The effect of ultrasound on human chromosomes in vitro

Human Genetics - Human peripheral blood and cultured human lymphocytes undergoing the first mitosis were exposed to ultrasound in order to find out whether ultrasound can induce chromosome...     

The effects of busulphan on the chromosomes of normal human lymphocytes

In vitro exposure of human lymphocytes to busulphan (BUS) produced an increase in chromosome aberrations and in sister-chromatid exchange (SCE) frequency. The distribution of chromosome breaks throughout the karyotype was non-random and they occurred mainly in the G-negative bands. Certain bands had a marked susceptibility to BUS and comparisons with the human chromosome-break distributions reported for a number of drugs revealed that some of these bands were equally susceptible to other alkylating agents. Both the number of chromosome gaps and breaks and the SCE frequency increased with BUS concentration, but only the SCE dose-response was a clearly defined linear relationship. Therefore a standard SCE dose-response curve was constructed for future comparison with the results of similar investigations of patients on BUS therapy.     

The effects of busulphan on the chromosomes of normal human lymphocytes

In vitro exposure of human lymphocytes to busulphan (BUS) produced an increase in chromosome aberrations and in sister-chromatid exchange (SCE) frequency. The distribution of chromosome breaks throughout the karyotype was non-random and they occurred mainly in the G-negative bands. Certain bands had a marked susceptibility to BUS and comparisons with the human chromosome-break distributions reported for a number of drugs revealed that some of these bands were equally susceptible to other alkylating agents. Both the number of chromosome gaps and breaks and the SCE frequency increased with BUS concentration, but only the SCE dose--response was a clearly defined linear relationship. Therefore a standard SCE dose--response curve was constructed for future comparison with the results of similar investigations of patients on BUS therapy.     

The location of four human satellite DNAs on human chromosomes.

In situ hybridisation was carried out with ³H-cRNAs transcribed from four human satellite DNAs. The human metaphase chromosomes used were stained with quinacrine and photographed prior to hybridisation. This allowed accurate karyotyping of the autoradiographs. A method of quantitative analysis of grain distribution permitted identification of minor sites of hybridisation which could be distinguished from purely random grains. The hybridisation patterns for each of the transcribed satellite cRNAs were similar, with the C-band of chromosome 9 and the Y chromosome being the most heavily labelled sites. Other detectable sites of hybridisation were the centromeric regions of chromosomes 1, 5, 7, 10, 12, 13, 14, 15, 17, 20, 21 and 22. The cRNA transcribed from DNA satellite II, however, was the only one to hybridise to the centromere region of chromosome 16. The evolution of the human satellite DNAs and the validity of current models of satellite DNA function are discussed in the light of the present results.     

The microtubular cytoskeleton and chromosomes of unfertilized human oocytes aged in vitro

Summary To detect structural alterations in human oocytes that may give rise to predisposition to aneuploidy, unfertilized human oocytes from an IVF programme were processed for indirect anti-tubulin immunofluorescence. The spindle of oocytes aged for 2 days is rather small, and bi- or multipolar. Chromosomes are no longer aligned at the spindle equator but are scattered all over the degenerating spindle. This implies that human oocytes aged for 2 days may no longer be able to develop into a chromosomally balanced, normal embryo. In oocytes aged for 3–4 days the chromosomes become more decondensed and form a restitution nucleus. Microtubules radiate out from the latter towards the cell periphery and form a network of fibres in the cytoplasm. A similar alignment of tubules is found in unfertilized, activated oocytes. Oocytes with an aberrant cytoskeleton and chromosomal array were predominantly obtained from aged females. They include two binucleated oocytes with two sets of chromosomes and two oocytes with displaced chromosomes one of which had a tripolar spindle.     

The effects of crude and purified human gonadotropin on in vitro stimulated human lymphocyte cultures.

Crude human chorionic gonadotropin (hCG) was found to be several fold more immunosuppressive than purified hCG in human peripheral blood lymphocyte cultures stimulated by phytohemagglutinin, pokeweed, purified protein derivative and allogeneic cells in vitro. Immunosuppression by crude hCG was consistently noted at levels less than 1000 IU/ml and usually 80% inhibition was achieved with doses of 5000–10,000 IU/ml, whereas 40–50% inhibition or less was observed by purified hCG at 10,000 IU/ml. In two crude hCG preparations subjected to Sephadex G-100 chromatography, the fractions that inhibited lymphocyte cultures appeared in the eluate after the major peak of hCG activity. These data indicate that inhibitory substance(s) other than hCG are responsible for most of the immunosuppressive properties of first trimester pregnancy urine. Both crude and purified hCG were stimulatory to human lymphocytes when used alone without mitogens when cultured in fetal calf serum.     

Radiation protection of human lymphocyte chromosomes in vitro by orientin and vicenin.

Orientin (Ot) and Vicenin (Vc), two water-soluble flavonoids isolated from the leaves of Indian holy basil Ocimum sanctum have shown significant protection against radiation lethality and chromosomal aberrations in vivo. In the present study the protective effect of Ot and Vc against radiation induced chromosome damage in cultured human peripheral lymphocytes was determined by micronucleus test. In order to select the most effective drug concentration, fresh whole blood was exposed to 4Gy of cobalt-60 gamma-radiation with or without a 30 min pre-treatment with 6.25, 12.5, 15.0, 17.5 or 20 microM of Ot/Vc. Micronucleus (MN) assay was done by cytochalasin induced cytokinesis block method. Radiation significantly increased the MN frequency (16 times normal). Pre-treatment with either Ot or Vc at all concentrations significantly (P<0.05-0.001) reduced the MN count in a concentration dependent manner, with the optimum effect at 17.5 microM. Therefore, fresh blood samples were incubated with/without 17.5 microM Ot/Vc for 30 min and then exposed to 0.5-4Gy of gamma-radiation. Radiation increased the MN frequency linearly (r(2)=0.99) with dose. Pre-treatment with Ot or Vc significantly (P<0.01-0.001) reduced the MN counts to 51-67% of RT alone values, giving DMFs of 2.62 (Ot) and 2.48 (Vc). Both the compounds showed significant antioxidant activity in vitro at the above concentrations, which was significantly higher than that of DMSO at equimolar concentrations. Thus, the results demonstrate that both the flavonoids give significant protection to the human lymphocytes against the clastogenic effect of radiation at low, non-toxic concentrations. The radioprotection seems to be associated with their antioxidant activity. The clinical potential of these protectors in cancer therapy needs to be investigated.     

Studies on the chromosomes of human lymphocytes treated with diazepam in vitro

Human lymphocytes were exposed in vitro to various concentrations of the psychotherapeutic agent diazepam, using two different techniques for culturing the lymphocytes, a whole-blood and a macro procedure. There was no evidence for a damaging effect of diazepam on the lymphoblast chromosomes at any concentration or exposure time studied with either technique of culture. The significance of results obtained in vitro on chromosome-breaking effects of chemical agents in commercial use is discussed, and the importance of some technical details in conducting such experiments is pointed out.