Carla Santana

Induced mitotic death of HeLa cells by abnormal expression of c-H-ras

When HeLa cells were selected for stable expression of a neo gene, linked either to mutated or wt c-H-ras genes, morphological examination of selected clones from several experiments revealed formation of giant multinucleated cells. These morphological alterations culminate in cell death, as a consequence of mitotic catastrophe (or mitotic death). Although clones expressing the mutated gene produced significantly larger numbers of these giant cells, those transfected with the normal allele were also found to produce significantly more giant multinucleated cells than non-transfected HeLa cells. Northern blot analysis of mRNA revealed overexpression of the normal H-ras gene in these clones. Chromatin structure analysis of these clones showed gross alterations, including the presence of micronuclei and heteroploid nuclei. Interestingly, odd numbers of nuclei were found in colonies of these giant cells. In addition, alterations in cell cycle parameters were observed, including the appearance of a subpopulation of cells with an abnormal content of DNA, probably representing dying cells. Our data support the notion that abnormal expression of H-ras contributes to mitotic catastrophe and death of a subpopulation of HeLa cells.

Protein tyrosine phosphorylation in leukocyte activation through receptors for IgG.

Membrane receptors for the Fc portion of immunoglobulin G (IgG) antibodies (FcγRs) are expressed on almost every type of hematopoietic cells, where they mediate a wide variety of effector functions. A high degree of structural heterogeneity exists among FcγRs. The biological significance of such heterogeneity is unknown, since the structural diversity does not appear to be reflected in the binding specificity nor in the effector functions that each distinct receptor is able to mediate. Recent work has emphasized the essential role of protein tyrosine phosphorylation in the initiation of trans‐membrane signaling by these receptors. In this article we review the role of protein tyrosine phosphorylation in signal transduction by the different types of FcγRs in order to assess to what extent the structural heterogeneity of this receptor family is related to different activation pathways utilized by each of its members. J. Leukoc. Biol. 60: 433–440; 1996.

Oncogenic H-ras induces cyclin B1 expression in a p53-independent manner

The role of p53 in controlling the G2 checkpoint, in part by repressing cyclin B1 transcription, has been well established. However, accumulating evidence indicate that p53-independent pathways may also play an important role. Ras proteins have been shown to regulate G1/S, but also G2/M transitions. Since cyclin B1/cdc2 complex is the key regulator controlling the G2/M checkpoint, we were interested in addressing if the H-ras oncogene could regulate cyclin B1 expression in a p53-independent manner. We observed an induction of cyclin B1 promoter activity in the presence of H-ras oncogene in SW480 cells, which contain null p53 alleles. In addition, HeLa cells known to express the HPV18 E6 oncogene that inactivates p53, exhibited increased levels of cyclin B1 mRNA and protein when transfected with the H-ras oncogene. Higher expression of cyclin B1 correlated with higher levels of cyclin B1/cdc2 complex and kinase activity that interestingly, showed no inhibition at G2/M after DNA damage. These data suggest that H-ras participates in pathways that regulate cyclin B1 expression and therefore controls the G2/M checkpoint in a p53-independent manner.

Anthracyclines: isolation of overproducing strains by the selection and genetic recombination of putative regulatory mutants of Streptomycespeucetius var. caesius

In Streptomyces peucetius var. caesius, the production of anthracyclines was suppressed either by 330 mM d-glucose or 25 mM phosphate. In addition, the anthracycline doxorubicin and the glucose analogue 2-deoxyglucose inhibited the growth of this microorganism at concentrations of 0.025 mM and 10 mM respectively. Spontaneous and induced mutants, resistant to the action of these compounds, were isolated, tested and chosen by their ability to overproduce anthracyclines. Genetic recombination between representative mutants was carried out by the protoplast fusion technique. Some recombinants carrying resistance to doxorubicin, phosphate and 2-deoxyglucose produced more than 40-fold greater levels of anthracyclines than those obtained with the parental strain. This improvement resulted in total antibiotic titres of more than 2 g/l culture medium at 6 days of fermentation.

