Strain variations in anti-sperm antibody responses and anti-fertility effects in inbred mice

This study provides evidence for polygenic controls of antisperm antibody levels in inbred male mice immunized with syngenic testis and epididymis. H2-linked and non-H2-linked genes were involved. Mice of H-2d haplotype were high responders, whereas those with H-2k haplotype were nonresponders; however, B10.D2/nSnJ mice (H-2d) were also nonresponders. In vitro fertilization inhibition by antisera correlated positively with the serum antisperm antibody levels, particularly with antibody of the immunoglobulin (Ig) G class. Inheritance of antibody response that inhibited in vitro fertilization (IVF) was an autosomal dominant trait, but this was not apparent for the control of antibody levels per se. Since IVF was inhibited by both IgG and fragment antigen-binding (Fab) isolated from immune sera, but not by immune IgG previously absorbed by sperm or testis, the biologic effect is antigen-specific and probably involved blockade of functional antigenic epitopes. Antisera to testis, caput sperm or cauda sperm were found to inhibit IVF to a similar degree. Inbred strains of mice that produced the highest levels of serum antisperm antibodies that inhibited IVF were A/J, SJL/J, DBA/1J and BALB/cByJ mice, and their antisera immunoprecipitated a common sperm antigen molecule of 35,000 to 40,000 Mr. In contrast, C57BL/6 and C57BL/10 mice produced significant antibody levels that had no effect on IVF, and their sera did not react with the 35,000- to 40,000-Mr peak. Moreover, among BALB/c H-2 congenic mice, only antiserum of responder BALB/cByJ (H-2d) mice immunoprecipitated the 35,000- to 40,000 Mr peak. Thus the 35,000- to 40,000-Mr protein may be of functional significance in the fertilization process.     

Membranotropic effects of the antibacterial agent Triclosan

Triclosan is a broad-spectrum hydrophobic antibacterial agent used in dermatological preparations and oral hygiene products. To gain further insight into the mode of action of Triclosan we examined its effects on membranes by performing leakage titrations of different oral bacteria and studying its interaction with model membranes through the use of different biophysical techniques. There was negligible efflux of intracellular material from Streptococcus sobrinus at the minimal inhibitory concentration of Triclosan; whatever leakage did occur commenced only at much higher concentrations. In contrast, no leakage was observed at even the minimal bactericidal concentration for Porphyromonas gingivalis. Triclosan decreased the onset temperature of the gel to liquid-crystalline phase transition of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-3-[phospho-rac-glycerol] membranes and was immiscible with these lipids in the fluid phase at concentrations greater than 5 mol%. Steady-state fluorescence anisotropy measurements of different phospholipid/Triclosan samples using 3-(p-6-phenyl-1,3,5-hexatrienyl)-phenylpropionic acid were consistent with the calorimetric data. Incorporation of increasing amounts of Triclosan into 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) vesicles induced the nonlamellar H(II) hexagonal phase at low temperatures and new immiscible phases at temperatures below the main transition of DEPE. Taking these results together suggests that the antibacterial effects of Triclosan are mediated at least in part through its membranotropic effects, resulting in destabilized structures which compromise the functional integrity of cell membranes without inducing cell lysis.     

Tubulin assembly, taxoid site binding, and cellular effects of the microtubule-stabilizing agent dictyostatin

