Enhancement of concurrent yield of spermatogonial, meiotic I, and meiotic II metaphase chromosomes from Chinese hamster testes

A procedure to enhance the concurrent yield of large numbers of spermatogonial, meiotic I, and meiotic II metaphases from Chinese hamster testes has been developed. Animals were injected intraperitoneally with 5, 10, 20, or 40 mg of colchicine/kg and killed 3 hr after the treatment. Both testes from each animal were excised; one testis was minced, and germ cells were isolated by using 0.1% trypsin solution; the other testis was minced, and germ cells were isolated by using a conventional procedure. Chromosomal preparations were made with a standard air-drying technique. Examination of slides revealed that with the trypsin-isolation procedure the concurrent yields of spermatogonial, meiotic I, and meiotic II metaphases were significantly higher (P less than .05) than the yields obtained with the conventional procedure. Furthermore, 40 mg of colchicine/kg produced maximum numbers of all three types of germ cell metaphases. At this maximum concentration, metaphases were stable with no evidence of structural aberrations, and the frequency of hyperploidy was not significantly different (P greater than .05) from hypoploidy.     

辐射致雄性小鼠不育症模型的建立及生精细胞损伤的初步分析

目的：构建辐射致雄性小鼠不育症模型，并对模型小鼠的生精细胞损伤进行初步分析。方法：不育症模型构建：65只雄性C57BL/6小鼠随机分为7组，正常对照组(A组)及6组60Co γ照射组，后者包括B组(12 Gy)、C组(6+6 Gy)、D组(10 Gy)、E组(6+4 Gy)、F组(8 Gy)、G组(4+4 Gy)。观察分析小鼠死亡情况(24w)及30、50、70 d受孕率。生精细胞损伤分析：72只雄鼠随机分为4组：正常对照组、6+4 Gy组、4+4 Gy组、6 Gy组。30、50、70 d观察睾丸组织切片(HE染色)并评估其生精功能( Johnsen评分)；对PLZF阳性标记的精原干细胞(免疫组化法)计数。结果：A、E、F、G组生存时间较长，B、C组生存时间较短。30、50、70 d，D、E组受孕率均为0%。各照射组Johnsen评分均低于对照组(P<0.05)；6+4 Gy组的PLZF阳性细胞计数呈逐渐上升趋势，但均明显低于对照组(P<0.05)。结论：6+4 Gy的60Co-γ射线照射7周龄的C57BL/6雄性小鼠，可以构建不育症模型。模型小鼠的PLZF标记的精原干细胞计数虽然低于对照组，但并非完全消失，提示不育症的原因并非精原干细胞的完全消亡。     

Neural tube and other developmental anomalies in the guinea pig following maternal hyperthermia during early neural tube development.

Guinea pigs were exposed to hyperthermia for 1 hr once or twice on day 11, 12, 13, or 14 (E11-E14) of pregnancy. The mean rectal temperatures were elevated by 3.4 degrees C-4.0 degrees C. This treatment resulted in a marked elevation of rates of resorption and developmental defects in embryos examined at day E23. The defects observed were those affecting the neural tube (NTD) (exencephaly, encephaloceles, and microphthalmia), kyphosis/scoliosis, branchial arch defects, and pericardial edema. Embryos with NTD and kyphosis/scoliosis have not been found among newborn guinea pigs to date following maternal heat exposure on days E12-E14. It appears that embryos with these defects are filtered out by resorption or abortion by days E30-E35.     

Dose-related clastogenic effects induced by benzene in bone marrow cells and in differentiating spermatogonia of Swiss CD1 mice.

