ReProComet: a new in vitro method to assess DNA damage in mammalian sperm.

The increasing request of chemical safety assessment demands for the validation of alternative methods to reduce the resort to animal experimentation. Methods that evaluate reproductive toxicity are among those requiring the largest use of animals. Presently, no validated in vitro alternative exists for the assessment of reproductive toxicity. Mammalian sperm are sensitive targets of DNA-reactive chemicals, which form premutagenic adducts. Here, we propose a new method based on comet assay to detect DNA damage induced by potential germ cell mutagens in bull sperm available from assisted reproduction practices. In somatic cells, chemical-induced adducts can be revealed by comet assay that detects DNA breaks produced during adduct repair. Mature sperm, however, are devoid of repair enzymes, and adducts are processed only after fertilization. For this reason, comet assay is not sensitive to detect DNA lesions induced in sperm by most chemicals. To overcome such limitation, we developed a modified comet assay based on the addition of a protein extract from HeLa cells to agarose-embedded sperm on microscopic slides. To test the method, sperm were treated in vitro with methyl methanesulfonate (MMS) or melphalan (MLP) and comet assay was conducted both with and without protein supplementation. No effect of MMS or MLP was detected without protein supplementation; on the contrary, a clear-cut dose-dependent effect was measured after addition of the cell extract. These results represent a proof of concept of a novel in vitro mutagenicity test on sperm that could offer a promising approach to complement previously validated in vivo germ cell genotoxicity assays.     

老年人根管治疗疗效的相关因素

目的:研究老年人根管治疗疗效及其影响因素.方法:对56例65岁以上老年人,根管治疗后2年以上的126颗牙齿进行检查,按治疗后年限分3组:2～3年组,4～5年组,6年以上组.应用根尖周指数(Periodicalindex)评定X线片和综合临床检查,对其治疗进行评价.结果:老年人根管治疗成功率为72.2%;三组间根管治疗成功率差异无显著性.恰填根管治疗的成功率(82.2%)比欠填和超填的成功率(47.2%)要高;恰填病例中,冠方修复体质量好的患牙比质量差的患牙根尖周炎发病率低(25.7%和58.4%);去除欠填和超填病例后,桩冠修复患牙的根管治疗成功率为58.8%,而未做桩冠修复的患牙成功率为87.5%.结论:根充质量、冠方修复体质量、桩冠修复是影响老年人根管治疗疗效的主要因素.     

葡立胶囊与抗骨增生胶囊联用治疗颈椎病的临床研究

目的 了解、观察葡立胶囊与抗骨增生胶囊联用治疗颈椎病对血清SOD含量及疼痛功能评分的影响。方法 对一组神经根型颈椎病患者，用葡立胶囊与抗骨增生胶囊联合应用治疗3个月，测定血清SOD含量及疼痛功能评分变化。结果 颈椎病患者治疗组血清SOD含量与治疗前相比明显升高，疼痛功能评分治疗前相比明显降低。结论 葡立胶囊与抗骨增生胶囊联合应用可明显缓解症状，提高血清SOD活力，延缓颈椎间盘退变，对治疗颈椎病有明显疗效。     

Exo-focal postischemic neuronal damage in the rat brain: alteration of [H]forskolin binding using in vitro autoradiography

