenol A (BPA) between rat and mouse after oral administration. METHODS: A total of 18 specific pathogen free (SPF) male rats and 18 mice were treated with 300 mg/kg BPA by oral administration, blood samples were taken from rats and mice after BPA administration at 0.5, 1.0, 12.0 h time points (n = 6 at each point). Serum BPA levels were quantified using fluorescence-high performance liquid chromatography (FL-HPLC) analysis. The rats and mice (n = 6, respectively) were perfused with 100 ml of 0.1 mmol/L BPA by intestinal absorption in situ, then the BPA levels of perfusion fluid at 0.5, 1.0, 2.0 h time points and serum at 2.0 h after BPA perfusion were determined by FL-HPLC analysis. The levels of UDP-glucuronosyltransferase 2B1 (UGT2B1) mRNA expression in the liver of rats and mice were analyzed by semi-quantitative RT-PCR and UGT2B1 enzymatic activity was determined by FL-HPLC method. The rats and mice (n = 6, respectively) were treated with 300 mg/kg BPA by oral administration after fasting 24 h, the feces were collected during 24 h and the levels of BPA in feces were determined by FL-HPLC analysis. RESULTS: At 0.5, 1.0, 12.0 h after oral administration at 300 mg/kg BPA, the levels of serum BPA in mice ((66.57 ± 14.95), (51.16 ± 16.06), (22.73 ± 5.00) µg/ml, respectively) were significantly higher than in rats ((15.63 ± 5.65), (18.34 ± 5.02), (7.65 ± 2.58) µg/ml, respectively) (F values were 50.660, 17.957, 8.420, respectively, P < 0.05), the rates of absorption in mice small intestine during 0 h-, 0.5 h-, 1.0 - 2.0 h ((10.20 ± 4.20), (1.49 ± 0.67), (1.31 ± 0.55) µg × cm(-2) × min(-1), respectively) were higher than that in rats ((1.87 ± 0.69), (0.47 ± 0.13), (0.36 ± 0.08) µg × cm(-2) × min(-1), respectively) (F values were 14.954, 8.877, 11.536, respectively, P < 0.05), the serum BPA levels in mice ((22.64 ± 4.35) µg/ml) were significantly higher than in rats ((4.13 ± 0.83) µg/ml) after 2 h perfusion with 0.1 mmol/L BPA (F = 74.643, P = 0.000), the levels of UGT2B1 mRNA expression and enzymatic activity in the rats liver were obviously higher than in the mice liver. After oral administration at 300 mg/kg BPA, the feces BPA levels of rats ((1.50 ± 0.32) mg/g) were significantly higher than that of the mice ((0.57 ± 0.35) mg/g) (F = 21.215, P = 0.001) during 24 h. CONCLUSION: The serum BPA level of mouse is significantly higher than the rat after oral administration at 300 mg/kg BPA, which may be caused by BPA high absorption rate of mouse small intestine and strong ability of BPA glucuronidation and excretion of the rat.

Soft-hydrothermal processing of red cedar bedding reduces its induction of cytochrome P450 in mouse liver.

Red cedar-derived bedding materials cause changes in cytochrome P450-dependent microsomal enzyme systems in laboratory animals. We examined the effect of essential oil of red cedar (EORC), as well as the effect of bedding from which it had been removed, on the hepatic expression cytochrome P450s in mice. EORC was obtained from liquid extracts of red cedar bedding by a soft-hydrothermal process and was administered orally to mice. Between days 1 and 2 after administration, hepatic P450s were significantly induced as follows: CYP3As, 7.1x; CYP1As, 1.6x; CYP2E1, 1.5x; CYP2Cs, 1.6x. A housing study of mice indicated that red cedar bedding increased the levels of these P450s in mouse liver, whereas mice housed in cedar bedding from which EORC had been removed (ST-cedar bedding) showed significantly lower levels of P450s, especially CYP3As, CYP1As and CYP2E1. Soft-hydrothermal processing partially removed many components of EORC. In particular, several volatile sesquiterpenes, naphthalene-derived aromatics and 4,4-dimethyl-13alpha-androst-5-ene were decreased in the ST-cedar bedding, suggesting that these may be responsible for P450 induction. This study demonstrated that the removal of these volatile compounds by soft-hydrothermal processing can decrease the hepatic P450-inducing effect of red cedar bedding.

