Prevalence of human papillomavirus types in North and Central regions of Mexico.

Human papillomavirus (HPV) is a DNA virus linked to mucosal and cutaneous carcinogenesis. More than 200 different HPV types exist. We carried out a transversal study to investigate the prevalence of HPV types in two regions of Mexico. A total of 724 genital and non-genital samples from women (F) and men (M) were studied; 241 (33%) from North-Eastern (NE) and 483 (66%) from South-Central (SC) Mexico. The overall prevalence was 87%. In genital lesions from females, the NE group showed a prevalence of HPV types 16 (37%), 6 (13%), 59 (6%), 11, 18 and 66 (5.4% each); and the SC group showed types 6 (17%), 16 (15%), 11 (14.5%), 18 (12%) and 53 (6%). In the genital lesions from males, NE group showed types 16 (38%), 6 (21%), 11 (13%) and 59 plus 31 (7.5%) and the SC group showed types 6 (25%), 11 (22%), 18 (17%) and 16 (11.5%). When the two regions were compared, a higher prevalence of low-risk HPV 6 and 11 was found in the SC region and of high-risk HPV 59, 31 and 66 (the latter can also be present in benign lesions) in the NE region. Our findings complement efforts to understand HPV demographics as a prerequisite to guide and assess the impact of preventive interventions.

Polymorphisms in GSTM1, GSTT1, GSTP1, and GSTM3 genes and breast cancer risk in northeastern Mexico.

Glutathione S-transferases (GSTs) are a family of phase II metabolizing enzymes involved in carcinogen detoxification and the metabolism of various bioactive compounds. Several genes that code for these enzymes are polymorphic in an ethnicity-dependent manner, with particular genotypes previously associated with an increased risk of breast cancer. The purpose of this study was to determine the frequencies of polymorphisms in the genes GSTM1, GSTT1, GSTP1, and GSTM3 and to investigate whether an association exists between these genes and breast cancer risk in subjects from northeastern Mexico. Genotypes were determined for 243 women with histologically confirmed breast cancer and 118 control subjects. Gene polymorphisms were analyzed using a DNA microarray. We found an increased breast cancer risk associated with the GSTM1 gene deletion polymorphism (OR = 2.19; 95%CI = 1.50-3.21; P = 0.001). No associations between the GSTT1, GSTP1, and GSTM3 genotypes and neoplasia risk were observed. In conclusion, we determined the genotype distribution of GST polymorphisms in control subjects and breast cancer patients from northeastern Mexico. The GSTM1 null genotype was associated with breast cancer risk. Our findings may be used to individualize breast cancer screening and therapeutic intervention in our population, which displays ethnic characteristics that differentiate it from other populations in Mexico.

Olfactomedin-like 3 (OLFML3) gene expression in baboon and human ocular tissues: cornea, lens, uvea, and retina.

BACKGROUND: Olfactomedin-like is a family of polyfunctional polymeric glycoproteins. This family has at least four members. One member of this family is OLFML3, which is preferentially expressed in placenta but is also detected in other adult tissues including the liver and heart. However, its orthologous rat gene is expressed in the iris, sclera, trabecular meshwork, retina, and optic nerve. METHODS: OLFML3 messenger amplification was performed by RT-PCR from human and baboon ocular tissues. The products were cloned and sequenced. RESULTS: We report OLFML3 expression in human and baboon eye. The full coding DNA sequence has 1221 bp, from which an open reading frame of 406 amino acid was obtained. The baboon OLFML3 gene nucleotidic sequence has 98% and amino acidic 99% similarity with humans. CONCLUSIONS: OLFML3 gene expression in human and baboon ocular tissues and its high similarity make the baboon a powerful model to deduce the physiological and/or metabolic function of this protein in the eye.

The human placental lactogen genes: structure, function, evolution and transcriptional regulation.

hPL is a member of an evolutionarily related gene family including hGH and hPRL. Expression of hPL is limited to the placenta but its physiological actions are far reaching. hPL has a direct somatotropic effect on fetal tissues, it alters maternal carbohydrate and lipid metabolism to provide for fetal nutrient requirements, and aids in stimulation of mammary cell proliferation. Two hPL genes (hPL3 and hPL4) encoding identical proteins are responsible for the production of up to 1-3 g PL hormone/day. Recent studies have characterized the regulatory controls of hPL expression. At the post transcriptional level, RNA stability may contribute to variable levels of hPL3 vs. hPL4 production. In addition, non-tissue-specific protein-promoter interactions involving the Sp1 transcription factor are necessary for hPL transcription initiation. A transcriptional enhancer located 3' to the hPL3 gene is responsible for the placenta-specific expression of this gene, while an additional enhancer may be located 3' to the hPl4 gene. The hPL enhancer is bound by multiple proteins including at least one placental specific protein that interacts with a TEF-1 motif. Therefore, enhancer-protein interactions most likely play a large part in the high levels of placenta-specific hPL expression.