We studied the alteration of intracellular signal transduction using quantitative autoradiography of the second messenger system in order to clarify the mechanisms of delayed neuronal damage in the remote areas of rat brain after transient focal ischemia. Chronological changes of [³H]forskolin binding sites were measured to demonstrate the striatal-nigral pathway after 90 min of right middle cerebral artery (MCA) occlusion and after such occlusion followed by 3 h, 6 h, 1 day, 3 days, 1 week, 2 weeks and 4 weeks of recirculation. [³H]Forskolin binding sites were found to be markedly decreased in the lateral segment of the caudate putamen supplied by the occluded MCA after 90 min of ischemia with no recirculation. On the contrary, there was no alteration on day 1, but 3 days after ischemic insult, marked reduction of [³H]forskolin binding sites was observed in the ipsilateral substantia nigra which lay outside the ischemic areas. This postischemic delayed phenomenon observed in the substantia nigra developed concurrently with ⁴⁵Ca accumulation, which was detected there in our previous study. The delayed reduction of [³H]forskolin binding sites in the substantia nigra observed in the present study indicates that striatonigral terminal degeneration at presynaptic sites is caused by precedent ischemic damage of the ipsilateral caudate putamen and that exo-focal postischemic neuronal death is caused by a transsynaptic process associated with the ischemic foci.     

Extensive pectoral muscle necrosis after defibrillation: nonthermal skeletal muscle damage caused by electroporation

A 37-year-old Italian male developed a myocardial infarct with subsequent ventricular fibrillation. He was defibrillated seven times with up to 360 Joules. Thirteen days later the patient died of recurrent myocardial infarct due to thrombotic occlusion of the left circumflex coronary artery. At autopsy, necrosis of the right pectoralis muscle was observed. Electroporation is the pathogenetic mechanism of skeletal muscle damage due to multiple defibrillations with high energy levels. Received: 8 January 1998 Accepted: 15 April 1998     

Laparoscopic treatment of Meckel's diverticulum

We present two cases in which complication of a Meckel's diverticulum were dealt with, and in one case diagnosed using the laparoscope. One was a large bleeding diverticulum containing ectopic gastric mucosa, with the diagnosis suggested preoperatively, confirmed laparoscopically, and the pathology resected extracorporeally. The second was a partial intermittent small-bowel obstruction due to torsion around the mesodiverticular band, diagnosed and resected via the laparoscope. The literature of Meckel's diverticula and complications is reviewed, with open and laparoscopic treatment options. Although uncommon, many cases of Meckel's diverticulum may be quite suitable for laparoscopic diagnosis and treatment.     

The effects of a 0.12% chlorhexidine-digluconate-containing mouthrinse versus a placebo on plaque and gingival inflammation over a 3-month period

Abstract Several previous studies have evaluated the effects of 0.12% chlorhexidine digluconate (ChD) mouthrinses on plaque and gingival inflammation. However, previously, none have been based in general dental practices. The aim of this study was to evaluate the potential to conduct controlled periodontal clinical trials in co-operation with general dental practitioners (gdps). The project took place in 5 general dental practices in the South of England. 121 healthy subjects (24 at 4 sites and 25 at the 5th). aged 18-65 years, mean 35 ± 12) years participated in a double-blind, randomised study during which they received full mouth assessments for plaque and gingival bleeding at baseline, 6 and 12 weeks. 60 subjects were randomly asigned to use the 0.12% ChD mouth wash and 6i the placebo. The assessments were carried out by 5 gpds, who had previously achieved inter-examiner κ scores of 0.78–0.85 (mean 0.81) for the plaque index (PlI), and of 0.73–0.94 (mean 0.87) for a modified gingival index (mGI), and who maintained κ scores of 0.51–0.90 for PII and of 0.73–1.00 for mGI during the 12 months required to complete the study. 98 subjects (48 ChD and 50 placebo) completed the study. Even though the baseline levels of plaque and gingivitis were low, by week 12, mean whole mouth piaque score of the ChD mouthwash users had fallen from 1.33 at baseline to 0.96 and was significantly lower (p < 0.001) than for the placebo users, 1.31 at baseline to 1.13. Whole-mouth gingival bleeding score fell from 0.56 to 0.42 in the ChD mouthwash group but was unchanged (0.54–0.55) in the placebo group. A subsidiary data analysis which considered the effects at sites indicated that within these overall differences, the ChD users experienced almost 2× the reduction from plaque score 2 at baseline at proximal molar sites over a 12-week period (50.6% ChD versus 27.6% placebo). It was concluded that 0.12% ChD mouthwash reduced plaque accumulation fay 28% and gingival inflammation by 25% over a 12–week period, that it is feasible for a group of gdps to maintain high levels of inter–examiner consistency in the use of PlI and mGI, that it is also feasible to carry out such a multicentre study in general dental practice, and that the use of mean mouth scores per subject to analyse the effects of mouthrinses may well mask variations in response throughout the mouth.     