Cancer hazard is due to genotoxic events in somatic cells and genetic hazard in the strict sense is due to mutagenic events in germ cells. The investigation of sensitivity differences between somatic and germinal cells is pertinent to the question whether a genotoxic carcinogen is also a germ cell mutagen. Cytogenetic damage induced by benzene in mice was evaluated by determining the frequencies of chromosomal aberrations in bone marrow and spermatogonial cells of male Swiss CD1 mice. First, the analysis was performed by administering 1 ml/kg (880 mg/kg) of benzene as a single oral dose and sampling either cell type after a wide range of times (6, 12, 18, 24, 30, 36, 42 and 48 h) to determine the time of maximum response. At this dose benzene showed high clastogenic activity in bone marrow cells with a peak between 24 and 30 h. In differentiating spermatogonia the frequency of aberrant cells was highest 24 h after treatment. The overall effect in spermatogonia was lower than in bone marrow cells. Second, the dose response was determined 24 h after treatment with two additional doses of benzene: 0.1 ml/kg (88 mg/kg) and 0.5 ml/kg (440 mg/kg) for bone marrow cells; 0.25 ml/kg (220 mg/kg) and 0.5 ml/kg (440 mg/kg) for differentiating spermatogonia. The clastogenic effect was dose dependent in both cell types. The frequencies of aberrant cells increased in a linear-quadratic manner in bone marrow and linearly in differentiating spermatogonia. Furthermore, the per cell damage was higher in bone marrow than in spermatogonial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of pulsed ultrasound on mandibular distraction

This study evaluated the effect of pulsed ultrasound on tissue repair and bone growth during mandibular osteodistraction. Twenty-one rabbits were divided into three groups of 7. The distraction started 72 h after surgically severing both sides of the mandible and proceeded at a rate of 1.5 mm/12 h for 5 days. Group 1 received pulsed ultrasound (nominally 200 s pulse of 1.5 MHz at a 1.1 kHz pulse repetition frequency, 30 mW/cm²) for 20 min on both sides of the mandible every other day (alternating sides). Group 2 received the same pulsed ultrasound treatment on one side of the mandible every day for 20 min. Group 3 did not receive any ultrasound treatment. Bone formation at the distraction site was assessed by photodensitometry on head radiographs, a vibratory coherence test across the distraction site, a postmortem three-point bending mechanical stiffness test, and a postmortem histological examination. Statistical analyses performed using analysis of variance revealed that pulsed ultrasound enhanced bone formation at the distraction site with a high level of significance when assessed by the increase in new bone photodensity (p=0.001), vibratory coherence (p=0.001), mechanical stiffness (p=0.003), and qualitative histological studies, especially when the pulsed ultrasound treatment was directly applied daily. © 2002 Biomedical Engineering Society. PAC2002: 8750Kk, 8763Df     

Apoptosis and necrosis induced by cyclic mechanical stretching in alveolar type II cells

Alveolar type II (ATII) cells are exposed to mechanical stretch during breathing and mechanical ventilation. Increased stretch may contribute to lung injury. The influence of three stretching patterns (characterized by frequency [min(-1)] - increase in surface area [%]: S40-13, S60-13, S40-30) on parameters of apoptosis, necrosis, and membrane integrity of rat ATII cells was compared with that in static cultures. The S40-13 stretching pattern simulated normal breathing. The other patterns were chosen to study increased amplitude and frequency. There were no significant differences between the S40-13 group and static cultures. Lactic acid dehydrogenase (LDH) release and early apoptotic cells were significantly increased in S60-13 and S40-30 in comparison with static cultures (LDH: 0.089 +/- 0.014 microg/ml and 0.177 +/- 0.050 microg/ml versus 0.050 +/- 0.011 microg/ml; early apoptosis: 17 +/- 3.5% and 23 +/- 3.1% versus 9.7 +/- 1.4%) at 24 h. Necrosis was significantly increased only in the S40-30 group (13 +/- 2.4% versus 6.1 +/- 0.9% in static culture at 24 h). Captopril as well as L-Arginine prevented apoptosis and reduced apoptotic cells to static culture levels in the S40-30 group, but did not influence necrosis and LDH release. Increased mechanical stretch may contribute to lung injury by induction of apoptosis and necrosis in ATII cells. Apoptosis induced by high-amplitude mechanical stretch is prevented by captopril and L-Arginine.     

