Tyrosinase Activity (TH,DO, PAGE-Defined Isozymes) and Melanin Production in Regenerating Hairbulb Melanocytes of Lethal Yellow (Ay/a), Black (a/a), Agouti (AwJ/AwJ) and Albino (a/a/c2J/c2J) Mice (C57BL/6J)

We compared tyrosinase activity (TH, DO, and native PAGE-defined isozymes) and melanin production in participate and soluble fractions of hairbulb melanocytes of lethal yellow (A^y/a C/C), nonagouti black (a/a C/C), and albino (a/a c^2J/c^2J) of 3-, 6-, 9-, and 12-day regenerating hairbulbs. With respect to tyrosine hydroxylase (TH) and dopa oxidase (DO) activities, A^y/a melanocytes possessed only 25-35% of the activity of a/a; there were no genotype differences in either the subcellular distribution of activity in soluble and particulate fractions or in the relative increases of activity over the 12-day developmental period. TH data on wild-type agouti (A^wJ/A^wJ) mice over the 3-11 day regeneration interval showed an activity intermediate between that of a/a and A^y/a; the rate of TH increase reflected black and yellow phases of the agouti hair cycle. Analyses of the number and densities of dopa-sensitive bands following native PAGE of 3-, 6-, 9-, and 12-day hairbulb fractions of a/a and A^y/a mice suggested stage-dependent patterns. A comparison of rates and amounts of melanin production in 3-, 6-, 9-, and 12-day fractions showed consistent melanin production in A^y/a to be 10-20% that of a/a; however, fold increases in melanin production over the four stages were similar between genotypes. Overall, tyrosinase activity data support the notion that agouti locus modification of tyrosinase activity is a graded or quantitative rather than a qualitative phenomenon.     

Differentiation of hairbulb pigment cell melanosomes in compound agouti and albino locus mouse mutants (Ay, a, c2J; C57BL/6J)

Our objective was to determine using electron microscopy how nonagouti (a), lethal yellow (Ay), and albino (c2J) genes affect the program of mouse hairbulb melanosome differentiation; 1,921 hairbulb melanosomes from four genotypes (a/a C/C = B,Ay/a C/C = Y, a/a c2J/c2J = BA, and Ay/a c2J/c2J = YA) were scored for developmental stage, length, and width. Qualitative and quantitative electron microscopy revealed the following. An albino locus-induced diminution of melanosome size suggests that the albino locus is involved in structural features of melanosomes not directly related to the synthesis and deployment of tyrosinase. Ratio data on melanosome length-to-width confirm that the agouti locus determines melanosome shape, either spherical or elliptical; melanization is not required for melanosomes to achieve their agouti-locus-determined shapes. YA (Ay/a c2J/c2J) melanosomes, characterized by poorly organized matrices, absence of active tyrosinase, unusually large membrane invaginations, and significantly smaller dimensions than those of BA (a/a c2J/c2J), showed additive effects of both Ay and c2J alleles. These data suggest that the albino locus plays a structural as well as functional (tyrosinase) role in the differentiation of mouse hairbulb melanosomes. The agouti locus, even in the absence of melanization, directs melanosome shape either via synthesis and deployment of agouti-locus-encoded matrix proteins or by other structural factors. The additive effects of Ay and c2J alleles in compound YA mutants document the importance of specific interactions both functional and structural between agouti and albino loci.     

Progressive infertility in female lethal yellow mice (Ay/a; strain C57BL/6J)

Obese Ay/a females of 120 days or older, when compared to age-matched a/a controls (strain C57BL/6J), exhibited abnormal oestrous cyclicity characterized by reduced frequencies of true oestrous-stage smears, decreased mating success to proven a/a males, lowered uterine weights, and depressed ovulation rates. Exogenous gonadotrophins (PMSG/hCG) partly restored ovulation in obese Ay/a females to near control levels, demonstrating the sensitivity of Ay/a ovarian tissues to FSH and LH, at least at superovulatory levels. Concentrations of endogenous gonadotrophins and/or sensitivity of ovarian target cells to gonadotrophins may therefore be impaired in obese Ay/a females. Aberrant copulatory behaviour, reduced uterine weights, and depressed conception rates strongly suggest ovarian steroid deficiencies, perhaps secondary effects of reduced endogenous gonadotrophin activity. As in other obese rodent syndromes e.g. ob/ob, db/db, and fa/fa), a possible fundamental Ay-induced hypothalamic lesion is consistent with our data.     

