Estrogen influences dolichyl phosphate distribution among glycolipid pools in mouse uteri

The steroid hormone 17 beta-estradiol dramatically induces uterine N-linked glycoprotein assembly [Dutt, A., Tang, J.-P., Welply, J. K., & Carson, D. D. (1986) Endocrinology (Baltimore) 118, 661-673]. To determine the role that dolichyl phosphate availability plays in this induction, we studied the effects of estrogen priming on the content of dolichyl phosphate and the distribution of dolichyl phosphate among various glycolipids in uteri. Dolichol-linked saccharides were metabolically labeled to equilibrium with either [3H]glucosamine or [3H]mannose and extracted from primary explants of uterine tissue. The amount of dolichol-linked saccharide was calculated from the specific radioactivity determined for the corresponding sugar nucleotides extracted from the tissues. The major dolichol-linked saccharides identified were mannosylphosphoryldolichol (MPD), oligosaccharylpyrophosphoryldolichol (OSL), and N,N'-diacetylchitobiosylpyrophosphoryldolichol (CBL). Estrogen increased the levels of MPD and OSL 4-fold; however, CBL levels did not change. After 3 days of treatment, the levels of these glycolipids were very similar to those in uteri from pregnant mice. Remarkably, MPD constituted 90-95% of dolichol-linked saccharides detected under all conditions. The tissue contents of total dolichyl phosphate and alkali-labile dolichyl phosphate, presumably MPD, were estimated by liquid chromatography. The levels of alkali-labile dolichyl phosphate determined in this way were in good agreement with the values estimated for MPD by metabolic labeling; moreover, alkali-labile dolichyl phosphate constituted 50-98% of the total dolichyl phosphate pool. The variations in MPD content depended upon the steroid hormone influence, most notably that of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cathepsin D stimulates DNA synthesis and mitosis in mouse liver in vivo

Effects of a single intraperitoneal injection of cathepsin D (CatD) on DNA synthesis and mitosis in the mouse liver and kidney were investigated. Twenty micrograms of catD induced a significant stimulation of DNA synthesis in the liver, but not in the kidney, in a dose-dependent fashion and with a peak activity at 38 h after the injection. CatD also stimulated liver mitosis, with a peak value at 44 h after the injection.     

Enzyme-catalyzed oligosaccharide synthesis.

Cell-surface carbohydrates and their conjugates are involved in many types of molecular recognition. This review describes recent developments in enzyme-catalyzed oligosaccharide synthesis, with particular focus on glycosyltransferase and glycosidase reactions. With the increasing availability of glycosyltransferases via recombinant DNA technology, glycosyltransferase-catalyzed glycosylation with in situ regeneration of sugar nucleotides appears to be the most effective method for large-scale stereocontrolled oligosaccharide synthesis.     

Human thymic stromal lymphopoietin preferentially stimulates myeloid cells.

The sequence of a novel hemopoietic cytokine was discovered in a computational screen of genomic databases, and its homology to mouse thymic stromal lymphopoietin (TSLP) suggests that it is the human orthologue. Human TSLP is proposed to signal through a heterodimeric receptor complex that consists of a new member of the hemopoietin family termed human TSLP receptor and the IL-7R alpha-chain. Cells transfected with both receptor subunits proliferated in response to purified, recombinant human TSLP, with induced phosphorylation of Stat3 and Stat5. Human TSLPR and IL-7Ralpha are principally coexpressed on monocytes and dendritic cell populations and to a much lesser extent on various lymphoid cells. In accord, we find that human TSLP functions mainly on myeloid cells; it induces the release of T cell-attracting chemokines from monocytes and, in particular, enhances the maturation of CD11c(+) dendritic cells, as evidenced by the strong induction of the costimulatory molecules CD40 and CD80 and the enhanced capacity to elicit proliferation of naive T cells.     

