In vivo and in vitro effects of LH-RH on cAMP content of anterior pituitary in rats.

Changes in the cAMP content of anterior pituitary of male rats were studied after in vivo administration of LH-RH or in vitro incubation in the presence of LH-RH. Although a significant rise was observed 5 min after in vivo injection of LH-RH, the cAMP level increased after only a two-hour lag period in vitro. Maximum level was reached in vivo with 1 microgram LH-RH/100 g b.w., but there was no maximal production in vitro even in the presence of 5 x 10(-6) M LH-RH. A summary of the available observations on the effect of LH-RH on pituitary cAMP level is given in the present paper.     

Effects of glucocorticoids and catecholamines on maturing rat heart

1. The negative force-frequency response of normal rat heart was accentuated when the animals were adrenalectomized. Treatment of adrenalectomized animals with dexamethasone restored the normal force-frequency response. 2. Total adrenalectomy increased the sensitivity of rat heart to calcium. 3. Adrenalectomized-dexamethasone-treated hearts were more responsive to epinephrine and ouabain. 4. Total adrenalectomy caused independent myocardial disturbances in calcium handling elements (glucocorticoid effect) and beta receptors (catecholamine effect).     

The effects of pregnancy on the mouse thymic epithelium

Changes in the murine thymus during pregnancy were studied using immunocytochemistry with monoclonal antibodies against thymic epithelial, neuroendocrine, and thymulin-producing cells, fibroblasts, blood vessels and connective tissue components. Extensive alterations occur in mid-pregnancy. The medulla was greatly enlarged in the involuted thymus, and there were greater numbers of epithelial cells. These epithelial cells had an altered distribution forming large structures surrounding spherical masses of mononulear cells, lacked epithelial cells and often contained a central blood vessel with fibroblasts and connective tissue. We have called these structures medullary epithelial rings (MERs). To our knowledge these structures have not been described before. Late in pregnancy the loss of the central mononuclear cells leaves collapsed structures in a smaller medulla that nevertheless retains many epithelial cells. In virgins and early-pregnancy, there are cortical channels free of epithelial cells that are very infrequent later in pregnancy. This may reflect the loss of steroid-sensitive thymocytes from the cortex. The influence of sex-steroids neurological impulses and immune activity in causing the changes are discussed, as are the possible consequences in pregnancy of a reduced, thymocyte-depleted cortex and an enlarged medulla that shows great complexity and activity.     

In vivo effects of lipopolysaccharide on lymphoid and non-lymphoid cells in the mouse spleen

Summary In the present study the effects of lipopolysaccharide (LPS) on the cellular composition and phagocytosis of India ink in the inner parts of the periarteriolar lymphocyte sheaths (PALS) are described.Staining for B-, T-lymphocytes, and reticulin fibers in the spleen of normal and LPS-injected mice shows that the B-dependent follicular area is increased in size after LPS administration. However, the number of T-lymphocytes in the inner PALS is reduced markedly and a relatively high number of B-lymphocytes can be found in this area. The significance of this phenomenon is discussed.In untreated mouse spleen, carbon particles become localized in strongly acid-phosphatase (AP)-positive macrophages of the red pulp, marginal zone and white pulp 24 h after an intravenous injection of India ink. All these macrophages contain numerous carbon particles. After LPS pretreatment, the phagocytosis of carbon particles in the inner PALS is dramatically diminished, although many strongly AP-positive macrophages can be found in this area. The phagocytosis of carbon particles in the other compartments of the spleen did not change. It appears that injection of 2 g LPS or more is sufficient to induce this phenomenon which is most significant when LPS is injected 24 or 48 h before exposure to India ink.Abbreviations LPS lipopolysaccharide - PALS periarteriolar lymphocyte sheath - AP acid phosphatase - IDC interdigitating cells     

The influence of glucocorticoids and catecholamines on the neuromuscular transmission

The review surveys the impact of “stress hormones”–glucocorticoids and catecholamines–on the functioning of the neuromuscular synapse. The review brings together the data on the influence of the main agents of stress–cortisol and norepinephrine–on the intensity and timing of the acetylcholine release, as well as signaling effect of its co-mediator ATP.     