Biodegradation kinetics of BTE-OX and MTBE by a diesel-grown biomass.

The biodegradation kinetics of BTE-oX and MTBE, mixed all together in the presence of diesel-grown bioaugmented bacterial populations as high as 885 mg/L VSS, was evaluated. The effect of soil in aqueous samples and the effect of Tergitol NP-10 on substrate biodegradation rates were also evaluated. Biodegradation kinetics was evaluated for 54 h, every 6 h. All BTE-oX chemicals followed a first-order two-phase biodegradation kinetic model, whereas MTBE followed a zero-order removal kinetic model in all samples. BTE-oX removal rates were much higher than those of MTBE in all samples. The presence of soil in aqueous samples retarded BTE-oX and MTBE removal rates. The addition of Tergitol NP-10 to aqueous samples containing soil had a positive effect on substrate removal rate in all samples. Substrate percent removals ranged between 64.8-98.9% for benzene, toluene and ethylbenzene. O-xylene and MTBE percent removals ranged between 18.7-40.8% and 7.2-10.3%, respectively.

HGH isoforms: cDNA expression, adipogenic activity and production in cell culture.

We have isolated, cloned and achieved functional expression of the cDNAs for both 22 kDa and 20 kDa human growth hormone (hGH) isoforms. A selective cDNA cloning strategy was used to preferentially and simultaneously obtain both hGH 22 kDa and hGH 20 kDa cDNAs. These were used to construct minigenes which were subcloned into two eukaryotic expression vectors and then introduced transiently in COS-7 cells and stably into CHO cells in culture. Transfection assays in COS-7 cells of both minigenes allowed the detection of the secreted hGH 22 kDa and hGH 20 kDa. These hGHs isoforms secreted into COS-7 medium were able to specifically promote differentiation of 3T3-F442A preadipocytes to adipose cells. Adipocyte differentiation was quantitated by Oil Red O triacylglycerol staining or glycerophosphate dehydrogenase activity. Furthermore, stable CHO cell lines have been derived that produce these hGH isoforms.

Growth hormone and placental lactogen: biology, medicine and biotechnology.

Molecular cloning gave us access to the gene members of the human growth hormone and placental lactogen multigene family. Genomic sequencing provided clues for the understanding of the origin, functioning and regulation of this family. It has also allowed us to develop new diagnostic approaches for deficiencies of these hormones and to make new biotechnological contributions.

Versatile plasmid vectors for use in studies of eukaryotic gene expression.

A new pair of plasmid vectors useful in transient expression experiments of genes from higher eukaryotes was constructed. The vectors have been developed as derivatives of pSEG, a cloning and expression vector that has been used in studies of gene promoter structure and function [Barrera-Saldaña et al., EMBO J. 4 (1985) 3839-3849]. pUANL1 and pUANL2 include in their configuration a complete rabbit beta-globin transcriptional unit as internal control for gene expression the simian virus 40 (SV40) enhancer sequences, conveniently located unique restriction sites, the gene for resistance to ampicillin as a selectable marker and a prokaryotic ori for propagation in Escherichia coli. The new pair of pUANL vectors facilitates the cloning of foreign genes, placing two copies of the enhancer at either the 3' or the 5' side of gene. Our vectors completely lack SV40 ori, promoter and upstream sequences, which renders them ideal for gene expression studies where enhancer sequences are required but promoter and upstream sequences may interfere. Finally, by carrying an internal beta-globin reference gene, they are of special value for the standardization of quantitative S1 nuclease mapping studies of gene promoters.

Cloning and sequencing of cDNA encoding the cat growth hormone.

A cDNA encoding cat growth hormone (Fc GH) has been isolated and sequenced. This is the first report of a feline GH nucleotide and deduced amino acid (aa) sequences. This cat pituitary cDNA resembles a typical mammalian pre-GH cDNA with its encoded mature hormone differing from dog GH only by a single aa residue.

A dog growth hormone cDNA codes for a mature protein identical to pig growth hormone.