低频超声开放大鼠血脑肿瘤屏障与TNF-α表达之间的关系

目的探讨肿瘤坏死因子-α(TNF-α)与低频超声开放大鼠血脑肿瘤屏障之间的关系。方法应用1MHz低频超声辐照C6胶质瘤大鼠,采用伊文思蓝(EB)法检测血脑肿瘤屏障通透性的变化,应用透射电镜观察脑微血管内皮细胞紧密连接的变化,应用酶联免疫吸附试验(ELISA)法检测脑组织中TNF-α含量的变化。结果低频超声辐照后大鼠血脑肿瘤屏障的通透性逐渐增加,在辐照后1.5h￣3h达高峰,12h恢复至正常水平;透射电镜显示低频超声辐照后,0.5h至9h血管内皮细胞的紧密连接均有不同程度的开放,至12h时关闭;低频超声辐照后大鼠胶质瘤侧脑组织中TNF-α含量逐渐增加,在1.5h￣3h达高峰,12h恢复至正常水平。结论低频超声辐照大鼠脑胶质瘤模型后引起TNF-α量的变化与血脑肿瘤屏障通透性变化的趋势及达到峰值的时间相一致。TNF-α的增加可能是低频超声辐照开放大鼠脑胶质瘤血肿瘤屏障的重要因素之一。     

Learned persistence at 11–12 days but not at 10–11 days in infant rats

In 2 experiments using milk-suckling from an anesthetized dam as the reinforcer, evidence is presented that the transitional age in rat pups for learning of persistence as a result of appetitive partial reinforcement is between 11 and 12 days of age. In Experiment I, pups 12–13 days of age showed the partial reinforcement extinction effect (PREE) whereas 10–11-day olds did not. In Experiment II, pups trained at 11–12 days and tested at Day 13 did show a PREE at Day 13 but those trained at 10–11 days did not.     

Sensory regulation of maternal behavior in rats: Effects of pup age

Maternal behavior (retrieving, crouching, and licking) was induced in Sprague-Dawley virgin female rats by constant exposure to pups aged 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, 13-14, or 15-16 days. The incidence of spontaneous components of maternal behavior, notably retrieving, was geater towards pups 1-8 days of age than towards older pups, whereas the occurrence of cannibalism did not differ as a function of pup age. With pups 1-2 through 13-14 days, the latency to onset of full maternal behavior was shortest with 1-2-day-old pups (2-day median) and longest with 13-14-day-old pups (7-day median). Females exposed to pups aged 3-4 through 11-12 days did not differ significantly in their latencies, the medians of which ranged from 4.0 to 5.5 days. Only 1 female out of 8 exposed to pups aged 15-16 days became fully maternal, but 5 more displayed components of maternal responsiveness. The optimal nature of neonates and the general attractiveness of a wider range of pup ages as stimuli for the elicitation of maternal behavior in rats, as well as comparisons to mice and hamsters, were discussed.     

Investigation of the genotoxic effect of microwave irradiation in rat bone marrow cells: in vivo exposure

An in vivo mammalian cytogenetic test (the erythrocyte micronucleus assay) was used to investigate the extent of genetic damage in bone marrow red cells of rats exposed to radiofrequency/microwave (RF/MW) radiation. Wistar rats (n = 40) were exposed to a 2.45 GHz continuous RF/MW field for 2 h daily, 7 days a week, at a power density of 5-10 mW/cm(2). The whole body average specific absorption rate (SARs) was calculated to be 1.25 +/- 0.36 (SE) W/kg. Four subgroups were irradiated for 4, 16, 30 and 60 h. Sham-exposed controls (n = 24) were included in the study. The animals of each treated subgroup were killed on the final day of irradiation. Bone marrow smears were examined to determine the extent of genotoxicity after particular treatment times. The results were statistically evaluated using non-parametric Mann-Whitney and Kruskal-Wallis tests. In comparison with the sham-exposed subgroups, the findings of polychromatic erythrocytes (PCE) revealed significant differences (P < 0.05) for experimental days 8 and 15. The frequency of micronucleated PCEs was also significantly increased on experimental day 15 (P < 0.05). Pair-wise comparison of data obtained after 2, 8 and 30 irradiation treatments did not reveal statistically significant differences between sham-exposed and treated subgroups. Under the applied experimental conditions the findings revealed a transient effect on proliferation and maturation of erythropoietic cells in the rat bone marrow and the sporadic appearance of micronucleated immature bone marrow red cells.     