Developmental change of alpha-gustducin expression in the mouse fungiform papilla.

Developmental changes in the distribution pattern of taste buds in newborn mouse have not been previously elucidated, and little work has been done to examine the postnatal alteration of the expression of alpha-gustducin in the mouse taste buds. In the present paper, we delineated the development and frequency distribution of the taste buds as well as the immunohistochemical expression of alpha-gustducin, a G protein closely related to the transduction of taste stimuli in the fungiform papilla from the birthday till the age of week 9. At birth, more than 45 taste buds (with or without pores) were observed on the fungiform papilla, then the number of mature taste buds increased rapidly, and resulted in 66% (80.2 +/- 0.6 of 122.2 +/- 1.3) of fungiform papilla taste buds containing taste pores at week 3. By age the total counts of pored taste buds continuously increased and their morphological features became quite discernible. They became ellipse in shape, characterized by distinct pores. Quantitative analysis of alpha-gustducin expression at different postnatal ages revealed a significant increase in the number of immunolabeled taste buds and alpha-gustducin-positive cells in single taste buds from week 1 to 7, by week 7, the number reached the value found in adults (99.3 +/- 0.9 and 8.3 +/- 0.3, respectively). These results indicated that taste buds within fungiform papilla play an important role in the detection of nutrients in the postnatal mouse.

Opioid system diversity in developing neurons, astroglia, and oligodendroglia in the subventricular zone and striatum: impact on gliogenesis in vivo.

Accumulating evidence, obtained largely in vitro, indicates that opioids regulate the genesis of neurons and glia and their precursors in the nervous system. Despite this evidence, few studies have assessed opioid receptor expression in identified cells within germinal zones or examined opioid effects on gliogenesis in vivo. To address this question, the role of opioids was explored in the subventricular zone (SVZ) and/or striatum of 2-5-day-old and/or adult ICR mice. The results showed that subpopulations of neurons, astrocytes, and oligodendrocytes in the SVZ and striatum differentially express mu-, delta-, and/or kappa-receptor immunoreactivity in a cell type-specific and developmentally regulated manner. In addition, DNA synthesis was assessed by examining 5-bromo-2'-deoxyuridine (BrdU) incorporation into glial and nonglial precursors. Morphine (a preferential mu-agonist) significantly decreased the number of BrdU-labeled GFAP(+) cells compared with controls or mice co-treated with naltrexone plus morphine. Alternatively, in S100beta(+) cells, morphine did not significantly decrease BrdU incorporation; however, significant differences were noted between mice treated with morphine and those treated with morphine plus naltrexone. Most cells were GFAP(-)/S100beta(-). When BrdU incorporation was assessed within the total population (glia and nonglia), morphine had no net effect, but naltrexone alone markedly increased BrdU incorporation. This finding suggests that DNA synthesis in GFAP(-)/S100beta(-) cells is tonically suppressed by endogenous opioids. Assuming that S100beta and GFAP, respectively, distinguish among younger and older astroglia, this implies that astroglial replication becomes increasingly sensitive to morphine during maturation, and suggests that opioids differentially regulate the development of distinct subpopulations of glia and glial precursors.

Alterations of tetrahydrobiopterin biosynthesis and pteridine levels in mouse tissues during growth and aging.