Although methods for purification of dog (Canis familiaris) growth hormone (cfGH) were described in the late Sixties, the cloning of its cDNA has not been achieved until now. In order to clone the cfGH cDNA, we capitalized on the high degree of nucleotide sequence conservation among mammalian GH genes to design a pair of consensus oligodeoxyribonucleotide primers. With these, and starting with dog pituitary gland total RNA, we specifically amplified the cfGH cDNA using the reverse transcription-polymerase chain reaction. Its coding sequence (651 bp), as well as its 3' untranslated region (101 bp), resemble those of a typical mammalian GH cDNA. Interestingly, its encoded mature protein is identical to pig growth hormone (pGH).

Sequence of a cDNA encoding horse growth hormone.

A cDNA encoding horse growth hormone (ecGH) was isolated and sequenced. The coding sequence resembles a typical mammalian GH pre-protein and contains a 3' untranslated region of 101 nucleotides carrying two contiguous polyadenylation signals.

New vectors for the efficient expression of mammalian genes in cultured cells.

We have constructed a new pair of plasmid vectors for the efficient expression of mammalian genes. The first of the new plasmids, pAVE1, was derived from pCMVcat [Foecking and Hofstetter, Gene 45 (1986) 101-105] by replacing the chloramphenicol acetyltransferase-encoding sequences in the latter for a multiple cloning site. Since it possesses the powerful enhancer-promoter unit of the immediate early gene of human cytomegalovirus, pAVE1 is ideal for the expression of mammalian genes. The second expression vector, pAVE2, resulted when the 3'-end flanking region from the human growth hormone-encoding gene (hGH) was incorporated in pAVE1. This region provides sequences for 3'-end processing and polyadenylation of primary transcripts. Thus, pAVE2 is suitable for expression of cDNAs in cultured cells, where introns have little effect on gene expression. To test our new vectors, we inserted the structural region of the chromosomal hGH gene into pAVE1, and its cDNA into pAVE2. By independently transfecting the resulting recombinant plasmids into COS-7 cells, we have achieved high levels of hGH transient expression with both vectors.

A general method to optimize the amount of enzyme in restriction and DNA modification reactions using the beta galactosidase blue-white plaques assay.

We propose a simple and economical method for assaying the activity of restriction and other modifying enzymes. The method involves assaying the use of the blue and white colored phenotypes of bacterial colonies obtained by digesting the polylinker sequence of M13 bacteriophage vectors followed by transformation in appropriate strains on X-gal/IPTG plates. In conjunction with restriction enzymes and DNA ligases, the method can evaluate polymerase activity and can be applied to test 3'...5' exonuclease activities such as that of T4 DNA polymerase, without having to use expensive radioisotopes. We describe its application in the assessment of restriction enzymes, DNA ligase and DNA polymerase activities.

Expression studies of transfected multigene families by homologous DNA mutagenesis.

A valuable approach for multigene family studies where the expression product of at least one gene member of the family is measurable is described. In such cases, the effect on gene expression of nucleotide sequence differences or mutations occurring in other members of the family or at alleles can easily be determined. This is achieved by a strategy called homologous DNA mutagenesis. It consists of the insertion of mutated regions from homologous genes into the context of the gene coding for the assayable product. Here we demonstrate the feasibility of this approach using gene members of the human growth hormone and human placental lactogen (hGH-hPL) multigene family.

Low-speed purification of human placental nuclei.

A simple method for the purification of human placental nuclei is described. Nuclei were isolated by homogenizing tissue in standard saline citrate solution in the presence of zinc chloride to stabilize the nuclear membranes, NP40 as non-ionic detergent and sodium bisulphite for inhibition of proteolytic activity. Nuclei purification was achieved by low-speed centrifugation through a discontinuous sucrose gradient. The purified nuclei were evaluated by morphological criteria using phase contrast and electron microscopy. The extent of contamination by cytoplasmic debris was estimated by Papanicolaou's staining technique. Biochemical criteria include measurements of alkaline phosphatase activity as a plasma membrane enzyme marker and DNA-dependent RNA polymerase activity for the functional integrity of nuclear components. Transcriptionally active nuclei were obtained but the yield of nuclei was low; however, this low yield is compensated by the high degree of purity, the simplicity of the method and the functional and morphological integrity of the purified nuclei.

Molecular diagnosis of deletions in the human multigene complex of growth and placental lactogen hormones.

The type IA of the isolated deficiencies (ID) of growth (HGH) and placental lactogen (HPL) hormones are frequently the consequence of deletions in their respective genes. To facilitate the diagnosis of these cases, we developed a rapid method for detecting deletions of the genes involved based on the polymerase chain reaction (PCR) technique. This method consist of the simultaneous amplification via consensus primers of the 5 genes which conform the hGH-hPL multigene family, followed by the identification of each of them in the amplification product by gene-specific patterns of restriction enzyme cuts evidenced by agarose gel electrophoresis. We demonstrate the effectiveness of our method by identifying patients with deletions in gene members of the hGH-hPL family.