(-)-Dictyostatin is a sponge-derived, 22-member macrolactone natural product shown to cause cells to accumulate in the G2/M phase of the cell cycle, with changes in intracellular microtubules analogous to those observed with paclitaxel treatment. Dictyostatin also induces assembly of purified tubulin more rapidly than does paclitaxel, and nearly as vigorously as does dictyostatin's close structural congener, (+)-discodermolide (Isbrucker et al. (2003), Biochem. Pharmacol. 65, 75-82). We used synthetic (-)-dictyostatin to study its biochemical and cytological activities in greater detail. The antiproliferative activity of dictyostatin did not differ greatly from that of paclitaxel or discodermolide. Like discodermolide, dictyostatin retained antiproliferative activity against human ovarian carcinoma cells resistant to paclitaxel due to beta-tubulin mutations and caused conversion of cellular soluble tubulin pools to microtubules. Detailed comparison of the abilities of dictyostatin and discodermolide to induce tubulin assembly demonstrated that the compounds had similar potencies. Dictyostatin inhibited the binding of radiolabeled discodermolide to microtubules more potently than any other compound examined, and dictyostatin and discodermolide had equivalent activity as inhibitors of the binding of both radiolabeled epothilone B and paclitaxel to microtubules. These results are consistent with the idea that the macrocyclic structure of dictyostatin represents the template for the bioactive conformation of discodermolide.     

Mode of action of alpha-chlorohydrin as a male anti-fertility agent. Inhibition of the metabolism of ram spermatozoa by alpha-chlorohydrin and location of block in glycolysis

1. The effect of alpha-chlorohydrin on the metabolism of glycolytic and tricarboxylate-cycle substrates by ram spermatozoa was investigated. The utilization and oxidation of fructose and triose phosphate were much more sensitive to inhibition by alpha-chlorohydrin (0.1-1.0mm) than lactate or pyruvate. Inhibition of glycolysis by alpha-chlorohydrin is concluded to be between triose phosphate and pyruvate formation. Oxidation of glycerol was not as severely inhibited as that of the triose phosphate. This unexpected finding can be explained in terms of competition between glycerol and alpha-chlorohydrin. A second, much less sensitive site, of alpha-chlorohydrin inhibition appears to be associated with production of acetyl-CoA from exogenous and endogenous fatty acids. 2. Measurement of the glycolytic intermediates after incubation of spermatozoal suspensions with 15mm-fructose in the presence of 3mm-alpha-chlorohydrin showed a ;block' in the conversion of glyceraldehyde 3-phosphate into 3-phosphoglycerate. alpha-Chlorohydrin also caused conversion of most of the ATP in spermatozoa into AMP. After incubation with 3mm-alpha-chlorohydrin, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase activities were decreased by approx. 90% and 80% respectively, and in some experiments aldolase was also inhibited. Other glycolytic enzymes were not affected by a low concentration (0.3mm) of alpha-chlorohydrin. Loss of motility of spermatozoa paralleled the decrease in glyceraldehyde 3-phosphate dehydrogenase activity. alpha-Chlorohydrin, however, did not inhibit glyceraldehyde 3-phosphate dehydrogenase or triose phosphate isomerase in sonicated enzyme preparations when added to the assay cuvette. 3. Measurement of intermediates and glycolytic enzymes in ejaculated spermatozoa before, during and after injection of rams with alpha-chlorohydrin (25mg/kg body wt.) confirmed a severe block in glycolysis in vivo at the site of triose phosphate conversion into 3-phosphoglycerate within 24h of the first injection. Glyceraldehyde 3-phosphate dehydrogenase activity was no longer detectable and both aldolase and triose phosphate isomerase were severely inhibited. Spermatozoal ATP decreased by 92% at this time, being quantitatively converted into AMP. At 1 month after injection of alpha-chlorohydrin glycolytic intermediate concentrations returned to normal in the spermatozoa but ATP was still only 38% of the pre-injection concentration. Motility of spermatozoa was, however, as good as during the pre-injection period. The activity of the inhibited enzymes also returned to normal during the recovery period and 26 days after injection were close to pre-injection values. 4. An unknown metabolic product of alpha-chlorohydrin is suggested to inhibit glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase of spermatozoa. This results in a lower ATP content, motility and fertility of the spermatozoa. Glycidol was shown not to be an active intermediate of alpha-chlorohydrin in vitro.     