Androgen receptor CAG polymorphism and sporadic and early-onset prostate cancer among Mexican men.

A short CAG repeat length in the gene encoding for the androgen receptor (AR) has been associated with prostate cancer (PC) risk and aggressiveness. In Latino men, information on this association is scarce. Hence, the aim of this study was to evaluate this association in Mexican males. Using fragment analysis by capillary electrophoresis, we determined the number of CAG repeats—(CAG)n—in AR gene from 158 incident PC cases and 326 age-matched healthy controls (±5 years), residing in Mexico City, Mexico. According to Gleason scale and age at diagnosis, cases were classified as high (⩾7) and low grade (<7), as well as early onset (<60 years) or late onset PC (⩾60 years). At diagnosis, 78% of cases were classified as high-grade and 26.6% as early onset. Men with sporadic (no family history of PC) and early-onset PC presented shorter CAG repeat length than controls (18.6±2.2 vs 19.5±2.5; P=0.02). Lower number of CAG repeats (CAG)⩽19 were associated with a greater risk for early-onset PC (odds ratio: 2.31; 95% confidence interval: 1.14–4.69). CAG repeat length could increase the risk for sporadic and early-onset PC. The best cutoff point for identifying at-risk subjects was (CAG)19. However, further studies are necessary to replicate our findings in subjects with a family history of PC and also to evaluate the association between CAG repeats length and disease progression.

Protocols for the Manual and Automatic Microinjection of Somatic Cells in Culture and for the Analysis of Microinjected Cells

Micromanipulation of living cells was first used in electrophysiology studies during the first half of this century. Reliable techniques for intracellular recording were developed in the late 1940s. Less than one decade later, similar techniques were applied to perform direct transfer of biological material into cells (see Chambers and Chambers 1961). Initially, microinjection was designed for transplantation of mammalian nuclei (Graessmann 1970) and chromosomes (Diacumakos 1973) into recipient cells in culture. Further refinement of materials and instruments, however, permitted intracellular transfer of solutions containing macromolecules of various origins, such as viral RNA (Graessmann and Graessmann 1976; Stacey et al. 1977), purified proteins (Mabuchi and Okuno 1977; Tjian et al. 1978), and viral DNA (Anderson et al. 1980; Capecchi 1980). Likewise, microinjection was used for DNA transfer into fertilized eggs and a cloned gene was soon reported to be expressed in mouse somatic tissues (Gordon et al. 1980). Later, recombinant genes were stably introduced into the mouse germ line (Brinster et al. 1981; Wagner et al. 1981). Since then, microinjection has been used routinely to generate transgenic animals, and applied to a wide variety of studies using living cells, such as gene expression, signal transduction, or cytoskeleton studies.

Association of vWA and TPOX Polymorphisms with Venous Thrombosis in Mexican Mestizos

Objective. Venous thromboembolism (VTE) is a multifactorial disorder and, worldwide, the most important cause of morbidity and mortality. Genetic factors play a critical role in its aetiology. Microsatellites are the most important source of human genetic variation having more phenotypic effect than many single nucleotide polymorphisms. Hence, we evaluate a possible relationship between VTE and the genetic variants in von Willebrand factor, human alpha fibrinogen, and human thyroid peroxidase microsatellites to identify possible diagnostic markers. Methods. Genotypes were obtained from 177 patients with VTE and 531 nonrelated individuals using validated genotyping methods. The allelic frequencies were compared; Bayesian methods were used to correct population stratification to avoid spurious associations. Results. The vWA-18, TPOX-9, and TPOX-12 alleles were significantly associated with VTE. Moreover, subjects bearing the combination vWA-18/TPOX-12 loci exhibited doubled risk for VTE (95% CI = 1.02–3.64), whereas the combination vWA-18/TPOX-9 showed an OR = 10 (95% CI = 4.93–21.49). Conclusions. The vWA and TPOX microsatellites are good candidate biomarkers in venous thromboembolism diseases and could help to elucidate their origins. Additionally, these polymorphisms could become useful markers for genetic studies of VTE in the Mexican population; however, further studies should be done owing that this data only show preliminary evidence.