Maintenance of local cerebral blood flow after acute neuronal death: possible role of non-neuronal cells

In brain, a major factor regulating local perfusion is local neuronal activity. However, we have recently discovered that, in rat, five days after selective neuronal destruction in the parietal cortex by local microinjections of the excitotoxin ibotenic acid, local cerebral blood flow, within the lesion, remains in the normal range. We studied whether proliferating non-neuronal cells and/or local changes in microvascular density participate to maintain local cerebral blood flow. Rats were anesthetized (halothane 1-3%), ibotenic acid (10 micrograms in 1 microliter) was locally microinjected in a restricted region of the parietal cortex, and animals were allowed to recover. Three, five, seven, 11, 30 days later local cerebral blood flow was measured autoradiographically under chloralose anesthesia (40 mg/kg, s.c.) by the [14C]iodoantipyrine technique. Cellular density or microvascular area were determined on sections stained with Thionine or processed for the endothelial marker alkaline phosphatase, respectively. Local neurons were destroyed by 24 h after microinjections of ibotenic acid. However, from three to 11 days after lesion local cerebral blood flow was unchanged (P greater than 0.05; n = 5), thereafter declining so that by 30 days blood flow was 48 +/- 6% of control (P less than 0.05; n = 5). Cellular density increased within the lesion by 17.5-fold at seven to 11 days (P less than 0.01) and declined to a 11.7-fold elevation above control at day 30 (P less than 0.01). New cells consisted of macrophages, endothelium and glial fibrillary acidic protein-positive astrocytes. The microvascular area increased 4.2-fold from three to 11 days (P less than 0.01). The patency of the presumably newly formed vessels was determined by the presence of intravascular red blood cells, which were revealed histochemically. The area occupied by red blood cells within cerebral microvessels, in contrast to microvascular area, did not increase until seven days after lesion, reaching a 3.2-fold increase at 11 days. Thus within the lesion, local cerebral blood flow remains constant during the phase in which cellular and microvascular density increases. The presumably newly formed vessels cannot contribute to maintain local cerebral blood flow since during this phase they are not patent; rather patency develops coincident with the decline in local cerebral blood flow. We conclude that non-neuronal cells, most likely activated macrophages, may be an important factor regulating local cerebral perfusion, after acute neuronal death.     

Seminiferous epithelium damage after short period of busulphan treatment in adult rats and vitamin B12 efficacy in the recovery of spermatogonial germ cells

Several different strategies have been adopted in attempt to recover from chemotherapy‐damaged spermatogenesis that is often seen in oncologic patients. In this study, we have evaluated the impact of short period of exposure to busulphan on the haemogram and seminiferous epithelium of adult rats, focusing on spermatogonial depletion and Sertoli cell (SC) integrity. We then examined whether vitamin B₁₂ supplementation improves the haematological parameters and spermatogonia number. The animals received 10 mg/kg of busulphan (BuG) or busulfan+vitamin B₁₂ (Bu/B₁₂G) on the first and fourth days of treatment. In H.E.‐stained testicular sections, the areas of the seminiferous tubule (ST) and seminiferous epithelium were measured. The number of spermatogonia in H.E‐stained and PCNA‐immunolabelled testicular sections was quantified. The frequency of tubules with abnormal SC nuclei or TUNEL‐positive SC was evaluated. Vimentin immunofluorescence in ST was also evaluated. In BuG and Bu/B₁₂G, the animals showed leukopenia and thrombocytopenia, but the body weight reduced only in BuG. The areas of ST and seminiferous epithelium decreased in Bu/B₁₂G and BuG. In BuG, the number of H.E.‐stained and PCNA‐immunolabelled spermatogonia reduced significantly. The frequency of tubules containing abnormal SC nuclei and TUNEL‐positive SC increased and the vimentin immunoexpression pattern changed. In Bu/B₁₂G, the number of H.E.‐stained or PCNA‐immunolabelled spermatogonia increased fourfold in comparison with BuG. The structural changes in ST after 6 days of busulphan exposure may be associated with the potential effect of this anti‐neoplastic agent on SC. The increased number of spermatogonia in the busulphan‐treated animals receiving vitamin B₁₂ indicates that this vitamin can be an adjuvant therapy to improve the fertility in male cancer patients.     

