Fibroblast growth factor 16 stimulates proliferation but blocks differentiation of rat stem Leydig cells during regeneration.

OBJECTIVES: We aim to investigate the effects of fibroblast growth factor 16 (FGF16) on Leydig cell regeneration in ethane dimethane sulphonate (EDS)-treated rat testis. METHODS: We intraperitoneally inject 75 mg/kg EDS to adult male Sprague Dawley rats and then intratesticularly inject FGF16 (0, 10 and 100 ng/testis/day) from post-EDS day 14 for 14 days. We investigate serum hormone levels, Leydig cell number, gene and protein expression in vivo. We also explore the effects of FGF16 treatment on stem Leydig cell proliferation in vitro. RESULTS: FGF16 lowers serum testosterone levels (21.6% of the control at a dose of 100 ng/testis) without affecting the levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) on post-EDS day 28 in vivo. FGF16 increases Leydig cell number at doses of 10 and 100 ng/mg without affecting Sertoli cell number, increases the percentage of PCNA-positive Leydig cells, and down-regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) and Sertoli cell genes (Fshr, Dhh and Sox9) and their proteins in vivo. FGF16 increases phosphorylation of AKT1 and AKT2 as well as EKR1/2 in vivo, indicating that it possibly acts via AKT1/ATK2 and ERK1/2 pathways. FGF16 also lowers medium testosterone levels and down-regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) but increases EdU incorporation into stem Leydig cells in vitro. CONCLUSIONS: These data suggest that FGF16 stimulates stem and progenitor Leydig cell proliferation but blocks their differentiation, thus lowering testosterone biosynthesis.

Effects of nafamostat mesilate, a protease Inhibitor, on ischemia/reperfusion-induced kidney injury in mice

Ischemia is induced when blood flow to an organ is interrupted, and re-establishing blood flow is essential to prevent ongoing hypoxic injury, although it paradoxically imparts further injury. Nafamostat mesilate (NM), a synthetic serine protease inhibitor, has been used in patients undergoing hemodialysis who are at a high risk of bleeding. To determine the protective effect of NM on ischemia-reperfusion injury (IRI) in a mouse renal IRI model, NM was administered as a pre- and post-treatment or during ischemia reperfusion and compared to a control group. Mice were bilaterally nephrectomized and subjected to 40 min of renal pedicle occlusion followed by 24 h reperfusion. NM (240 μg/kg) significantly improved kidney function and lowered serum creatinine and blood urea nitrogen levels. Consistently, NM inhibited collagen formation in kidney tissues. NM treatment attenuated the effects of ischemia/reperfusion on kidney tissues and significantly inhibited activation of Toll-like receptor 4, nuclear factor kappa-light-chain-enhancer of activated B cells-phospho-65 (NF- kB-p65), phospho-inhibitor of NF-κβa, and inducible nitric oxide synthase (iNOS). NM treatment also decreased expression of Bcl-2, Caspase-3 and Bax in kidney tissues, which has been linked with induction of apoptosis in kidney tissues. Our studies suggest that NM may be a novel therapeutic agent to prevent and treat kidney IRI, in which iNOS and/or NF-κβ are upregulated. The exact regulatory mechanism and its functional significance require further elucidation

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Identification of amino acid residues contributing to the ATP-binding site of a purinergic P2X receptor.

P2X receptor subunits have intracellular N and C termini, two membrane-spanning domains, and an extracellular loop of about 280 amino acids. We expressed the rat P2X(2) receptor in human embryonic kidney cells, and used alanine-scanning mutagenesis on 30 residues with polar side chains conserved among the seven rat P2X receptor subunits. This identified a region proximal to the first transmembrane domain which contained 2 lysine residues that were critical for the action of ATP (Lys(69) and Lys(71)). We substituted cysteines in this region (Asp(57) to Asp(71)) and found that for S65C and I67C ATP-evoked currents were inhibited by methanethiosulfonates. At I67C, the inhibition by negatively charged ethylsulfonate and pentylsulfonate derivatives could be overcome by increasing the ATP concentration, consistent with a reduced affinity of ATP binding. The inhibitory action of the methanethiosulfonates was prevented by pre-exposure to ATP, suggesting occlusion of the binding site. Finally, introduction of negative charges into the receptor by mutagenesis at this position (I67E and I67D) also gave receptors in which the ATP concentration-response curve was right-shifted. The results suggest that residues close to Ile(67) contribute to the ATP-binding site.