In vivo effects of epidermal and fibroblast growth factors on DNA replication in mouse skin

A method was developed for measuring in vivo DNA synthesis after exposure to epidermal growth factor (EGF) or fibroblast growth factor (FGF) in a local area of mouse skin using ring-shaped forceps in combination with autoradiography. The technique should be useful for analysing the effects of growth factors on individual cells of the skin in vivo. EGF induced semisynchronized DNA synthesis in basal cells of the epidermis dose-dependently, but FGF did not. Time course study showed that EGF-induced DNA synthesis in basal cells increased with time for 24 h, and then decreased rapidly. EGF-induced DNA synthesis in basal cells was proportional to the time exposed to EGF (0-60 min). FGF and EGF both had little effect on dermal fibroblastic cells. The discrepancy between in vivo observations and those with cultured mammalian cells is discussed.     

In vivo and in vitro inhibition of JE gene expression by glucocorticoids

Glucocorticoids are potent anti-inflammatory agents which affect cell growth and migration in a wide variety of systems and have profound effects on monocytes, decreasing their circulating number as well as inhibiting their accumulation at sites of inflammation and injury. Although the mechanisms by which glucocorticoids regulate gene induction have been established, the mechanisms by which they inhibit inflammation or cell growth and migration have yet to been determined. JE is one of the most abundant genes induced by platelet-derived growth factor (PDGF) in vitro and is also induced in vivo in response to ischemia or injury. JE encodes a low molecular weight glycoprotein that functions in part as a monocyte chemotactic factor and thus may be important in recruiting monocytes to sites of tissue injury and/or inflammation. We report that glucocorticoids block the induction of JE mRNA by serum or PDGF in cultured vascular smooth muscle cells. The effect of glucocorticoids appears largely due to destabilization of JE mRNA and has specificity for JE, in that other "early" PDGF-inducible genes are not inhibited by glucocorticoids. The effect of glucocorticoids also occurs in vivo: methyl prednisolone blocks the constitutive expression and inhibits the ischemia-induced elevation of JE mRNA levels in rat kidneys. The inhibition of JE mRNA accumulation by glucocorticoids may be related to the anti-inflammatory effects of these agents and defines JE as a member of what may be a group of PDGF-inducible genes that are responsive to corticosteroids.     

In vivo effects of arsenite on meiosis, preimplantation development, and apoptosis in the mouse

Inorganic arsenic, an environmental contaminant, produces a variety of stress responses in mammalian cells, including metabolic abnormalities accompanied by growth inhibition and carcinogenesis. Much of the toxicity of arsenic is known to stem from its uncoupling effects on mitochondria. Because previously we had shown that mitochondrial dysfunction can disrupt oocyte and embryo development, we investigated effects of arsenite on meiotic progression and early embryo development in mice. Six-week-old CD-1 mice were treated with 0 (solvent as control), 8 mg/kg (a dose previously established in mice as the maternal no-observed-adverse-effect level), and 16 mg/kg doses of sodium arsenite every 2 days for a total of seven i.p. injections ver a period of 14 days. The incidence of meiotic anomalies, characterized by spindle disruption and/or chromosomal misalignment, was significantly increased in arsenite-treated groups (25% after 8 mg/kg and 62.5% after 16 mg/kg), compared to normal metaphase II in control oocytes. Further, arsenite treatment significantly decreased cleavage rates of zygotes at 24 h, morula formation at 72 h, and development to blastocysts at 96 h in a dose-dependent manner. The total cell number in developed blastocysts did not differ significantly between the 8 mg/kg arsenite treatment and control groups, but was significantly reduced in the 16 mg/kg arsenite treatment group. Moreover, the percentage of apoptotic nuclei was significantly increased in blastocysts following 16 mg/kg arsenite treatment. These data suggest that arsenite causes meiotic aberrations, which may contribute to decreased cleavage and preimplantation development, as well as increased apoptosis.     

Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturationin vivo

Thymic medullary type epithelial cell line (MTEC1), which expressed H-2D^d and Ia^d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H-2^b mice reconstituted with H-2^bxd F1bone marrow cells. Two months later, the injected MTEC1 cells were found to be still present in the recipient thymus. Splenocytes from chimeric mice, inin vitro functional assays, were analyzed to investigate whether the MTEC1 cellsin vivo could induce the production of H-2^d restricted antigen-specific T cells. The H-2^d restricted VSV-antigen specific proliferating and IL-2 producing T cells as well as H-2^d restricted influenza virus specific cytotoxic T cells were found in chimeric mice injected with MTEC1 cells, and these cells were shown to be tolerant to H-2^d selfantigen. On the contrary, H-2^d restricted antigen-specific and H-2^d self-antigen tolerant T cells were not shown in control mice injected with saline. These results suggest that intrathymically injected MTEC1 cells could induce T lineage cell development and functional maturation in the intact thymus. A hypothesis of “second thymic selection” in thymic medulla has been postulated and its implication discussed.     

Effects of catecholamines on early development of the chick embryo: relationship to effects of calcium and cAMP

Catecholamines (dopamine or norepinephrine) injected under the blastoderm of the unincubated chick embryo produced a thickened primitive streak and prevented the migration of axial mesoblast after 24 h. The mesoblastic cells that accumulated in the primitive streak contained many intracytoplasmic yolk granules. After 48 h, neural tube, notochord, and somites were severely affected, and their cells appeared loaded with yolk inclusions. Heart, lateral plates, blood cells, and blood vessels differentiated normally. At the onset of gastrulation, the level of glycogen was fivefold lower in catecholamine-treated embryos than in control embryos. Injection of glucose plus dopamine, at equimolar concentrations resulted in normal development both at 24 h and at 48 h. Because adrenergic stimulation of glycogenolysis in differentiated cells is usually mediated by cAMP and/or by calcium, we attempted to determine whether these substances could reproduce the effects of catecholamines. Only calcium was able to produce, to a limited extent, the same morphogenetic disturbances as those produced by catecholamines, whereas the chelating agent, ethylenediamine tetraacetic acid, when administered with dopamine, partially inhibited the effects of catecholamines. An increase in the number of yolk granules was the only common finding among embryos treated with cAMP and catecholamines. Blood and a well differentiated, gastrular endoderm always developed, independently of the nature of the substance with which the embryos had been treated. Morphogenetic disturbances caused by exogenous catecholamines could be due to depletion of glucose. Alternatively, a different metabolic commitment might exist within the diverse populations of cells that constitute the mesoblastic layer.     

Immunologic effects of neonatal infection with mouse thymic virus.

Mouse thymic virus is a herpesvirus that causes extensive thymic necrosis when given to newborn mice. During the time of acute infection spleen cells have markedly diminished reactivity to T cell phytomitogens and to allogeneic cells and are incapable of effecting a primary in vitro response to a "T-dependent" antigen; responses to B cell mitogens and to a T-independent antigen are unimpaired. Spleens from acutely infected mice have low theta antigen normal numbers of immunoglobulin-bearing cells. Surprisingly, despite widespread necrosis and cellular depletion, thymic cell reactivity to mitogens is unimpaired. However, the ability to thymocytes to proliferate and to generate cytotoxic killer cells in response to allogeneic cells is diminished.     

In vivo and in vitro stimulatory effects of Cordyceps sinensis on testosterone production in mouse Leydig cells

The in vivo and in vitro effects of Cordyceps sinensis (CS) and its extracted fractions on the secretion of testosterone in mice were studied. CS, F2 (water soluble protein), and F3 (poorly water soluble polysaccharide and protein) significantly stimulated in vitro testosterone production in purified mouse Leydig cells. However, F1 (water soluble polysaccharide) had no effect (p>0.05). F2 and F3 stimulated in vitro testosterone production in dose- and time-dependent relationships with maximal responses at 3 mg/ml for 3 h (p<0.05). An in vivo study illustrated that testosterone levels in plasma were significantly increased by CS, F2, and F3, respectively (p<0.05). Because CS, F2, and F3 stimulated both in vitro and in vivo testosterone secretions in mice, it is possible that CS might contribute to an alternative medicine for the treatment of some reproductive problems caused by insufficient testosterone levels in human males.     