Effects of reciprocal ovary transplantation on reproductive performance of lethal yellow mice (Ay/a; C57BL/6J)

This study was conducted to determine whether reproductive failures in ageing, obese lethal yellow (Ay/a) females are due primarily to defects within Ay/a ovaries or to systemic defects which may operate outside the ovaries. Reciprocal ovary transplantation between control (a/a) and lethal yellow (Ay/a) females provided an experimental system to test the reproductive potential of not only Ay/a ovaries in control (a/a) females but also control (a/a) ovaries in mutant (Ay/a) females. Results on reproductive performance of all four combinations of grafts between Ay/a and a/a mice proved that Ay-induced reproductive failures are not due to intrinsic ovarian lesions but rather to defects operating extrinsically to the ovary. The hypothalamo-pituitary axis is a likely site for this reproductive lesion.     

GABA(A) receptor beta(2) subunit mRNA content is differentially regulated in ethanol-dependent DBA/2J and C57BL/6J mice

Chronic ethanol treatment is known to alter gene expression and function of γ-aminobutyric acid type-A (GABA_A) receptors. Here we focus on the β₂ subunit which is widely expressed in the mammalian brain, and plays a key role in the GABA binding site. Previous studies using rodent models of ethanol dependence show either increased or no change of β₂ subunit mRNA and peptide content following chronic ethanol administration. In humans, polymorphism at the β₂ subunit is associated with ethanol dependence in some, but not all, populations. In the present study we measured mRNA content in the cerebellum and cerebral cortex using ethanol-naive and ethanol-dependent DBA/2J and C57BL/6J mice. The DBA/2J strain displays severe ethanol withdrawal severity, while the C57BL/6J strain shows milder withdrawal reactions. RNase protection analysis demonstrated that the DBA/2J strain is more sensitive to ethanol-induced increases in β₂ subunit mRNA content in the cerebellum, showing significant increases at lower blood ethanol concentrations than C57BL/6J mice. The ethanol-induced regulation in C57BL/6J mice appears to be more complex, with decreases in β₂ subunit mRNA content at low blood ethanol concentrations, and increases at higher concentrations. These data suggest that differences between C57BL/6J and DBA/2J mice in the degree of physical dependence (withdrawal) on ethanol may be related to differential sensitivity to ethanol regulation of β₂ subunit expression.     

Differential expression and regulation of myristoylated alanine-rich C kinase substrate (MARCKS) in the hippocampus of C57/BL6J and DBA/2J mice

The myristoylated alanine-rich C kinase substrate (MARCKS) is a major protein kinase C (PKC) substrate in brain that binds the inner surface of the plasma membrane, calmodulin, and cross-links filamentous actin, all in a PKC phosphorylation-reversible manner. MARCKS has been implicated in hippocampal-dependent learning and long-term potentiation (LTP). Previous studies have shown DBA/2 mice to exhibit poor spatial/contextual learning, impaired hippocampal LTP, and hippocampal mossy fiber hypoplasia, as well as reduced hippocampal PKC activity and expression relative to C57BL/6 mice. In the present study, we assessed the expression (mRNA and protein) and subcellular distribution (membrane and cytolsol) of MARCKS in the hippocampus and frontal cortex of C57BL/6 and DBA/2 mice using quantitative western blotting. In the hippocampus, total MARCKS mRNA and protein levels in C57BL/6J mice were significantly lower ( approximately 45%) compared with DBA/2J mice, and MARCKS protein was observed predominantly in the cytosolic fraction. MARCKS expression in frontal cortex did not differ significantly between strains. To examine the dynamic regulation of MARCKS subcellular distribution, mice from each strain were subjected to 60 min restraint stress and MARCKS subcellular distribution was determined 24 h later. Restraint stress resulted in a significant reduction in membrane MARCKS expression in C57BL/6J hippocampus but not in the DBA/2J hippocampus despite similar stress-induced increases in serum corticosterone. Restraint stress did not affect cytosolic or total MARCKS levels in either strain. Similarly, restraint stress (30 min) in rats also induced a significant reduction in membrane MARCKS, but not total or cytosolic MARCKS, in the hippocampus but not in frontal cortex. In rats, chronic lithium treatment prior to stress exposure reduced hippocampal MARCKS expression but did not affect the stress-induced reduction in membrane MARCKS. Collectively these data demonstrate higher resting levels of MARCKS in the hippocampus of DBA/2J mice compared to C57BL/6J mice, and that acute stress leads to a long-term reduction in membrane MARCKS expression in C57BL/6J mice and rats but not in DBA/2J mice. These strain differences in hippocampal MARCKS expression and subcellular translocation following stress may contribute to the differences in behaviors requiring hippocampal plasticity observed between these strains.     