In the present study, we investigated age-related changes in pteridine levels and enzymatic activity responsible for tetrahydrobiopterin biosynthesis in mouse tissues. Until about 15 weeks after the birth, the remarkable change of tetrahydrobiopterin (BH4) was observed in all tissues tested. Between 20 and 50 weeks after the birth, pteridines levels were almost constant in all of the tissues. Total biopterin levels were decreased and levels of pterin and neopterin were increased in the period exceeding 50 weeks in all of the tissues. Activities of guanosine triphosphate (GTP) cyclohydrolase I, pyrvoyltetrahydropterin synthase, and the production of BH4 were recognized by specific biochemical assays, and we investigated the age-related changes in mouse tissues. The alteration of these enzymatic activities was indicated to be similar to that described in the change of pteridine levels.

Effects of Lentinus edodes, Grifola frondosa and Pleurotus ostreatus administration on cancer outbreak, and activities of macrophages and lymphocytes in mice treated with a carcinogen, N-butyl-N-butanolnitrosoamine.

ICR mice were treated with a carcinogen, N-butyl-N'-butanolnitrosoamine BBN), every day for 8 consecutive weeks and the effects of oral administration of edible mushrooms on the induction of urinary bladder carcinoma and on the activities of macrophages and lymphocytes were studied. Bladder carcinoma were found in all 10 mice (100%) treated with BBN alone, while we observed carcinoma only in 9 of 17 mice (52.9%), in 7 of 15 mice (46.7%) and 13 of 20 mice (65.0%) treated with Lentinus edodes, Grifola frondosa and Pleurotus ostreatus, respectively. Chemotactic activity of macrophages was suppressed in mice treated with BBN alone but maintained almost the normal level in mice treated with BBN plus Lentinus, Grifola or Pleurotus. Lymphocytes collected from mice treated with BBN plus each mushroom showed almost normal blastogenic response against concanavalin A, although those from mice treated with BBN alone completely retarded their response. Cytotoxic activity of lymphocytes against Yac-1 cells was also maintained at a normal level in mice treated with BBN plus each mushroom. Whereas in mice treated with BBN alone significant depression of NK cell activity occurred. Significantly higher cytotoxic activity against P-815 cells was observed in lymphocytes from mice treated with BBN plus each mushroom than that in lymphocytes from normal mice or mice treated with BBN alone.

Basal expression and induction of glutamate decarboxylase and GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus.

The excitatory, glutamatergic granule cells of the hippocampal dentate gyrus are presumed to play central roles in normal learning and memory, and in the genesis of spontaneous seizure discharges that originate within the temporal lobe. In localizing the two GABA-producing forms of glutamate decarboxylase (GAD65 and GAD67) in the normal hippocampus as a prelude to experimental epilepsy studies, we unexpectedly discovered that, in addition to its presence in hippocampal nonprincipal cells, GAD67-like immunoreactivity (LI) was present in the excitatory axons (the mossy fibers) of normal dentate granule cells of rats, mice, and the monkey Macaca nemestrina. Using improved immunocytochemical methods, we were also able to detect GABA-LI in normal granule cell somata and processes. Conversely, GAD65-LI was undetectable in normal granule cells. Perforant pathway stimulation for 24 hours, which evoked population spikes and epileptiform discharges in both dentate granule cells and hippocampal pyramidal neurons, induced GAD65-, GAD67-, and GABA-LI only in granule cells. Despite prolonged excitation, normally GAD- and GABA-negative dentate hilar neurons and hippocampal pyramidal cells remained immunonegative. Induced granule cell GAD65-, GAD67-, and GABA-LI remained elevated above control immunoreactivity for at least 4 days after the end of stimulation. Pre-embedding immunocytochemical electron microscopy confirmed that GAD67- and GABA-LI were induced selectively within granule cells; granule cell layer glia and endothelial cells were GAD- and GABA-immunonegative. In situ hybridization after stimulation revealed a similarly selective induction of GAD65 and GAD67 mRNA in dentate granule cells. Neurochemical analysis of the microdissected dentate gyrus and area CA1 determined whether changes in GAD- and GABA-LI reflect changes in the concentrations of chemically identified GAD and GABA. Stimulation for 24 hours increased GAD67 and GABA concentrations sixfold in the dentate gyrus, and decreased the concentrations of the GABA precursors glutamate and glutamine. No significant change in GAD65 concentration was detected in the microdissected dentate gyrus despite the induction of GAD65-LI. The concentrations of GAD65, GAD67, GABA, glutamate and glutamine in area CA1 were not significantly different from control concentrations. These results indicate that dentate granule cells normally contain two "fast-acting" amino acid neurotransmitters, one excitatory and one inhibitory, and may therefore produce both excitatory and inhibitory effects. Although the physiological role of granule cell GABA is unknown, the discovery of both basal and activity-dependent GAD and GABA expression in glutamatergic dentate granule cells may have fundamental implications for physiological plasticity presumed to underlie normal learning and memory. Furthermore, the induction of granule cell GAD and GABA by afferent excitation may constitute a mechanism by which epileptic seizures trigger compensatory interictal network inhibition or GABA-mediated neurotrophic effects.