Analysis of 16 cystic fibrosis mutations in Mexican patients.

We carried out molecular analysis of 80 chromosomes from 40 unrelated Mexican patients with a diagnosis of cystic fibrosis. The study was performed in two PCR steps: a preliminary one to identify mutation delta F508, the most frequent cause of cystic fibrosis worldwide, and the second a reverse dot-blot with allele-specific oligonucleotide probes to detect 15 additional common mutations in the Caucasian population. A frequency of 45% for delta F508 was found, making it the most common in our sample of Mexican patients. Another five mutations (G542X, 3,849 + 10 kb C-->T, N1303K, SN549N, and 621 + 1 G-->T) were detected, and those accounted for 11.25%. The remaining mutations (43.75%) were undetectable with the methodology used.

CFTR mutations in three Latin American countries.

We analyzed 192 cystic fibrosis (CF) alleles in three Latin American countries: Mexico, Colombia, and Venezuela. Mutation screening was performed by polymerase chain reaction (PCR) and a reverse dot blot detection kit that enables determination of 16 of the most common CF mutations worldwide. Mutations were detected in 47.9% of the screened CF alleles. The most prevalent CF allele was DeltaF508 (39. 6%). The remaining 16 non-DeltaF508 detectable mutations represented 8.3% of the CF alleles. Among them, the G542X, N1303K, and 3849+10kb C>T were the most common. Although the frequency of DeltaF508 described here is lower than that reported for Caucasian populations, including in Spain, it is remarkable that mutation prevalences found in this study resemble those observed in Spain. Two of these mutations, G542X and 3849+10kb C>T, that were relevant in this analysis, have a particularly high incidence in Spanish communities. The low frequency of DeltaF508 described here may be explained by the Amerindian, Caucasian, and Black admixture that occurred in Latin America after the discovery of the New World, and also by the probable occurrence of mutations contributed by the original natives, which were undetectable in this analysis.

The stimulatory effect of estradiol 17-beta on prolactin mRNA is inhibited by anti-calmodulin drugs.

The stimulatory action of estrogens on prolactin (PRL) secretion and synthesis is well known; on the other hand, anti-calmodulin drugs have recently been shown to inhibit prolactin in vitro release induced by estrogens. Based on these data, we decided to evaluate the in vivo effect of anti-calmodulin drugs (trifluoperazine and W7) on basal and estradiol-17 beta stimulated levels of PRL mRNA in anterior pituitary lobes obtained from adult male rats. Total RNA was isolated from pooled pituitaries recovered from animals under the same treatment and, from it, hybridizable PRL mRNA was detected. Estradiol-17 beta consistently stimulated PRL mRNA levels by 3-4 fold. The utilization of either trifluoperazine or W7, invariably inhibited estradiol-17 beta stimulated PRL mRNA. Metoclopramide, a drug with antidopaminergic activity, potentiated the stimulatory effect of estradiol-17 beta on PRL mRNA levels. These results suggest that anti-calmodulin drugs have an in vivo antiestrogenic effect on PRL mRNA levels confirming previous in vitro studies. Although, it is difficult to be conclusive about the mechanism through which these drugs act, one possibility is that the calcium-calmodulin system may be involved.

Expression of human placental lactogen and variant growth hormone genes in placentas.

Previous studies comparing the expression levels of human placental lactogen (hPL) genes have shown varying results, due to, perhaps, the fact that in all of them only one placenta was being analyzed. Here, the expression of hPL and growth hormone variant (hGH-V) genes in fifteen term placentas was comparatively analyzed at the RNA level, using reverse transcription coupled to polymerase chain reaction (RT-PCR). The abundance of the combined RNA transcripts derived from these genes varied from one placenta to another. The authors found that hPL-4 transcripts were more abundant than those of hPL-3 in most samples (ratios from 1:1 to 6:1), transcripts from the putative hPL-1 pseudogene were more abundant at the unprocessed stage while those of the hGH-V gene were mostly processed. Again, the authors of this study observed wide variation from placenta to placenta in the abundance of both of these types of transcripts. The same was observed when a group of six placentas from abortuses and nine from pregnancies complicated by preclampsia, diabetes and hypertension was studied. The authors conclude that the disagreeing results reported in the literature which are not in agreement concerning the expression levels of hPL genes could be explained by normal variations of their expression levels among the different placentas analyzed.