Cholera Toxin B-Mediated Targeting of Lipid Vesicles Containing Ganglioside GM1 to Mucosal Epithelial Cells

Purpose. To determine whether the non-toxic pentameric B subunit of Cholera toxin (CTB) binding to ganglioside GM1 on both the lipid vesicles and epithelial cells may provide a means to target lipid vesicles to mucosal cells expressing surface GM1. Methods. Sonicated lipid vesicles containing ganglioside GM1 were prepared. Inter-vesicle cross-linking due to pentameric CTB binding to these GM1 vesicles was determined with a sub-micron particle analyzer. Association of CTB to GM1 vesicles was analyzed with continuous sucrose gradient centrifugation. CTB-mediated binding of GM1 vesicles to human mucosal epithelial cells (Caco-2 and HT-29), mucous membranes of mouse trachea, and nasal tissues were detected with fluorescent labeled vesicles. Results. An increase in lipid particle size due to binding of CTB to lipid vesicles and inter-vesicles cross-linking was detected. At a 30-to-1 mole ratio of membrane-bound GMl-to-CTB, optimum increase in GM1 vesicle aggregation, was detected. Under such conditions, all the added CTB molecules were associated with GM1 vesicles. Time course analysis showed that inter-vesicles cross linking by CTB was detectable within 10 min. and reached a maximum value at 60 min. CTB associated GM1-vesicles bind to mucosal epithelial cells HT-29 and Caco-2 with similar affinity [K_d = 7.8 × 10^–4 M lipid (Caco-2) and 7.6 × 10^–4 M lipid (HT-29)]. GM1 mediated binding specificity was demonstrated by blocking with anti-GMl antibody and the insignificant degree of CTB-associated GM1 vesicle binding to GM1 deficient C6 cells. Conclusions. The CTB-mediated GM1 binding to multiple membrane surfaces provides selective localization of GM1 vesicles to GM1 expressing mucosal cells and tissues. The strategy may be useful in localizing drugs and proteins to gut and respiratory tract mucosa.     

Development of intestinal epithelial cell lines using a transgenic mouse

The epithelial cells of the colonic mucosa of the animal have proved impossible to culture using standard tissue culture techniques. Immortalization of adult colonic epithelial cells has been unsuccessful due to the lack of DNA synthesis in these cells once they are isolated from the tissue. Recently an unique transgenic mouse bearing a temperature sensitive mutant of the known immortalizing gene, SV40 large T has become available. The advantage of this mouse is that the SV40 large T gene is expressed in every cell. Active immortalizing protein is produced in each cell at the permissive temperature. We have used colonic mucosa from these mice to initiate cultures of epithelial cells from the colon of adult mice. The cells grow readily at the permissive temperature but die within 7 days at the non-permissive temperature. The methods used to develop these cultures are described.     

恒河猴坐姿着陆冲击内耳损伤的观察

为研究机体及各组织器官对冲击力的耐受限值，探讨致伤原因及提出防护措施，利用着陆冲击塔，使实验动物恒河猴的坐姿状态下，进行冲击试验，试验后１ｈ左右处死，作大体解剖观察并取心，肝，脾，肺，颞骨等组织，固定，切片，镜检。同时重点观察了内耳的损伤。结果表明，冲击载荷不大（平台过载值为≤２５Ｇ）时内脏虽然有轻度损伤，但内耳可避免受员，当冲击力加大到一定程度（平台过载值为３０～３５Ｇ）导致内脏中等损伤时，内耳