Age-associated changes in tumor necrosis factor alpha receptor on peripheral blood monocytes and bone marrow macrophages from ICR mice and Wistar rats.

We examined the age-associated changes in tumor necrosis factor alpha (TNF alpha) receptor on peripheral blood monocytes and bone marrow macrophages obtained from slc:Wistar rats and slc:ICR mice. The TNF alpha receptor on monocytes and macrophages in both rats and mice showed high and low affinity sites. The Kd values of those sites gradually increased with age in both types of cells from both species. Binding capacities (receptor number) were low in 52-week-old animals. These results suggest that the TNF alpha receptor on monocytes and macrophages of these species might decrease with age in both quality and quantity.

Microvasculature in the mouse ovarian follicle demonstrated by a lectin angiography method.

A lectin angiography method was applied to identify ovarian vasculature in 32-34 days old mice treated with gonadotropins. Blood was washed out by perfusion until the inferior vena cava became translucent. Horseradish-peroxidase (HRP)-conjugated Concanavalin A (Con A) solution (10-15 ml) was perfused into the systemic circulation via the left ventricle at the rate of 2-3 ml/min. The animals were left for a 30 min reaction interval. The lumina of the blood vessels were flushed with 5-10 ml of phosphate buffered saline (pH 7.4). The ovaries were excised and fixed by immersion in 4% paraformaldehyde and sectioned serially at thickness of 100, 200 or 400 microm using a Microslicer. The binding of HRP-conjugated-Con A to endothelial cells was visualized by using 3,3'-diaminobenzidine-4 HCl (DAB-4 HCl) reaction. By examining various sections the three-dimensional architecture of the vascular networks of the preovulatory Graafian follicles and corpora lutea can be established. Capillary networks of preovulatory Graafian follicles were identified in sections of ovaries removed 11 h after hCG injection. Then, capillary networks of the Graafian follicles increased due to the hypertrophic growth of the theca interna and extension of capillary branches into the follicles. Well-developed capillary networks of corpora lutea were found in ovaries removed 24 h after hCG injection. For these observations the 200 microm-sections were the most useful. The present modified lectin angiography is a useful method for visualizing the microvasculature of mouse ovaries.

Expression of the metabotropic glutamate receptor mGluR1 alpha and the ionotropic glutamate receptor GluR1 in the brain during the postnatal development of normal mouse and in the cerebellum from mutant mice.