In vitro and in vivo effects of naringin on cytochrome P450-dependent monooxygenase in mouse liver

In vitro and in vivo effects of naringin on microsomal monooxygenase were studied to evaluate the drug interaction of this flavonoid. In vitro addition of naringin up to 500 microM had no effects on benzo(a)pyrene hydroxylase (AHH) activity of mouse liver microsomes. In contrast, the aglycone naringenin at 300 to 500 microM decreased AHH activity by 50% to 60%. Analysis of Lineweaver-Burk and Dixon plots indicated that naringenin competitively inhibited AHH activity with an estimated Ki of 39 microM. Naringenin at 100 microM also reduced metabolic activation of benzo(a)pyrene to genotoxic products as monitored by umuC gene expression response in Salmonella typhimurium TA1535/pSK1002. In the presence of equimolar naringenin and benzo(a)pyrene, umuC gene expression presented as beta-galactosidase activity was reduced to a level similar to the control value. Administration of a liquid diet containing 10 mg/ml naringin for 7 days caused 38% and 49% decreases of AHH and 7-methoxyresorufin O-demethylase activities, respectively. In contrast, the administration had no effects on cytochrome P450 (P450)-catalyzed oxidations of 7-ethoxyresorufin, 7-ethoxycoumarin, N-nitrosodimethylamine, nifedipine, erythromycin and testosterone. Microsomal P450 and cytochrome b5 contents and NADPH-P450 reductase activity were not affected. Immunoblot analysis using MAb 1-7-1, which immunoreacted with both P450 1A1 and 1A2, revealed that the level of P450 1A2 protein was decreased by 38%. These results demonstrate that naringenin is a potent inhibitor of AHH activity in vitro and naringin reduces the P450 1A2 protein level in vivo. These effects may indicate a chemopreventive role of naringin against protoxicants activated by P450 1A2.     

Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturation in vivo

Thymic medullary type epithelial cell line (MTEC1), which expressed H-2D^d and Ia^d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H-2^b mice reconstituted with H-2^bxd F1bone marrow cells. Two months later, the injected MTEC1 cells were found to be still present in the recipient thymus. Splenocytes from chimeric mice, inin vitro functional assays, were analyzed to investigate whether the MTEC1 cellsin vivo could induce the production of H-2^d restricted antigen-specific T cells. The H-2^d restricted VSV-antigen specific proliferating and IL-2 producing T cells as well as H-2^d restricted influenza virus specific cytotoxic T cells were found in chimeric mice injected with MTEC1 cells, and these cells were shown to be tolerant to H-2^d selfantigen. On the contrary, H-2^d restricted antigen-specific and H-2^d self-antigen tolerant T cells were not shown in control mice injected with saline. These results suggest that intrathymically injected MTEC1 cells could induce T lineage cell development and functional maturation in the intact thymus. A hypothesis of “second thymic selection” in thymic medulla has been postulated and its implication discussed. Project supported by the National Natural Science Foundation of China (Grant No. 39230320).     

活体电转化技术在新生小鼠视网膜中的应用

活体电转化技术是在高电压的脉冲作用下,瞬态增加细胞膜的渗透性从而将外源基因高效导入细胞的方法。与病毒载体等其他方法相比,活体电转化技术具有安全、高效、快速、稳定及应用范围广等优点,近年来在很多组织和器官中得到广泛使用,包括在眼科研究领域。文章介绍了活体电转化技术在新生小鼠视网膜中的应用,通过新生小鼠视网膜下注射的方法,经几次高电压的脉冲,将高浓度的绿色荧光蛋白表达质粒导入新生小鼠视网膜细胞内。通过冰冻切片观察绿色荧光蛋白在视网膜中的表达。结果表明绿色荧光蛋白在视网膜外核层高表达,证实了活体电转化技术可以将外源基因高效、快捷的导入视网膜,从而为研究视网膜发育及功能提供一种有效的手段。