Responsiveness of testis morphology to chemotherapy in childhood leukemia

The appearance of seminiferous tubules and interstitial cells of children, aged 2.5 to 13 years, affected by acute lymphoblastic leukemia was analyzed in sections. The testicular biopsies were performed at the end of therapy (vincristine, prednisone, L-asparaginase, 6-mercaptopurine, intrathecal methotrexate), which was affected for the same period and at the same doses. Three age groups were considered (I, 2.5 to 5 years; II, 6 to 9 years; III, 12 to 13 years). Age groups I and II presented damage of some tubules (25-35%) and areas of degeneration. Histometric analysis performed for A type spermatogonial population gave a mean value corresponding to controls in age group I and a mean value significantly lower with respect to controls in age group II. Moreover, age group II presented a lack of increase in tubular cross section. These results suggest that there is a vulnerability both of whole tubules and of some areas of Sertoli cells and germ cels to cytotoxic-induced damage. Leydig cells appear to be the cells least sensitive to drugs, and hormonal data indicate that the hypothalamic pituitary function appears to be intact, despite chemotherapy. Long-term prospective studies of reproductive function in children receiving cancer chemotherapy are needed to determine the magnitude and duration of damage resulting from therapeutic treatment.     

Influence of strong magnetic fields on genetic endpoints in Tradescantia tetrads and stamen hairs

Inflorescences from Tradescantia clones 4430 and 02 were subjected to mean magnetic field intensiteis of 0.16 or 0.76 to 0.78 tesla for 6 to 11 days. Following exposures to both intensities, damage to pollen mother cell chromosomes at early prophase was determined by scoring the frequency of micronuclei in early tetrads. Pink mutations in stamen hair cells of clone 4430 were scored for 15 days after a 6-day exposure to the higher intensity level while clone 02 was scored during and for 6 days after an 11-day exposure to the 0.16 tesla field. Comparison of micronuclei results from exposed groups to those for control groups that were scored concurrently showed that there was no significant difference in frequency for either clone or either field intensity (P values in contingency table analyses were between 0.42 and 0.6); similarly, pink mutation frequencies for clone 4430 exposed and control groups did not differ significantly (a contingency test yielded χ² = 1.4 and P = 0.26). Comparisons of pink mutation frequencies during the first 6 days of clone 02 exposure to those in the period when an effect should be greatest (days 7 through 17 revealed no significant difference (χ² = 2.6 and P = 0.1) between the two scoring periods, through frequencies were higher during the early scoring period.     

枸杞多糖对精原干细胞体外增殖的影响

背景：精原干细胞移植对不育具有潜在的临床应用价值,体外建立精原干细胞的培养系统获得数量较多的精原干细胞,仍是目前研究中亟待解决的问题。 目的：观察枸杞多糖对精原干细胞体外增殖的影响。 方法：采用两步酶消化法获取出生4~6 d雄性C57BL/6小鼠睾丸Sertoli细胞与精原干细胞,将精原干细胞接种在Sertoli细胞饲养层上,再加入枸杞多糖或联合细胞因子添加到细胞培养液中。1周后以流式细胞仪检测细胞周期及细胞活性率,并检测各组精原干细胞GFRa-1、Thy-1、c-kit的阳性率。 结果与结论：单独加入枸杞多糖后精原干细胞数量明显增加,增殖明显,联合加入胶质细胞源性神经营养因子与白血病抑制因子精原干细胞增殖更加明显(P < 0.05)。并发现体外培养1周后的精原干细胞仍保持其睾丸组织内的精原干细胞特征,大多仍维持在未分化状态。表明在枸杞多糖或枸杞多糖联合胶质细胞源性神经营养因子及白血病抑制因子作用下,可促进精原干细胞体外增殖。     