TNF-TNFR2 interactions are critical for the development of intestinal graft-versus-host disease in MHC class II-disparate (C57BL/6J-->C57BL/6J x bm12)F1 mice

TNF-TNFR2 interactions promote MHC class II-stimulated alloresponses while TNF-TNFR1 interactions promote MHC class I-stimulated alloresponses. The present studies were designed to evaluate whether TNF-TNFR2 interactions were involved in the in vivo generation of CD4(+) T cell-mediated intestinal graft-versus-host disease (GVHD) in the (C57BL/6J (hereafter called B6) --> B6 x B6.C-H-2(bm12) (bm12))F(1) GVHD model. Briefly, 5 x 10(6) splenic CD4(+) T lymphocytes from B6.TNFR2(-/-) or control B6 mice were transferred with 1--2 x 10(6) T cell-depleted B6 bone marrow cells (BMC) to irradiated MHC class II-disparate (bm12 x B6)F(1) mice. Weight loss, intestinal inflammation, and the surface expression of CD45RB (memory marker) on intestinal and splenic lymphocytes were assessed. IL-2 and IFN-alpha mRNA levels in intestinal lymphocytes were assessed by nuclease protection assays. A significant reduction in weight loss and intestinal inflammation was observed in recipients of the TNFR2(-/-)CD4(+) SpC. Similarly, a significant decrease was noted in T cell numbers and in CD45RB(low) (activated/memory) expression on intestinal but not CD4(+) T cells in recipients of TNFR2(-/-)CD4(+) spleen cells. IL-2 and IFN-alpha mRNA levels were reduced in the intestine in the recipients of TNFR2(-/-) splenic CD4(+) T cells. These results indicate that TNF-TNFR2 interactions are important for the development of intestinal inflammation and activation/differentiation of Th1 cytokine responses by intestinal lymphocytes in MHC class II-disparate GVHD while playing an insignificant role in donor T cell activation in the spleen.     

Anti-obesity and anti-diabetic effects of Yerba Mate (Ilex paraguariensis) in C57BL/6J mice fed a high-fat diet

Yerba Mate, derived from the leaves of the tree, Ilex paraguariensis, is widely-used as a tea or as an ingredient in formulated foods. The aim of the present study was to evaluate the effects of Yerba Mate extract on weight loss, obesity-related biochemical parameters, and diabetes in high-fat diet-fed mice.To this end, by using in vivo animal models of dietary-induced obesity, we have made the interesting observations that Yerba Mate has the ability to decrease the differentiation of pre-adipocytes and to reduce the accumulation of lipids in adipocytes, both of which contribute to a lower growth rate of adipose tissue, lower body weight gain, and obesity. Our data from in vivo studies revealed that Yerba Mate treatment affects food intake, resulting in higher energy expenditure, likely as a result of higher basal metabolism in Yerba Mate-treated mice. Furthermore, in vivo effects of Yerba Mate on lipid metabolism included reductions in serum cholesterol, serum triglycerides, and glucose concentrations in mice that were fed a high fat diet. In conclusion, Yerba Mate can potentially be used to treat obesity and diabetes.     

Neuroprotective effect and brain receptor binding of taltirelin,a novel thyrotropin-releasing hormone (TRH) analogue,in transient forebrain ischemia of C57BL/6J mice

Thyrotropin-releasing hormone (TRH) and some of its stable analogues have been shown to improve neurologic dysfunctions such as brain trauma in both animals and humans. Our previous study revealed that taltirelin, a novel orally active TRH analogue, binds to rat brain TRH receptors in vivo. The present study was undertaken to investigate whether taltirelin has neuroprotective effects in transient brain ischemia of C57BL/6J mice induced by bilateral carotid artery occlusion (2VO). Neuronal cell density in the hippocampal CA1 region of C57BL/6J mice was significantly (39.9%) decreased 1 week after 2VO-reperfusion, compared to the case of the sham group, and this reduction of hippocampal neuronal density was significantly suppressed by an intravenous (i.v.) injection of taltirelin (0.3 mg/kg). The i.v. injection of taltirelin at this dosage produced a significant increase in the dissociation constant (Kd) of specific [3H]MeTRH binding in sham and 2VO-reperfusion groups (33.6 and 51.4%, respectively) compared with the vehicle-treated group. These results indicate that the intravenously injected taltirelin bound to TRH receptors in the ischemic brain. There was little difference in the brain-to-plasma concentration ratio (Kp) of [14C]sucrose between the sham and 2VO groups of C57BL/6J mice, indicating that the tight junction of the blood-brain barrier may be intact in the ischemic brain.In conclusion, the study has shown that taltirelin may have a significant neuroprotective effect on the ischemic brain.     

