Analysis of sister chromatid exchanges in lymphocytes cultured from 71 healthy men

Sister chromatid exchanges (SCE) were analyzed in peripheral blood lymphocytes from a select group of 71 healthy men, 56 nonsmokers and 15 cigarette smokers. In addition to estimating baseline SCE, data were examined to seek relationships of SCE frequencies to age and smoking. The baseline value of 7.53 SCE per cell from the 56 nonsmokers was within the range (5.60 to 9.10 SCE/cell) reported for other human populations. No relationship was found between the mean SCE frequency per cell and age. However, a significant increase in the SCE mean value was observed in smokers as compared to nonsmokers. The results of this study are compared with those of other reports on SCE effects of age and smoking.Abbreviations BUdR 5-bromo,2-deoxyuridine - SCE sister chromatid exchange     

Evaluation of culture media for effects on cell cycle kinetics and incidence of chromosomal aberrations in human blood cells

Peripheral blood samples from 17 apparently healthy male volunteers were set up in duplicate cultures using three commercially available media: Eagle's MEM, RPMI 1640, and TC 199. BUdR (5-bromo,2-deoxyuridine) (10 micrograms/mL) was added to one of the cultures from each person in each medium after 24 h of culture initiation. All cultures were harvested at 72 h of incubation in the presence of colcemid. RPMI 1640 stimulated the highest mitotic activity in both BUdR-treated and untreated cultures. Higher numbers of first division metaphases corresponded with the higher frequency of chromosome-type aberrations in cultures with Eagle's MEM as compared with RPMI 1640 media. On the other hand, higher numbers of chromatid-type aberrations were present in cultures with TC 199 as compared with those with Eagle's MEM. When the chromosome- and chromatid-type aberration data were pooled to score total cytogenetic abnormalities, an influence of the medium was demonstrable. While cultures with Eagle's MEM and TC 199 had the greater number of first division cells, third of subsequent division cells were most prevalent in RPMI 1640 cultures. It is inferred that the length of the cell cycle, the mitotic index, and to some degree the incidence of spontaneous cytogenetic abnormalities are variable attributes of culture media.     

T cell induction of precursor B cells: Temporal separation of helper T cell functional activities

Summary The temporal relationships involved in T cell induction of immunoglobulin-secreting B cells have been studied by employing a pulse label technique, in vitro. It was shown that addition of rabbit thymocytes or splenic T cells to B cell-enriched splenocyte populations at the time of initiation of cultures resulted in a marked enhancement in induction of immunoglobulin-secreting cells. However, even a two-hour delay in the addition of thymus cells was sufficient to reduce substantially the extent of induction when measured 70 hours later. Besides this early requirement for thymocytes, a late requirement was also detectable. Thus, thymus cells and splenocyte populations upon being mixed, subsequent to being cultured separately for 72 hours, yielded a several-fold enhancement in [³H]-immunoglobulin secreted during the course of a 90-minute labeling period with [³H]-leucine. Moreover, both the early and late thymocyte effects were lost after treatment with anti-thymocyte serum and complement.The thymocyte-mediated enhancement of immunoglobulin secretion by splenocytes that occurs late in the induction process was detected with spleen cells cultured for two or three days but not with freshly-isolated splenocytes. Although the rate of appearance of extracellular immunoglobulins was markedly enhanced by fresh thymus cells, the rate of appearance of intracellular immunoglobulins in such spleen cells was unchanged. The secretion-stimulating (secretagogue) activity of thymocytes appeared to be specific in that thymus cells were without effect on the rate of secretion of serum albumin by liver cells.In regard to the induction of immunoglobulin-secreting cells, both B and T cell-enriched population's were sensitive to mitomycin C treatment performed before initiation of cell culture, indicating that not only B cells but also T cells undergo some form of differentiation or maturation prior to functioning in the induction of immunoglobulin-producing cells. It should be noted in this context that the late T cell requirement was unaffected by prior mitomycin C treatment of thymocytes. On the other hand, thymocytes heated at 60°C for 5 minutes did not enhance immunoglobulin secretion when added at any time and the late thymocyte requirement could not be replaced with medium in which thymocytes had been previously cultured.     

Mutagenic effects of sodium azide in Drosophila melanogaster.

