Effects of retinoids on the in vitro development of Capra hircus embryos to blastocysts in two different culture systems.

The objective of this study was to evaluate the effect of retinol (RT) and retinoic acid (RA) on the in vitro development of pre-implantation goat embryos cultured in potassium simplex optimized medium or synthetic oviduct fluid or cocultured in oviductal cells monolayer either in potassium simplex optimized medium or synthetic oviduct fluid. A total of 2407 cumulus-oocyte complexes were aspirated from 2 to 6 mm ovarian follicles from slaughtered animals. Selected cumulus-oocyte complexes were subjected to in vitro maturation in TCM 199 for 24 h at 39 °C in an atmosphere of 5% (v/v) CO(2) in humidified air. In vitro fertilization was performed in modified defined medium. Eighteen hours after in vitro fertilization, cumulus cells were removed and presumptive zygotes were randomly distributed into experimental groups. In Experiment 1, presumptive zygotes were cultured in potassium simplex optimized medium, potassium simplex optimized medium + RT, potassium simplex optimized medium + retinoic acid, synthetic oviduct fluid, synthetic oviduct fluid + RT and synthetic oviduct fluid + RA at 39 °C in a humidified atmosphere of 5% (v/v) CO(2), 5% (v/v) O(2) and 90% (v/v) N(2). In Experiment 2, presumptive zygotes were cocultured in potassium simplex optimized medium + oviductal cells monolayer, potassium simplex optimized medium + RT + oviductal cells monolayer, potassium simplex optimized medium + RA + oviductal cells monolayer, synthetic oviduct fluid + oviductal cells monolayer, synthetic oviduct fluid + RT + oviductal cells monolayer and synthetic oviduct fluid + RA + oviductal cells monolayer in an atmosphere of 5% (v/v) CO(2) in humidified air. In both experiments, media were partially changed on day 2 after in vitro fertilization and unfertilized oocytes were excluded from the experiment. Embryos were cultured or cocultured for 8 days. In Experiment 1, there was no effect of RT or RA supplementation on the proportion of oocytes that reached the morula or blastocyst stages. By contrast, Experiment 2 demonstrated that the addition of 0.28 µg/ml RT and 0.5 µm RA to the embryo culture media stimulated (p < 0.05) development to the morula and blastocyst stages under the coculture conditions tested. In conclusion, retinoids play an important role in pre-implantation development of goat embryos and can be used to enhance in vitro embryo production.

Anti-melanoma monoclonal antibody HMB-45 on enhanced chemiluminescence-western blotting recognizes a 30-35 kDa melanosome-associated sialated glycoprotein.

HMB-45 is an anti-melanoma monoclonal antibody widely used in diagnostic pathology owing to its great specificity in identifying poorly differentiated melanomas. In this study, by a series of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblots with the enhanced chemiluminescent (ECL) detection method on the HU-214 melanoma cell line, we identified the antigen of HMB-45 in a protein or proteins of 30-35 kDa. Although this result is in discrepancy with the previous literature which identified the antigen as a protein of 7 or 10 kDa, a family of proteins of 25-70 kDa of as a protein of 100 kDa (gp100), the present data indicate that the antigen signal we found might be specific. Furthermore, immunoblots on neuraminidase-treated cell lysates show, in agreement with already published data, that the antigen might be a sialated glycoprotein with the sialic acid involved in the epitope. Immunoblots on partially purified melanosomes confirmed the presence of the antigen in these organelles.

Molecular analysis of six variant Philadelphia chromosome translocations in chronic myeloid leukemia.

In 420 Philadelphia positive (Ph+) chronic myeloid leukemia (CML) patients karyotyped at diagnosis in our laboratory, 26 Ph variants (6.2%) were observed. Twelve of them are reported. Five cases are "simple" variants without detectable involvement of band 9q34, and seven are "complex," since a third chromosomal band is involved in the Ph formation. Two translocations [t(7;22)(q36;q11) and t(9;22;12)(q34;q11;q11)] are reported for the first time. Six cases were characterized molecularly, and bcr-abl rearrangement was demonstrated, confirming involvement of 9q34 band also in the cases in which chromosomes 9 appear cytogenically normal. Chimeric mRNAs in which M-BCR exon 3 is joined to abl exon 2 (type b3-a2) were detected in four of six cases; one case showed a DNA breakpoint in zone III, which may also give rise to the same transcript. In one case, mRNA junction was b2-a2. The frequency of the b3-a2 junction occurs more frequently in CML patients with a Ph variant than in patients with the standard translocation, suggesting a preferential correlation between this type of transcript and the involvement of other chromosomes in Ph formation.

Clinical value of PCR in diagnosis and follow-up of leukaemia and lymphoma: report of the third Workshop of the Molecular Biology/BMT study group.

The proceedings of the third workshop of the molecular biology/bone marrow transplantation (BMT) study group held in January 1991 in Verona, Italy. This workshop was convened to review progress in the application of molecular techniques to the diagnosis and follow-up of patients with chronic myeloid leukaemia (CML) as well as other haematologic malignancies. The results of polymerase chainreaction studies in 157 CML patients 1-90 months post BMT suggest that leukaemia is frequently detectable for the first 12 months but rarely detected thereafter except in patients known to have a high risk of relapse. In the acute leukaemias and lymphomas there is a rapidly increasing number of leukaemia-specific as well as clone-specific molecular markers now available for the detection of minimal disease. It may be possible to coordinate multi-center prospective studies to investigate the role of these markers in the diagnosis and follow-up of haematologic malignancies.

An assessment of chimeric transcript detection in CML patients after bone marrow transplantation.

The Polymerase Chain Reaction (PCR) was used to evaluate minimal residual disease in 21 Ph+ CML patients at various intervals after allogeneic bone-marrow transplantation (ABMT) by amplification of bcr-abl cDNA. All patients were cytogenetically Ph- at the moment of molecular analysis. Of these 76% were PCR negative, 24% positive for bcr-abl transcripts. 100% of the Cyclosporine A/Methotrexate treated patients (7/7) were negative. Severe chronic GvHD was twice as frequent in PCR positive patients (60%) than in negative ones (31%). The only patient who relapsed during follow up was PCR positive. The two longest survivors were PCR negative. These data are still insufficient for assessing the predictive value of PCR analysis in CML. Patients. 25 patients with Ph+ CML at diagnosis were enrolled in this study. Two died soon after BMT because of infection for failure of engraftment/early relapse, two were Ph chromosome positive and PCR+, and were therefore dismissed from this study. All remaining 21 patients were cytogenetically Ph- at the time of molecular analysis and underwent ABMT from matched donors. All patients were conditioned with cyclophosphamide and TBI: 330 cGy the three days prior to transplantation (990 cGy total, treatment B), or 200 cGy two times daily for three days (1200 cGy total, treatment A). In 3 cases the marrow was treated for GvHD prophilaxis with Campath alone or Campath plus BT 5/9 monoclonal antibodies (1). All patients were treated with Cyclosporin A (CS) 5 mg/kg i.v. from the day prior to transplantation until 25-30 days after; 9 of these were treated with CS plus Methotrexate (MTX).