Programmable reactivity-based one-pot oligosaccharide synthesis

A detailed protocol is described for the application of a programmable one-pot oligosaccharide synthesis methodology to the synthesis of fucosyl GM1. This serves as a general example of the application of this method to the synthesis of any desired oligosaccharide. The method relies on a large database of relative reactivities for differentially protected tolyl thioglycoside donor molecules and a computer program to suggest the best order of addition for assembly of the oligosaccharide in optimal yield and with the fewest operations. The product is a protected form of the desired oligosaccharide isolated in 47% yield, which is then deprotected using standard procedures to provide fucosyl GM1 oligosaccharide (1) in 44% yield. The total time for synthesis of 1 from building blocks 3, 4 and 5 is approximately 4 d, whereas synthesis of the same compound by traditional stepwise procedures would take significantly longer. Protocols for the synthesis of thioglycoside building blocks 3 and 4 are also described.     

Estrogen synthesis in the hippocampus

Estradiol plays essential roles in the modulation of synaptic plasticity and neuroprotection in males as well as in females, as has been shown particularly in the hippocampus. Although it has long been known that aromatase, the final enzyme in estrogen synthesis, is expressed in the hippocampus, a new paradigm emerged when it was shown that estradiol is actually synthesized de novo in this part of the brain. Increasing evidence indicates that hippocampus-derived estradiol plays a role in synaptic plasticity and neuroptrotection, rather than estradiol originating from the gonads. In recent years, a number of in vivo and in vitro studies have shown that hippocampus-derived estradiol substantially contributes to hippocampal function, in particular to structural synaptic plasticity.     

Thioglycosides as glycosylating agents in oligosaccharide synthesis

A review is given of the use of thioglycosides as glycosyl donors in oligosaccharide synthesis. Both indirect use, by conversion of the thioglycoside into a glycosyl halide and direct use, by electrophilic activation of the thioglycoside, are discussed.Abbreviations DMTST dimethyl(methylthio)sulfonium trifluoromethanesulfonate - Bz benzoyl - Bn benzyl - pNBz p-nitrobenzoyl - Phth phthallyl - Ph phenyl     

热休克蛋白70在妊娠早期小鼠子宫中的表达及雌激素的诱导作用

采用Western印迹、免疫组织化学、免疫电镜和图像分析技术研究了妊娠早期小鼠子宫热休克蛋白70(Heat shock protein,HSP70)的表达变化以及雌二醇对子宫HSP70表达的影响。结果表明：(1)与正常小鼠相比,孕鼠HSP70含量显著增多,且随妊娠日龄的增加而增加(P＜0．01);(2)小剂量(0．28μg／g体重)和大剂量(1．10μg／g体重)雌二醇均可诱导小鼠子宫HSP70免疫反应阳性细胞数显著增加(P＜0．01),但不表现剂量依赖关系;(3)Western印迹显示雌二醇使子宫HSP70蛋白谱带发生改变,正常小鼠仅有73kDl条蛋白带,小剂量组检出68kD、72kD、73kD3条蛋白带,大剂量组检出72kD、73kD2条蛋白带;(4)电镜下,HSP70免疫阳性反应定位于子宫内膜基质细胞胞浆与细胞核。这些结果提示,HSP70可能与蜕膜反应中基质细胞增殖密切相关,雌二醇对子宫HSP70的表达具有明显的诱导作用[动物学报49(3)：345—352,2003]。     

Estrogen receptors in the wobbler mouse

Recent research has raised the interesting possibility that the neurological mutant mouse, wobbler (wr/wr), possesses an estrogen receptor deficit analogous to the androgen receptor deficiency found in androgen-resistant mice with testicular feminization. In the present report we examined estrogen-binding activity in cytosolic extracts of kidney, liver, and brain from wobbler mice, littermate control animals, and C57BL/6J mice, using DNA-cellulose chromatography. Estrogen binding components exhibiting properties of estrogen receptors were present in all tissues examined. Estrogen receptors adhered to DNA, displayed characteristic elution profiles from DNA-cellulose, and showed high affinity and limited capacity for estradiol, in contrast to non-receptor entities which bind estradiol. The qualitative elution patterns for estrogen receptors did not differ among groups within each tissue studied, and were similar to those reported previously in mouse kidney and brain. While estrogen receptors have been shown in mouse liver by other techniques, this is the first demonstration of putative estrogen receptors in mouse liver by DNA-cellulose chromatography. No consistent deficits in estrogen receptor concentration were found in wobblers compared to littermates. Thus, the data do not support the hypothesis that the wobbler mouse is an estrogen receptor-deficient mutant.     