左炔诺孕酮和炔雌醚复合物(EP-1)及组分对鼠类不育效果的研究进展

由于当前传统灭杀带来的一系列问题,鼠害不育控制逐渐受到重视。自张知彬等2004年率先报道左炔诺孕酮和炔雌醚复合物(EP-1)对野生鼠类不育效果的研究以来,国内多个研究团队对EP-1及其组分的不育效果、剂量、饵料制作、环境安全性、行为与分子机理等方面开展了大量的验证和完善研究,取得了许多重要的进展。这些结果进一步说明EP-1及组分对鼠类具有很好的不育效果,并具有两性不育、高效低量、可持续、相对环保与安全、野外投放方便、经济可行等特点。EP-1或炔雌醚毒饵的不育剂量约为10-50μg/m L(0.001%-0.005%),甚至更低;其组分在土壤和水体的半衰期约为5-16 d和小于3 d。在鼠类繁殖的早期,采取野外一次饱和投放即可有效降低全年鼠类的繁殖,效果可延续至第二年。可见,EP-1及其组分有望成为鼠害控制的一个新手段。建议今后进一步加强野外不育与跨年持续效应的验证、环境安全性评价和行为生态学机制等研究工作。     

Unexpected beta adrenergic effects of the antitumor agent, cyclocytidine, on rat salivary glands.

In addition to its potent antileukemic properties, cyclocytidine has a sialogogue action that depends on stimulation of beta adrenergic ereceptors of salivary glands. Furthermore, when chronically administered (for 3 days), cyclocytidine caused enlargement of parotid and submaxillary glands and heart that resembled the hypertrophy caused by chronic isoproterenol administration. The salivas evoked by cyclocytidine also closely resembled those evoked by isoproterenol, and were extremely viscous, and high in K+, (121 plus or minus 5.6, for submaxillary, and 42 plus or minus 2.9, for parotid), low in flow rate (0.007 mg/min times mg) and parotid saliva contained high concentrations of amylase (805 plus or minus 33 mg/mg gland). Cyclocytidine also caused marked emptying of parotid gland amylase. The cyclocytidine-induced salivary flow and gland emptying of amylase were prevented for 90 min when propranolol (but not dibenzyline or atropine) was administered prior to injection of the cyclocytidine. In addition, when the superior cervical ganglion was acutely removed, administration of cyclocytidine elicited salivary flow from the denervated as well as the innervated glands. These findings suggest that cyclocytidine does not affect salivary glands through indirect central or ganglionic actions. Cyclocytidine action does not exclusively involve beta receptors, since even in the presence of propranolol, secretory flow was evident after 90 min but when dibenzyline was given with the propranolol, complete blockade of cyclocytidine-stimulated saliva was effected. The dominant effect is, however, a beta adrenergic one. The undesirable side effects of cyclocytidine (parotid pain, postural hypotension, and cardiac hypertrophy) probably stem chiefly from its beta adrenergic properties and might be eliminated (or at least modified) by administration of propranolol with the cyclocytidine.     

Membrane ordering effects of the anticancer agent VM-26

The effect of the anticancer agent VM-26 on acyl chain order of cellular and model membranes was examined by electron spin resonance techniques. The order parameter for the paramagnetic probe 5-doxyl stearate was increased when VM-26 was incorporated into the bilayer of fluid-phase dimyristoylphosphatidylcholine (DMPC) or gel-phase dipalmitoylphosphatidylcholine (DPPC) liposomes at concentrations up to 4.8 mol%. The ordering effect of VM-26 in DMPC was greater than that of cholesterol on an equimolar basis. The less cytotoxic congener of VM-26, VP-16, was only one-third as active as VM-26 in its ordering effects on DMPC. Higher order parameters for 5-doxyl stearate were also noted in asolectin liposomes, Ehrlich ascites tumor cells, and CCRF-CEM cells treated with VM-26. We conclude that VM-26 has significant membrane associated activity in addition to its previously recognized nuclear effects.     

