Characterization of bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, as determined by 16S rDNA analysis

The bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, was studied in 16S rDNA clone libraries from three pulque samples. Sequenced clones identified as Lactobacillus acidophilus, Lactobacillus strain ASF360, L. kefir, L. acetotolerans, L. hilgardii, L. plantarum, Leuconostoc pseudomesenteroides, Microbacterium arborescens, Flavobacterium johnsoniae, Acetobacter pomorium, Gluconobacter oxydans, and Hafnia alvei, were detected for the first time in pulque. Identity of 16S rDNA sequenced clones showed that bacterial diversity present among pulque samples is dominated by Lactobacillus species (80.97%). Seventy-eight clones exhibited less than 95% of relatedness to NCBI database sequences, which may indicate the presence of new species in pulque samples.     

Synthesis and cardiovascular activity of metoprolol analogues

The synthesis of four novel analogues of metoprolol, a well-known beta1-blocker used to reduce arterial blood pressure, is described. The preparation of (2S,2'S)-7, (2R,2'S)-7, (2R,2'R)-8, and (2S,2'R)-8 was based on the reaction of racemic 2-[4-(2'-methoxyethyl)-phenoxymethyl]-oxirane (4) with (R)- or (S)-2-amino-1-butanol. Salient characteristics of analogues 7 and 8 relative to metoprolol are the incorporation of an additional stereogenic center, as well as a methyl group and a hydroxyl function on the nitrogen-containing chain. These novel derivatives present significant hypotensive and bradycardiac activity, although no blocking action toward beta1 and beta2 adrenergic receptor.     

Long-distance colonization,isolation by distance,and historical demography in a relictual Mexican pinyon pine (Pinus nelsonii Shaw) as revealed by paternally inherited genetic markers (cpSSRs)

Pinus nelsonii is a relictual pinyon pine distributed across a wide altitudinal range in semiarid zones in Mexico near the border between the States of Nuevo León and Tamaulipas. It also occurs in small patches in the State of San Luis Potosí. Pinus nelsonii is classified in the monotypic subsection Nelsoniae, separated from other pinyon pines (subsection Cembroides), because it possesses several distinctive characters including persistent fascicle sheaths, connate needles, and a distinctive wood anatomy. In the present study, chloroplast simple sequence repeats (cpSSRs) were used to estimate genetic variation in most known populations (nine) of P. nelsonii. The genetic variation (HT = 0.73; 27 haplotypes in 256 individuals) is moderate when compared to other pine species. Population differentiation ranged between low and moderate (FST = 0.13 and RST = 0.05), as did the Nei and Goldstein genetic distances between populations. However, this pattern varied depending on whether the infinite alleles or stepwise mutation model was used. In the former case a significant isolation by distance was found, but not in the latter. A significant association between geographical and genetic structure in one clade, through a nested clade analysis, was found, which suggested long-distance colonization between 125000 and 309000 years ago. We found weak evidence for a population expansion. A mismatch distribution suggests that P. nelsonii populations underwent an expansion 4.25 times their size between 59000 and 146000 years ago. On the other hand, the populations' star-like phylogeny and a slight parabolic relationship between coalescence times and lineage number also suggest weak population expansion. Overall, this species appears to have been in demographic stasis for a large proportion of the time detected by the markers used.     

Cytogenetical prenatal diagnosis by amniocentesis during the II and III gestation trimesters in Costa Rica

The identification of fetal abnormal chromosomes in high risk pregnancies allows proper pediatric and obstetric management of the cases as well as genetic counseling. The results of 842 genetic amniocentesis from 1986 to 1999 are reported. All procedures were performed transabdominally and under ultrasound guidance, in hospitals of the social security system and in private facilities. There were two main reasons for referral: abnormal ultrasound assessment (48% of cases) and advanced maternal age (35%). Most procedures (66%) were performed during the second trimester of pregnancy and 34% during the third trimester. Fetal cells were closed cultured and suspension harvested. Median turn around time was 14 days. In 217 amniotic fluid samples no diagnosis could be obtained, mainly due to absence of cell growth in late gestation samples or because of blood contamination. Of 625 fetal karyotypes 55 (9%) were abnormal, due to 33 trisomies (including a Robertsonian translocation trisomy 13), eight cases of monosomy X, three mosaics (including a mosaic trisomy 22), balanced and unbalanced translocations, extra structurally abnormal chromosomes and other defects. Pseudomosaicism was detected in five cases. Taking into account the reason for referral, cases studied as a result of abnormal ultrasound assessment exhibited 17% abnormal karyotypes, in contrast to 2.5% cytogenetic defects in pregnancies of women 35 years or older. Prenatal cytogenetic and sonographic findings correlated with the phenotype of the newborn in 211 cases available for follow-up. Prenatal diagnosis of fetal defects allowed genetic counseling as well as better obstetric management and pediatric care. Normal results of both tests provided reassurance to prospective parents.     

Transcription corepressor CtBP is an NAD(+)-regulated dehydrogenase

The Lcd1p/Mec1p complex is crucial for normal S phase progression and for signaling DNA damage. We show that Lcd1p/Ddc2p and Mec1p in cell extracts bind to DNA ends. Although Lcd1p binds DNA independently of Mec1p, recruitment of Mec1p to DNA requires Lcd1p. DNA binding by Lcd1p is also independent of Rad9p, Rad17p, and Rad24p. Recombinant Lcd1p binds DNA, and this is impaired by Lcd1p mutations that abrogate its in vivo functions. Furthermore, Mec1p is recruited to cdc13-induced DNA damage and HO endonuclease-induced double-strand breaks in vivo. This requires Lcd1p, and recruitment of Lcd1p/Mec1p to cdc13-induced damage is abolished by Lcd1p mutations that abrogate its in vivo functions. Recruitment of Lcd1p to these lesions is independent of Mec1p and Rad9p/Rad24p. Thus, recruitment of Mec1p to DNA lesions by Lcd1p is crucial for the DNA damage response.     