Brain hypoxia preferentially stimulates genioglossal EMG responses to CO2

Although the dominant respiratory response to hypoxia is stimulation of breathing via the peripheral chemoreflex, brain hypoxia may inhibit respiration. We studied the effects of two levels of brain hypoxia without carotid body stimulation, produced by inhalation of CO, on ventilatory (VI) and genioglossal (EMGgg) and diaphragmatic (EMGdi) responses to CO2 rebreathing in awake, unanesthetized goats. Neither delta VI/delta PCO2 nor VI at a PCO2 of 60 Torr was significantly different between the three conditions studied (0%, 25%, and 50% carboxyhemoglobin, HbCO). There were also no significant changes in delta EMGdi/delta PCO2 or EMGdi at a PCO2 of 60 Torr during progressive brain hypoxia. In contrast, delta EMGgg/delta PCO2 and EMGgg at a PCO2 of 60 Torr were significantly increased at 50% HbCO compared with either normoxia or 25% HbCO (P less than 0.05). The PCO2 threshold at which inspiratory EMGgg appeared was also decreased at 50% HbCO (45.6 +/- 2.6 Torr) compared with normoxia (55.0 +/- 1.4 Torr, P less than 0.02) or 25% HbCO (53.4 +/- 1.6 Torr, P less than 0.02). We conclude that moderate brain hypoxia (50% HbCO) in awake, unanesthetized animals results in disproportionate augmentation of EMGgg relative to EMGdi during CO2 rebreathing. This finding is most likely due to hypoxic cortical depression with consequent withdrawal of tonic inhibition of hypoglossal inspiratory activity.     

Cisatracurium stimulates testosterone synthesis in rat and mouse Leydig cells via nicotinic acetylcholine receptor

As a cis‐acting non‐depolarizing neuromuscular blocker through a nicotinic acetylcholine receptor (nAChR), cisatracurium (CAC) is widely used in anaesthesia and intensive care units. nAChR may be present on Leydig cells to mediate the action of CAC. Here, by Western blotting, immunohistochemistry and immunofluorescence, we identified that CHRNA4 (a subunit of nAChR) exists only on rat adult Leydig cells. We studied the effect of CAC on the synthesis of testosterone in rat adult Leydig cells and mouse MLTC‐1 tumour cells. Rat Leydig cells and MLTC‐1 cells were treated with CAC (5, 10 and 50 μmol/L) or nAChR agonists (50 μmol/L nicotine or 50 μmol/L lobeline) for 12 hours, respectively. We found that CAC significantly increased testosterone output in rat Leydig cells and mouse MLTC‐1 cells at 5 μmol/L and higher concentrations. However, nicotine and lobeline inhibited testosterone synthesis. CAC increased intracellular cAMP levels, and nicotine and lobeline reversed this change in rat Leydig cells. CAC may increase testosterone synthesis in rat Leydig cells and mouse MLTC‐1 cells by up‐regulating the expression of Lhcgr and Star. Up‐regulation of Scarb1 and Hsd3b1 expression by CAC was also observed in rat Leydig cells. In addition to cAMP signal transduction, CAC can induce ERK1/2 phosphorylation in rat Leydig cells. In conclusion, CAC binds to nAChR on Leydig cells, and activates cAMP and ERK1/2 phosphorylation, thereby up‐regulating the expression of key genes and proteins in the steroidogenic cascade, resulting in increased testosterone synthesis in Leydig cells.     