Water activity,temperature, and pH effects on growth of the biocontrol agent Pantoea agglomerans CPA-2

The growth response of the biocontrol agent Pantoea agglomerans to changes in water activity (a(w)), temperature, and pH was determined in vitro in nutrient yeast extract-sucrose medium. The minimum temperature at which P. agglomerans was able to grow was 267-272 kelvins (-6 to -1 degrees C), and growth of P. agglomerans did not change at varying pH levels (4.5-8.6). The minimum a(w) for growth was 0.96 in media modified with glycerol and 0.95 in media modified with NaCl or glucose. Solute used to reduce water activity had a great influence on bacterial growth, especially at unfavourable conditions (e.g., low pH or temperature). NaCl stimulated bacterial growth under optimum temperatures but inhibited it under unfavourable pH conditions (4.5 or 8.6). In contrast, the presence of glucose in the medium allowed P. agglomerans to grow over a broad range of temperature (3-42 degrees C) or pH (5-8.6) regimes. This study has defined the range of environmental conditions (a(w), pH, and temperature) over which the bacteria may be developed for biological control of postharvest diseases.     

Reduction of carcinogen-induced breast cancer in rats by an anti-fertility drug

The DMBA (dimethylbenz[a]anthracene)-induced mammary cancer systems seemed to be an ideal model for the study of the influence of oral contraceptives (OCs) on endocrine-related cancers. Groups of 20 female Sprague-Dawley rats, age 40-45 days, weight approximately 150 g, were given 3.0 or 0.3 mg of crystalline Enovid suspended in 1 ml of sesame oil per dose for 45 days. The controls consisted of a group of 20 rats given a single dose of 15 mg of DMBA, of 2 groups of 10 rats administered daily doses of 3.0 and 0.3, respectively, of Enovid for 45 days, and of 1 group of 20 rats on sesame oil. In addition to these 6 groups, 3 additional sets of 5 rats each were fed 3.0 or 0.3 mg Enovid per rat, or 0.3 mg Enovid plus the subsequent single dose of 15 mg DMBA. The animals were weighed weekly and thoroughly examined for the appearance of mammary tumors or other grossly apparent lesions. The dosage of Enovid was selected on the basis of preliminary experiments. With doses of 0.005 mg per 150 g rat the estrus cycle was usually but not always arrested 5 days after the treatment began. At doses of 0.05 and 0.5 mg the cycle was interrupted 3-5 days and at 1 day later. In the final studies with doses of 0.3 and 3.0 mg, the microscopic examination of the smears indicated that the estrus cycle was effectively halted in all rats throughout the Enovid, or Enovid plus carcinogen feeding period but returned to normal shortly after the cessation of Enovid administration. DMBA failed to restore the estrus cycle suggesting that Enovid exerts a pronounced effect on the endocrine system. A single dose of 15 mg DMBA yielded an increasing incidence of mammary tumors which reached 100% after about 6 months. The multiplicity also rose and reached an average of 5.8 masses per rat at the end of the planned experiment period of 9 months. Histopathologic examination of the tumors indicated that there were several types, and even the same tumor nodule exhibited areas of varying morphologic aspect. Administration of the lower level of Enovid tended to inhibit slightly the incidence of mammary cancer. Of the 20 sesame oil control animals, only 1, killed after 237 days, had a large fibroadenoma.     

Efficacy of animal anti-fertility compounds against sea lamprey (Petromyzon marinus) spermatozoa

Sterile-male-release technique is currently used to control the sea lamprey (Petromyzon marinus) population in the Great Lakes. The chemosterilant (bisazir) used in this program is extremely hazardous; special safety measures are necessary when handling this chemical. Therefore, replacement of bisazir with safer agents is desirable. In this study, we examined the effects of low-toxicity compounds with previously described spermicidal activity (mainly against mammalian sperm) on motility and fertilizing ability of sea lamprey spermatozoa. Nonoxynol-9, benzalkonium chloride, zinc acetate, cupric chloride, cysteamine, tannic acid and propranolol were able to inhibit both sperm motility and fertilizing ability. Effective concentrations of these spermicides ranged from 0.15 to 1%. Therefore, they can be potentially used in further study directed at in vivo sterilization of male sea lampreys.