Gene therapy of erectile dysfunction in the rat with penile neuronal nitric oxide synthase

Gene transfer to the penile corpora cavernosa of constructs of the inducible and endothelial nitric oxide synthase (NOS) cDNAs ameliorates erectile dysfunction in aged rats. In this study, we investigated whether the neuronal NOS (nNOS) variant responsible for erection, penile nNOS (PnNOS), can exert a similar effect, and whether the combination of electroporation with a helper-dependent adenovirus (AdV) improves gene transfer. PnNOS and beta-galactosidase cDNAs were cloned in plasmid (pCMV-PnNOS; pCMV-beta-gal) and "gutless" AdV (AdV-CMV-PnNOS; AdV-CMV-beta-gal) vectors, and injected into the penis of adult (beta-gal) or aged (PnNOS) rats, with or without electroporation. Penile erection was measured at different times after PnNOS cDNA injection, by electrical field stimulation of the cavernosal nerve. The expression of beta-galactosidase or PnNOS was estimated in penile tissue by either histochemistry and luminometry or Western blot, and the effects of AdV-CMV-PnNOS on mRNA expression were examined by a DNA microarray. We found that electroporation increased pCMV-beta-gal uptake, and its expression was detectable at 56 days. In the aged rats treated with pCMV-PnNOS and electroporation, the maximal intracavernosal:mean arterial pressure ratios were elevated for 11 and 18 days when compared with those in controls. Electroporation intensified penile uptake of as few as 10(6) viral particles (vp) of AdV-CMV-beta-gal, and with 10(7) vp beta-galactosidase was still detectable at 60 days. Electroporated AdV-CMV-PnNOS (10(7) vp) was effective at 18 days in stimulating the erection of aged rats, without inducing the expression of cytotoxic genes. In conclusion, intracavernosal gene therapy with PnNOS cDNA corrected the aging-related erectile dysfunction for at least 18 days when given by electroporation in a helper-dependent AdV at low viral loads.     

Angiotensin II modulates ion transport in rat proximal tubules through CYP metabolites

To assess the effect of angiotensin II on ion transport in rat isolated proximal tubules and establish the arachidonic acid cytochrome P450 metabolites' role mediating angiotensin II effect and to analyze whether corticosteroids play a role modulating this effect, we studied the effect of low (10 and 100 pM) and high (0.1-1 microM) angiotensin II concentrations on proximal tubule ion transport, measured as (86)Rb uptake. Low angiotensin II produced a stimulation on the (86)Rb uptake (195.79 +/- 35, 377.9 +/- 81, and 300 +/- 49 pg (86)Rb/microg protein/2 min, for control and 10 and 100 pM angiotensin II, respectively). High angiotensin II concentration inhibited ion transport (0.1 microM, 57.9 +/- 5 and 1 microM, 47.3 +/- 4 pg (86)Rb/microg protein/2 min), this effect was prevented by 17-ODYA and by losartan, while indomethacin had no effect. Dexamethasone treatment increased angiotensin II-induced (86)Rb uptake inhibition and arachidonic acid metabolism (19-, 20-HETE and 12-HETE), while adrenalectomy partly prevented angiotensin II-induced inhibition and decreased cytochrome P450-dependent arachidonic acid metabolism. In conclusion, high doses of angiotensin II produce inhibition of ion transport in rat isolated proximal tubules; this effect is mediated by AT(1) receptors, involves cytochrome P450-dependent arachidonic acid metabolites, and is upregulated by corticosteroids.     

Monomeric midkine induces tumor cell proliferation in the absence of cell-surface proteoglycan binding

Midkine (MK), a retinoic acid-inducible heparin-binding protein, is a mitogen which initiates a cascade of intracellular protein tyrosine phosphorylation mediated by the JAK/STAT pathway after binding to its high affinity p200(+)/MKR cell surface receptor in the G401 cell line [Ratovitski, E. A. (1998) J. Biol. Chem. 273, 3654-3660]. In this study, we determined the biophysical characteristics of purified recombinant murine MK and analyzed the requirements for ligand multimerization and cell surface proteoglycan binding for the G401 cell mitogenic activity of MK. Our studies indicate that the secreted form of MK (M = 13 kDa) exists in solution as an asymmetric monomer with a frictional coefficient of 1. 48 and a Stokes radius of 23.7 A. By constructing bead models of MK using the program AtoB and the program HYDRO to predict the hydrodynamic properties of each model, our data suggest that MK has a dumb-bell shape in solution composed of independent N- and C-terminal domains separated by an extended linker. This asymmetric MK monomer is a biologically active ligand with mitogenic activity on G401 cells in vitro. Neither heparin-induced formation of noncovalent MK multimers nor tissue transglutaminase II covalent multimerization of MK enhanced MK mitogenic activity in this system. Since neither heparin competition nor cell treatment with chondroitinase ABC or heparinase III abolished the mitogenic effects of MK on G401 cells, cell-surface proteoglycan binding by MK does not appear to be a requirement for its observed mitogenic effects. These results provide strong evidence that the MK-specific p200(+)/MKR has distinctive biochemical properties which distinguish it from the receptor tyrosine phosphatase cell-surface proteoglycan PTPzeta/RPTPbeta and support the hypothesis that the diverse biological effects of MK are mediated by multiple cell-specific signal transduction receptors.