A monoclonal antibody that recognizes the GPIIb/IIIa antagonist DMP 728. Reversal of the effects of DMP 728 on platelet aggregation and bleeding time in the dog.

Since hemorrhagic events represent a major safety concern associated with the use of new antithrombotic therapies such as glycoprotein (GP) IIb/IIIa receptor blockade, we evaluated the ability of a monoclonal antibody recognizing DMP 728 (cyclic [D-2-aminobutyryl-N2-methyl-L-argininyl-glycyl-L-aspartyl-3- aminomethyl-benzoic acid] methanesulfonic acid salt), a potent GPIIb/IIIa receptor antagonist, to reverse the pharmacological actions of DMP 728 in the dog. DC11 was chosen for in vivo evaluation based on its ability to inhibit the binding of [3H]DMP 728 to activated platelets and to attenuate the inhibition of ADP-induced aggregation on platelet-rich plasma ex vivo by DMP 728. After anesthesia mongrel dogs were given DMP 728 (20 micrograms/kg body wt IV) infused into the femoral vein, bleeding times were determined using a Simplate device from incisions on the backside of the tongue, and platelet aggregation was determined ex vivo. Nearly complete inhibition of platelet aggregation was observed for the dogs treated with DMP 728 (20 ug/kg IV) for up to 210 minutes, and bleeding times were prolonged > 15 minutes for 2 hours and remained elevated for more than 4 hours. DC11 (0.2 or 1.0 mg/kg body wt IV) given to dogs 10 minutes after DMP 728 resulted in 50% attenuation of the effect of DMP 728 on aggregation at 3 hours. Approximately 34% inhibition of the DMP 728-mediated bleeding time was achieved at 1 hour with the 0.2 mg/kg dose, whereas approximately 50% inhibition of the bleeding time was observed for the 1 mg/kg dose at 1 hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemopreventive effects of tamoxifen in ethyl methanesulphonate-induced rat mammary carcinogenesis.

Ethyl methanesulphonate (EMS), an alkylating agent and a potent mutagen, has been shown to be an effective carcinogen for the induction of mammary carcinoma in female Wistar King A rats. We therefore utilized this new system to assess the effects of tamoxifen (TAM) on mammary carcinogenesis. In Group A rats, given EMS orally for a period of 12 weeks, mammary carcinomas were first detected at the 13th week and were found in all surviving rats at the 20th week. The concomitant administration of TAM for 4 weeks, in Group B rats, retarded the development of the tumors significantly. There was a significant reduction in the incidence of estrogen receptor (ER)-positive tumors in the rats previously exposed to TAM; 100% in Group A versus 50% in Group B. Neither the progesterone receptor (PgR) nor androgen receptor (AR) status of the tumors were significantly different between these two groups. The inhibitory effects of TAM on tumor induction was also observed when TAM treatment started after EMS administration, though the intensity was smaller than that in Group B. These findings suggest the preventive action of TAM on EMS-induced mammary carcinogenesis, and indicate that this tumor system may provide a feasible model for research on chemoprevention and hormone therapy using an antiestrogen for human mammary carcinoma.

[Study on ethane dimethane sulphonate (EDS)-induced spermatogenic damage and protective drugs against this damage in the rat].