Interleukin 4 and Interferon Gamma Production in Restimulated CD4+ and CD8+ Cells Indicates Memory Type Responsiveness

Interleukin 4 (IL-4) and interferon gamma (IFN-gamma) production was analysed in murine spleen cells during primary and secondary mitogen stimulation in vitro. The kinetics, frequency and phenotype of single lymphokine-producing cells were studied by combining intracytoplasmatic immunofluorescence and surface staining. Both IL-4 and IFN-gamma was produced by CD4+ as well as CD8+ cells, however 75-80% of IL-4 producers were CD4+ and 90% of IFN-gamma+ cells were CD8+. In primary stimulations, concanavalin A (Con A) activation or anti-CD3 antibody together with phorbol 12-myristate 13-acetate (PMA) induced different patterns of lymphokine production. Approximately the same frequency of IFN-gamma+ cells was induced by both stimulation procedures but the kinetics was different with a peak at 30 h using Con A and at 52 h using anti-CD3 and PMA. IL-4 production peaked at 52 h, but the frequency of IL-4+ cells was 8-10 times higher after stimulation by anti-CD3 and PMA than after Con A stimulation. During restimulation of the mitogen activated cells, lymphokines were rapidly produced; both IL-4 and IFN-gamma production peaked at 8-11 h. Only a small increase in the frequency of IL-4+ cells was seen, at most two to three times. No evidence for a major shift of lymphokines produced between primary and secondary stimulations could be found. Instead, the pattern of lymphokine production induced by the primary stimulus was dominant also in secondary cultures irrespective of stimulation condition.     

Dose-dependent immune response to Mycobacterium bovis BCG vaccination in neonates

In 10-week-old infants vaccinated at birth with Japanese Mycobacterium bovis BCG, the number of dermal needle penetrations correlated positively with frequency of proliferating CD4(+) T cells in whole blood following BCG stimulation for 6 days but did not correlate with secreted cytokine levels after 7 h or interferon CD4(+) T-cell frequency after 12 h of BCG stimulation.     

Differential effects of satellite RNA on the accumulation of cucumber mosaic virus RNAs and their encoded proteins in tobacco vs zucchini squash with two strains of CMV helper virus

The presence of cucumber mosaic virus (CMV) satellite RNA usually reduces the yield of accumulated helper virus, although more so in solanaceous than in cucurbit hosts. The accumulation of viral RNA and viral-encoded proteins of two strains of CMV (Fny- and Sny-) known to differ in their ability to support satellite RNA in zucchini squash was examined in squash and tobacco to determine the effect of satellite RNA on the accumulation of viral-associated components. In the absence of satellite RNA, Fny- and Sny-CMV showed similar levels of accumulation of RNA at 7 days postinoculation (p.i.), but by 14 days p.i. the Fny-CMV RNAs accumulated to lower levels than did both strains at 7 days p.i., in either host. The levels of accumulated Sny-CMV-encoded proteins were higher than those encoded by Fny-CMV in tobacco, but not squash plants, at 7 days p.i. At 14 days p.i., for Fny-CMV vs Sny-CMV, there were differences in the levels of accumulation of most CMV-encoded proteins in both hosts, more exacerbated in tobacco vs squash. The effect of satellite RNA was to intensify these differences; that is, by 7 days p.i., satellite RNA reduced the accumulation of Fny-CMV RNAs 1 and 2 and their encoded proteins in both tobacco and squash but had little or no effect on the accumulation of Sny-CMV RNAs or encoded proteins. By 14 days p.i., the levels of accumulation of all Fny-CMV RNAs and encoded proteins were severely reduced in both hosts, and the levels of accumulation of Sny-CMV RNAs 1 and 2 and their encoded proteins were also reduced in tobacco, but not squash. Sny-CMV did not support satellite RNA accumulation in squash plants or protoplasts. Satellite RNA did not appear to have a direct effect on the movement of either CMV strain. Rather, accumulation studies in tobacco protoplasts indicated that the difference in response of Fny-CMV vs Sny-CMV to satellite RNA in tobacco was due to the extent to which satellite RNA affected the levels of RNA 1, and to a lesser extent RNA 2, and their encoded proteins, 1a and 2a, both components of the CMV replicase.     