Growth hormone-releasing peptide-2 stimulates secretion and synthesis of adrenocorticotropic hormone in mouse pituitary

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides which induce strong GH release in both animals and humans. Among them, GHRP-2 is known to stimulate GH release by acting at both hypothalamic and pituitary sites, but also induces adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRP-2 may stimulate ACTH release directly via GHRP receptor type 1a in ACTH-producing tumors. GHRP-2 increases ACTH secretion in rat in vivo, but not ACTH release from rat primary pituitary cells. In the present study, in order to elucidate the mechanism underlying ACTH secretion by GHRPs, mouse pituitary cells were stimulated by GHRP-2. GHRP receptor mRNA was expressed in the mouse pituitary, and GHRP-2 directly stimulated secretion and synthesis of ACTH in the mouse anterior pituitary cells. GHRP-2 increased intracellular cyclic AMP production. H89, a potent protein kinase A (PKA) inhibitor, and bisindolylmaleimide I, a selective protein kinase C (PKC) inhibitor, inhibited the GHRP-2-induced ACTH release, and that H89, but not bisindolylmaleimide I, inhibited the GHRP-2-induced proopiomelanocortin mRNA levels. Together, the GHRP-2-induced ACTH release was regulated via both PKA and PKC pathways in the mouse pituitary cells, while ACTH was synthesized by GHRP-2 only via the PKA pathway.     

Phorbol myristate acetate stimulates RNA and casein synthesis in cultured mouse mammary gland tissues

Prolactin and phorbol myristate acetate (TPA) stimulate the rate of [3H]uridine incorporation in cultured mouse mammary gland explants in a similar fashion. Both the time-courses and magnitude of responses were the same; in addition, maximum stimulatory concentrations of TPA and prolactin elicited a nonadditive response when tested together. Nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, abolished both the TPA and prolactin effects on [3H]uridine incorporation. TPA also effected an enhanced rate of [3H]leucine incorporation into a casein-rich phosphoprotein fraction, but only if the explants were also cultured with spermidine.     

Solid-phase oligosaccharide synthesis and related technologies

Research efforts directed at the development of methodologies effective for solid-phase synthesis of oligosaccharides have resulted in a number of impressive achievements. In addition, closely related technologies, such as soluble polymer-supported synthesis and fluorous synthesis of the same class of molecules, have proved to be quite promising.     

Modelling of oligosaccharide synthesis by dextransucrase

Dextransucrase catalyses the formation of dextran, but also of numerous oligosaccharides from sucrose and different acceptors, if appropriate conditions are chosen. Much experimental work has been carried out and a scheme of reactions and a mathematical model have been developed to describe the complex kinetic behaviour of the enzyme. A computer program was used to calculate the parameters of the model from a broad range of experimental data, investigating a large number of kinetic tests with the acceptors maltose and fructose. The results lead to design considerations for a continuous reactor system with immobilized dextransucrase to produce leucrose, a disaccharide of industrial interest.     

Excess tyrosine stimulates eumelanin and pheomelanin synthesis in cultured slaty melanocytes from neonatal mouse epidermis

The mouse slaty (Dct(slt)) mutation is known to reduce the activity of dopachrome tautomerase (DCT). The reduced DCT activity inhibits melanosome maturation and reduces the melanin content in the skin, hair and eyes. It is not known whether eumelanin and pheomelanin synthesis in slaty melanocytes is modulated by melanogenic factors. In this study, to address this point, epidermal melanocytes derived from 0.5-, 3.5- and 7.5-day-old wild-type mice (Dct(+)/Dct(+) at the slaty locus) and from congenic mice mutant (Dct(slt) /Dct(slt) at that locus) were cultured in serum-free primary culture with or without additional L-tyrosine (Tyr). The content of melanin was measured by high-performance liquid chromatography in the cultured melanocytes as well as culture supernatants in serum-free primary culture. L-Tyr was found to increase the content of pheomelanin in addition to eumelanin in cultured slaty melanocytes and cuture supernatants at all ages tested. The eumelanin and pheomelanin contents in culture supernatants were greater than in cultured melanocytes. The eumelanin and pheomelanin contents in culture supernatants from 7.5-day-old slaty melanocytes in the presence of L-Tyr were greater than those from wild-type melanocytes. These results suggest that the inhibition of eumelanin synthesis by the slaty mutation can be partly restored by the addition of excess L-Tyr. Eumelanin and pheomelanin may accumulate with difficulty in slaty melanocytes and be easily released from them during skin development. L-Tyr may stimulate this release.