The effects of a single administration of ethane dimethane sulphonate (EDS), which has a direct cytotoxic effect on Leydig cells, was assessed for its spermatogenic damage and intratubular androgen level in SD male adult rats. The protective effect of human chorionic gonadotropins (hCG) (s.c.), testosterone propionate (TP) (s.c.) and intratesticular administration of testosterone microcrystal suspension (Tmcs) against the spermatogenic damage in rats EDS given was also evaluated. EDS caused a decrease of the seminiferous tubular diameter and impaired spermatogenesis remarkably; moreover, it also caused significant decreases in intratubular androgen levels. These results suggest that EDS-treated SD male adult rats may be suitable as a model for hormone dependent infertility. The administration of hCG and intratesticular Tmcs prevented tubular damage and increased the intratubular T level. On the other hand, the administration of TP prevented tubular damage while remarkably decreasing intratubular androgen level. In this connection, it was inferred that priming of rats with TP caused an increase in intratubular androgen binding protein, which would stimulate spermatogenesis. The fact that a single injection of Tmcs caused no tubular damage suggests that intratubular T level is one of the factors playing an important role in spermatogenesis and that an intratesticular injection of Tmcs may be useful for the treatment of some cases of idiopathic male infertility.

Specific destruction of Leydig cells in mature rats after in vivo administration of ethane dimethyl sulfonate.

Effects of ethane dimethyl sulfonate (EDS) on Leydig cells have been studied using the following parameters: morphology, histochemistry of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and esterase, quantitative activity of esterase, testosterone concentrations in plasma, and steroid production by isolated interstitial cells in vitro. Degenerating Leydig cells were observed within 16 h after the injection of mature rats with EDS (75 mg/kg body weight). At that time the testosterone concentration in plasma and the specific activity of esterase in testis tissue were decreased to approximately 35% and 60% of the control value, respectively. At 48 h after EDS only a few normal Leydig cells were left and the plasma testosterone concentration was less than 5% of the control value. The specific activity of esterase in total testis tissue was similar to the activity of dissected tubules from untreated rats. At 72 h no Leydig cells could be detected and no 3 beta-HSD and esterase-positive cells were present. At that time macrophages were still present in the interstitium and the appearance of the spermatogenic epithelium was normal, but 1 wk after EDS the elongation of spermatids was disturbed, probably due to a lack of testosterone. In some of the animals the cytotoxic effects of EDS on Leydig cells could be partly inhibited by human chorionic gonadotropin treatment. The basal steroid production by interstitial cells from mature rats 72 h after EDS was not significant and no stimulation by LH was observed, whereas no effect of EDS could be detected on steroid production by interstitial cells isolated from immature rats and mice 72 h after treatment. Other compounds with similar structures, such as butane dimethyl sulfonate (busulfan) and ethane methyl sulfonate (EMS) had no effect on Leydig cells from mature rats. It is concluded that EDS specifically destroys Leydig cells in mature rats.

interactions of ionizing radiation, nitrosamines, sulfonoxyalkanes and antioxidants as they affect carcinogenesis and survival in mice.

Butylated hydroxytoluene (BHT), pretreatment of 8-wk-old BALB/c mice for 28 days protected against 30-day lethality by X-rays, diethylnitrosamine (DEN), dimethylnitrosamine (DMN) and ethyl methanesulfonate (EMS) but not against methyl methanesulfonate (MMS). In X-ray survivors, neither mean survival nor leukemia incidences were affected by BHT. However, BHT (lifetime), given alone or with DEN (300 MG/KG BW in H2O) or 250 R X-rays, increased irradiated survivors at 18 months but not leukemia or tumor incidences. BHT + DEN treated females had increased survival and a decreased frequency of forestomach squamous carcinomas compared with DEN alone; no differences were seen in males or in pulmonary adenomas. In X-ray + DEN treated females, effect upon survival was additive, but the specific diseases causing this effect were not identified.

A protective effect of caffeine on drosophila larvae treated with methyl methanesulfonate.

Lethality induced in larval populations of Drosophila melanogaster was recorded after treatment with (1) caffeine, (2) MMS or (3) caffeine plus MMS. The mixture of caffeine plus MMS was less toxic than expected from the effects observed after treatment with either substance individually. It is postulated that in the combined treatment the caffeine, by inhibiting semiconservative DNA replication, allows for some additional time for repair of alkylated DNA by a repair pathway which is not sensitive to caffeine, possibly excision repair.