Immunohistochemical staining of non-Hodgkin's lymphoma with monoclonal antibodies specific for the leucocyte common antigen

Forty cases of non-Hodgkin's lymphoma (NHL) have been stained with monoclonal antibodies (F8-11-13, F-10-89-4 and Dako LC) to the Leucocyte Common Antigen (LC) in both cryostat and paraffin sections with an immunoperoxidase technique. In cryostat sections all B-cell lymphomas (25/25) reacted with F10-89-4 and Dako LC, whilst the majority (23/25) stained with F8-11-13; of the T-cell lymphomas studied, all reacted with F10-89-4 (6/60 and Dako LC (4/4), however 2/6 did not react with F8-11-13. Similar variability in reaction was observed in malignant histiocytosis where 1/3 did not react with F8-11-13 whilst all three reacted with F10-89-4 and Dako LC. In paraffin sections (using the two MCabs F8-11-13 and Dako LC) three of the 25 B-cell lymphomas failed to stain with either F8-11-13 or Dako LC (one lymphocytic lymphoma and two lymphomas showing plasmacytic differentiation). The remainder of the B-cell lymphomas reacted with both antibodies. Six out of 12 T-cell lymphomas did not stain with either F8-11-13 or Dako LC, using our standard immunoperoxidase procedure. Staining with Dako LC was however detected in all cases of T-cell lymphoma when incubation with primary antibody was extended from thirty minutes (standard) to overnight. This study confirmed that LC can be detected in all NHL in cryostat sections, and that in the majority of B-cell NHL the higher molecular weight component of LC was demonstrable using F8-11-13. Difficulty in detecting LC determinants after tissue processing for paraffin sections in a number of cases of NHL, especially those of T-cell type or showing plasmacytic differentiation, suggests that lack of reaction with these antibodies does not always preclude the diagnosis of lymphoma.     

Early induction of genetic instability and apoptosis by arsenic in cultured Chinese hamster cells

In order to assess at what time from the beginning of exposure inorganic arsenic can give rise to genetic instability and trigger apoptosis, V79-C13 Chinese hamster cells were treated with 10 microM sodium arsenite for 24 h. Under these conditions, cell survival was >70% and cells showed neither an increase in chromosome aberration frequency nor a delay in cell cycle progression. Investigations, which were carried out every 6 h during the treatment, revealed an early appearance of genetically unstable cells, namely micronucleated, multinucleated and mononucleated 'giant' cells, as well as apoptotic cells. Indirect immunostaining using anti-beta-tubulin antibody showed severe alterations in spindle morphology after only 6 h treatment, when cells with small spindles whose poles were inside the metaphase plate appeared, and after 12 h treatment, when cells in which spindle assembly had completely failed were observed. These cells, unable to complete mitosis, underwent apoptosis. In fact, cells which turned out to be positive in the TdT-FragEL test had condensed chromatin arranged in metaphase-like plates; their maximum frequency was reached after 24 h treatment. A cytogenetic study was conducted at the end of the period of exposure to arsenic and after post-treatment incubation in fresh medium for up to 5 days. It showed that the percentage of cells with 21 chromosomes (modal number of the cell line) decreased, making way for aneuploid cells. Arsenic, therefore, induced early genetic instability or apoptosis in dividing cells. However, while apoptosis tended to cease when arsenic was removed from the culture medium, the acquired instability remained and propagated within the cell population.