PF 9601N [N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine], a new MAO-B inhibitor,attenuates MPTP-induced depletion of striatal dopamine levels in C57/BL6 mice

Monoamine oxidase isoform B (MAO-B) is involved in Parkinson's disease (PD) induced by the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxin (MPTP) in human and non-human-primate. MAO-B inhibitors, such as L-deprenyl have shown to prevent against MPTP-toxicity in different species, and it has been used in Parkinson therapy, however, the fact that it is metabolized to (-)-methamphetamine and (-)-amphetamine highlights the need to find out new MAO-B inhibitors without a structural amphetaminic moiety. In this context we herein report, for the first time, anywhere a novel non-amphetamine-like MAO-B inhibitor, PF 9601N, N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine. This attenuates the MPTP-induced striatal dopamine depletion in young-adult and adult-old C57/BL mice, using different schedules of administration, and which behave "ex vivo" as a slightly more potent and selective MAO-B inhibitor than L-deprenyl, assayed for comparative purposes in the same experimental conditions. The MAO-B ID(50) values were calculated from the total MAO-B activity measured against [14C] phenylethylamine (22 microM) as substrate, at each inhibitor concentration. The MAO-B ID(50) values resulted to be 381 and 577 nmol/kg for PF 9601N and L-deprenyl, respectively. The intraperitoneally (i.p.) co-administration to young-adult C57/BL6 mice of MPTP (30 mg/kg), with different concentrations of PF 9601N or L-deprenyl (29.5-0.357 micromol/kg) showed a dose-dependent protective effect against striatal dopamine depletion, measuring the dopamine contents and its metabolites by HPLC. The ED(50) value proved to be 3.07 micromol/kg without any significant differences between either MAO-B inhibitor. Nevertheless, lower doses of PF 9601N (1.5 micromol/kg) were necessary to get almost total protection, without any change in the DOPAC and HVA content, when administered 2 h before MPTP (30 mg/kg), whereas partial protection (45%) against dopamine depletion was observed in the case of L-deprenyl. In both cases, MAO-B inhibition was a necessary condition in order to observe the protective effect. When adult-old (8-10 months) C57/BL6 mice were used, MPTP (25 mg/kg) administration induced 25 days later, an irreversible dopamine depletion. In these conditions, chronic administration with 0.15 micromol/kg of PF 9601N, before the toxin, every 24 h for 10 days, rendered almost total protection of dopamine depletion, whereas L-deprenyl yielded only 50% protection of the dopamine content, assayed in the same conditions. It is worth remarking, that in both cases MAO-B was not affected. From these results, it can be concluded that PF 9601N attenuates MPTP neurotoxicity "in vivo" better than L-deprenyl through different mechanisms, with special relevance to the protective effect, independent of MAO-B inhibition, observed in the irreversibly MPTP-lesioned adult-old mice. Therefore, this novel non-amphetamine MAO-B inhibitor could be potentially effective in PD therapy.     

Leptin Receptor (Lepr) Is a Negative Modulator of Bone Mechanosensitivity and Genetic Variations in Lepr May Contribute to the Differential Osteogenic Response to Mechanical Stimulation in the C57BL/6J and C3H/HeJ Pair of Mouse Strains

This study investigated the role of leptin receptor (Lepr) signaling in determining the bone mechanosensitivity and also evaluated whether differences in the Lepr signaling may contribute to the differential osteogenic response of the C57BL/6J (B6) and C3H/HeJ (C3H) pair of mouse strains to mechanical stimuli. This study shows that a loading strain of ∼2,500 μϵ, which was insufficient to produce a bone formation response in B6 mice, significantly increased bone formation parameters in leptin-deficient ob⁻/ob⁻ mice and that a loading strain of ∼3,000 μϵ also yielded greater osteogenic responses in Lepr-deficient db⁻/db⁻ mice than in wild-type littermates. In vitro, a 30-min steady shear stress increased [³H]thymidine incorporation and Erk1/2 phosphorylation in ob⁻/ob⁻ osteoblasts and db⁻/db⁻ osteoblasts much greater than those in corresponding wild-type osteoblasts. The siRNA-mediated suppression of Lepr expression in B6 osteoblasts enhanced (but in osteoblasts of C3H (the mouse strain with poor bone mechanosensitivity) restored) their anabolic responses to shear stress. The Lepr signaling (leptin-induced Jak2/Stat3 phosphorylation) in C3H osteoblasts was higher than that in B6 osteoblasts. One of the three single nucleotide polymorphisms in the C3H Lepr coding region yielded an I359V substitution near the leptin binding region, suggesting that genetic variation of Lepr may contribute to a dysfunctional Lepr signaling in C3H osteoblasts. In conclusion, Lepr signaling is a negative modulator of bone mechanosensitivity. Genetic variations in Lepr, which result in a dysfunctional Lepr signaling in C3H mice, may contribute to the poor osteogenic response to loading in C3H mice.