The mutagenic effects of sodium azide (NaN3) were studied at low pH in male Drosophila melanogaster using the sex-linked recessive-lethal test. No significant increase in the mutation frequency was observed after abdominal injection of azide solutions buffered at either pH 3.8 OR 4.6. However, a weak mutagenic effect was noticed in the flies fed for 3 days on 0.1 mM azide (pH 4.6) solution.     

A novel early effect of concanavalin A on thymocytes.

Concanavalin A, added to freshly isolated rabbit thymocytes, markedly enhanced the extracellular appearance of non-immunoglobulin proteins. Time course studies revealed that the onset of enhancement occurred virtually without delay. The effect appeared to be restricted only to certain of the thymus-derived cells because thymocytes obtained from rabbits treated with hydrocortisone, as well as splenocytes derived from untreated rabbits essentially did not exhibit the enhancement. Stimulation by concanavalin A was specific in that pokeweed mitogen and lipopolysaccharide were without effect and also in that α-methyl-mannoside, but not galactose, abrogated the concanavalin A-mediated enhancement. Experiments with mouse thymocytes demonstrated that the cells which responded to concanavalin A were primarily cells that bear the θ-antigen on their surface (T-cells).     

Focused ultrasound stimulation on meibomian glands for the treatment of evaporative dry eye

Current treatments for meibomian gland dysfunction have several limitations, creating a necessity for other advanced treatment options. The purpose of this study is to determine the effectiveness of focused ultrasound stimulation for the treatment of dry eye disease caused by meibomian gland dysfunction. An in vivo study of nine Dutch Belted rabbits was conducted with focused ultrasound stimulation of the meibomian glands. A customized line-focused ultrasonic transducer was designed for treatment. Fluorescein imaging, Schirmer’s test, and Lipiview II ocular interferometer were used to quantify outcomes from three aspects: safety, tear production, and lipid layer thickness. Both tear secretion and lipid layer thickness improved following ultrasound treatment. Five to 10 min after the ultrasound treatment, the mean values of lipid layer thickness increased from 55.33 ± 11.15 nm to 95.67 ± 22.77 nm (p < 0.05), while the mean values measured with the Schirmer’s test increased from 2.0 ± 2.3 to 7.2 ± 4.3 (p < 0.05). Positive effects lasted more than three weeks. Adverse events such as redness, swelling, and mild burn, occurred in two rabbits in preliminary experiments when the eyelids sustained a temperature higher than 42°C. No serious adverse events were found. The results suggest that ultrasound stimulation of meibomian glands can improve both tear production and lipid secretion. Ultimately, ultrasound stimulation has the potential to be an option for the treatment of evaporative dry eye disease caused by meibomian gland dysfunction.     

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.     

WNT1-inducible signaling pathway protein-1 activates diverse cell survival pathways and blocks doxorubicin-induced cardiomyocyte death

The anthracycline antibiotic doxorubicin (DOX) is a potent cancer chemotherapeutic agent that exerts both acute and chronic cardiotoxicity. Here we show that in adult mouse cardiomyocytes, DOX activates (i) the pro-apoptotic p53, (ii) p38MAPK and JNK, (iii) Bax translocation, (iv) cytochrome c release, and (v) caspase 3. Further, it (vi) inhibits expression of anti-apoptotic Akt, Bcl-2 and Bcl-xL, and (vii) induces internucleosomal degradation and cell death. WNT1-inducible signaling pathway protein-1 (WISP1), a CCN family member and a matricellular protein, inhibits DOX-mediated cardiomyocyte death. WISP1 inhibits DOX-induced p53 activation, p38 MAPK and JNK phosphorylation, Bax translocation to mitochondria, and cytochrome c release into cytoplasm. Additionally, WISP1 reverses DOX-induced suppression of Bcl-2 and Bcl-xL expression and Akt inhibition. The pro-survival effects of WISP1 were recapitulated by the forced expression of mutant p53, wild-type Bcl-2, wild-type Bcl-xL, or constitutively active Akt prior to DOX treatment. WISP1 also induces the pro-survival factor Survivin via PI3K/Akt signaling. Overexpression of wild-type, but not mutant Survivin, blunts DOX cytotoxicity. Further, WISP1 stimulates PI3K–Akt-dependent GSK3β phosphorylation and β-catenin nuclear translocation. Importantly, WISP1 induces its own expression. Together, these results provide important insights into the cytoprotective effects of WISP1 in cardiomyocytes, and suggest a potential therapeutic role for WISP1 in DOX-induced cardiotoxicity.