Expression of the metabotropic glutamate receptor type 1 alpha (mGluR1 alpha) and the non-N-methyl-D-aspartate (NMDA) ionotropic glutamate receptor type 1 (GluR1) in mouse brain was investigated using the antibodies raised against the synthetic peptides corresponding to their C-terminal amino acid sequences. Both receptor proteins are glycosylated predominantly in an asparagine-linked manner, and are abundant in post-synaptic membranes. We showed that mGluR1 alpha and GluR1 expression within the first 3 postnatal weeks undergoes dramatic changes in time and space, i.e., in the hippocampus and cerebellum. These spatio-temporal expression patterns appear to be correlated with the postnatal ontogenesis and establishment of the glutamatergic neurotransmission system in the hippocampus and cerebellum, cell migration, dendritic and axonal growth, spine formation, and synaptogenesis. In the adult cerebellum, mGluR1 alpha is intensely expressed in Purkinje neurons and GluR1 in Bergmann glial cells. Both receptors are expressed to a fair degree in weaver mutant cerebellum despite granule cell degeneration. However, the intrinsic expression levels of both mGluR1 alpha and GluR1 are markedly reduced in the cerebellum of the Purkinje cell-deficient and underdeveloped mutant mice, Purkinje-cell-degeneration, Lurcher, and staggerer, suggesting that GluR1 expression in Bergmann glia cells may be correlated with the sustained interaction with adjacent Purkinje neurons.

Beneficial effects of a calcium antagonist, nifedipine, on death and cardiovascular calcinosis induced by dietary magnesium deficiency in adult mice.

The effects of nifedipine on the survival rate and the cardiovascular calcinosis were investigated in dietary magnesium (Mg)-deficient mice. Male mice aged 8 to 10 weeks were fed with a diet which was either normal (0.07%) or deficient (0.007%) in Mg for 30 d. Nifedipine was added to the Mg-deficient diet for 30 d in a concentration of 1000 ppm. About 65% of Mg-deficient mice died within 30 d. In contrast, all the nifedipine-treated mice survived for 30 d. Mg-deficient mice showed hypomagnesaemia and hypocalcaemia which were not influenced by nifedipine treatment. Aortic and cardiac calcium contents increased in Mg-deficient mice, and these increments were normalized by nifedipine treatment. These results suggest that the life-saving effect of nifedipine in Mg-deficient mice might relate, at least in part, to the prevention of a deleterious increase in Ca in cardiovascular tissues.

Expression and modification of Hox 2.1 protein in mouse embryos.

A polyclonal antibody, alpha Hox 2.1a, has been generated and used to immunolocalize Hox 2.1 protein in mouse embryos. Protein is present in nuclei of all tissues previously shown to express Hox 2.1 RNA. In addition, protein is seen in somites and proximal regions of the limb buds, tissues in which Hox 2.1 RNA expression was not clearly detected previously by in situ hybridization. At the 7 somite stage, protein is detectable in the neural tube up to the level of somite 1, but later retracts to a more posterior position. Immunoblot, in vitro translation, and immunoprecipitation experiments were carried out to characterize the Hox 2.1 protein. The results show that the Hox 2.1 gene produces at least two related phosphorylated proteins present in different proportions in different tissues.

Response of lymphoid cells to thymic hormonal factors isolated from mouse thymus.

Thymic hormonal factors were isolated from mouse thymus by two methods. (1) Thymic cytosols in phosphate buffer saline were filtered through Sephadex G100 with 0.1 M NH4HCO3 (pH 8.0) as buffer and the protein peaks were collected. (2) Protein having thymosin activity (F5) was isolated from thymic cytosols after heat inactivation, salt fractionation and desalting on Sephadex G25. Molecular weights of all the proteins were determined on SDS-PAGE. Biological activity of thymic proteins was studied by in vitro and in vivo assays, using synthetic thymosin alpha 1 as the standard. Thymocytes treated with different thymic proteins showed maximum stimulation at 16 h of incubation period. Preincubation of the thymocytes with the thymic proteins and subsequent incubation with Con-A decreased the stimulation index. Incubation of spleen lymphocytes with thymic proteins increased the percentage of Tdt+ cells. The antitumor effects of thymic proteins carried out on animals having leukemia, showed statistically significant results. Clinically however, the antitumor effects of the thymic proteins alone and in combination chemotherapy were negligible at 1 mg/kg body weight dose level.