Association of vitamin D receptor polymorphisms with osteoporosis in mexican postmenopausal women

It has been reported that Vitamin D receptor polymorphisms are associated with osteoporosis, particularly those demonstrated by the BsmI and FokI restriction enzymes. Herein we report the results of a case-control study performed in postmenopausal Mexican women. We studied 65 osteoporotic women (< or = -2.5 SD bone mineral density [BMD] of young normal females) and 57 controls (over 90% > or = -1.5 SD BMD of young normal females. Restriction enzymes BsmI and FokI were used to identify polymorphisms. Odds ratios and their 95% confidence intervals were calculated, and analysis was performed controlling for age as a covariate. The BsmI genotypes revealed a higher frequency of the bb genotype in cases than in controls, contradicting much of the literature that suggests this genotype protects females against osteoporosis. Regarding the FokI genotypes, we were unable to confirm that the FF genotype has a protective effect against osteoporosis. The inconsistencies found in the literature and the results obtained in the present work suggest to us that other genetic and nongenetic factors are involved in the occurrence of osteoporosis, confounding the results of the possible association of osteoporosis and VDR polymorphisms.     

Disruption of sensitization to methylphenidate by a single administration of MK-801

Blockade of sensitization to methylphenidate by a single injection of MK-801 was investigated using a computerized activity monitoring system. Male Sprague-Dawley rats were housed in test cages and motor activity was recorded continuously for 16 days. After 2 days of baseline recording and a saline injection on day 3, the rats were randomly divided into four experimental groups. All received 2.5 mg/kg of methylphenidate (s.c.) once a day from days 4 to 9, then after five days of no treatment, they were re-challenged with 2.5 mg/kg of methylphenidate on day 15. One group received only methylphenidate, while the other three groups also received a single i.p. injection of MK-801 (0.30 mg/kg) either 24 h (day 3) or 1 h prior to the first of the six methylphenidate injections (day 4), or 1 h prior to the second methylphenidate injection (day 5). A single injection of MK-801 on day 4 (1 h prior to methylphenidate) blocked the development of sensitization to methylphenidate, since a sensitized response could not be elicited six days after cessation of repeated methylphenidate administration (day 15). However, sensitization to methylphenidate still occurred in the groups receiving MK-801 (0.30 mg/kg) on day 5, indicating that the mechanism by which a single injection of MK-801 disrupts sensitization to methylphenidate is sensitive to timing and is not a direct long-term effect. In conclusion, a single injection of MK-801 persistently blocks the development of sensitization to methylphenidate only if it is given with methylphenidate on the first day of the repetitive treatment phase.     

A haplotypic approach to founder-origin probabilities and outbred QTL analysis

Founder-origin probability methods are used to trace specific chromosomal segments in individual offspring. A haplotypic method was developed for calculating founder-origin probabilities in three-generation outbred pedigrees suited to quantitative trait locus (QTL) analysis. Estimators for expected founder-origin proportions were derived for a linkage group segment, an entire linkage group and a complete haplotype. If the founders are truly outbred, the haplotypic method gives a close approximation when compared with the Haley et al. (1994) method that simultaneously uses all marker information for QTL analysis, and it is less computationally demanding. The chief limitation of the haplotypic method is that some information in two-allele intercross marker-type configurations is ignored. Informativeness of marker arrays is discussed in the framework of founder-origin probabilities and proportions. The haplotypic method can be extended to more complex pedigrees with additional generations.     

A novel connection between the yeast Cdc42 GTPase and the Slt2-mediated cell integrity pathway identified through the effect of secreted Salmonella GTPase modulators

Modulation of host cellular GTPases through the injection of the effector proteins SopE2 and SptP is essential for Salmonella typhimurium to enter into non-phagocytic cells. Here we show that expression of the guanine nucleotide exchange factor for Cdc42 SopE2 in Saccharomyces cerevisiae leads to the activation of Fus3 and Kss1 MAPKs, which operate in the mating and filamentation pathways, causing filamentous growth in haploid yeast cells. Furthermore, it promotes the activation of the cell integrity MAPK Slt2. Cdc42 activation by removal of its putative intrinsic GTPase-activating proteins (GAPs), Rga1, Rga2, and Bem3, also results in the phosphorylation of Kss1, Fus3, and Slt2 MAPKs. These data support the role of these GAP proteins as negative regulators of Cdc42, confirm the modulating effect of this GTPase on the filamentation and mating pathways and point to a novel connection between Cdc42 and the cell integrity pathway. Cdc42-induced activation of Slt2 occurs in a mating and filamentation pathway-dependent manner, but it does not require the function of Rho1, which is the GTPase that operates in the cell integrity pathway. Moreover, we report that Salmonella SptP can act as a GAP for Cdc42 in S. cerevisiae, down-regulating MAPK-mediated signaling. Thus, yeast provides a useful system to study the interaction of bacterial pathogenic proteins with eukaryotic signaling pathways. Furthermore, these proteins can be used as a tool to gain insight into the mechanisms that regulate MAPK-mediated signaling in eukaryotes.     

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of ¹⁵N-¹⁵N and ¹H-¹⁵N trans-hydrogen bond scalar couplings, ^2h J _NN and ^1h J _HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models.     

Markovian chemicals "in silico" design (MARCH-INSIDE), a promising approach for computer aided molecular design II: experimental and theoretical assessment of a novel method for virtual screening of fasciolicides

A novel method for in silico selection of fluckicidal drugs is introduced. Two QSARs that permit us to discriminate between fasciolicide and non-fasciolicide drugs (the first) and to outline some conclusions about the possible mechanism of action of a chemical (the second) are performed. The first model correctly classified 93.85% of compounds in the training series and 89.5% of the compounds in the predicting one. This model correctly classified 87.7, 93.8, 92.2 and 93.9% of compounds in leave- n-out cross validation procedures when n takes values from 2 to until 6. The model seems to be stable in around 92% of good classification in leave- n-out cross validation analysis when n>6. The second model correctly classified 70% of non-fasciolicide compounds, 85.71% of beta-tubulin inhibitors and 100% of proton ionophores in the training set. This model recognizes as proton ionophores 100% of any nitrosalicylanilides in the predicting series. Both models have a low p-level <0.05. Finally, the experimental assay of six organic chemicals by an in vivo test permit us to carry out an assessment of the model with a fairly good 100% agreement between experiment and theoretical prediction.     

Revision of the taxonomic position of the xylanolytic Bacillus sp. MIR32 reidentified as Bacillus halodurans and plasmid-mediated transformation of B. halodurans

Bacillus sp. MIR32 has been isolated using xylan as the only carbon source, and one of its xylanolytic enzymes has been extensively studied. Biochemical analysis first related this strain to Bacillus amyloliquefaciens, but further studies based on a comparison of 16S rDNA sequences, G+C content, and DNA-DNA hybridization showed that strain MIR32 should be classified as a member of the species Bacillus halodurans. This change is also supported by the typical phenotype observed and by the results of PCR amplification directed toward spacers in rDNA and tDNA genes, which were assayed and compared with those of B. halodurans DSM 497(T). Although among alkaliphilic bacilli competence development has not been experimentally demonstrated, in this work both B. halodurans MIR32 and DSM 497(T) were transformed according to a simple procedure developed in our laboratory, reaching 10(2)-10(3) stable transformants per microgram of plasmid DNA.     

Monitoring Azospirillum-wheat interactions using the gfp and gusA genes constitutively expressed from a new broad-host range vector

To monitor the colonization of wheat roots by Azospirillum brasilense, we constructed several plasmids based on the pBBR1 replicon expressing the gfp and gusA genes constitutively. Both genes were placed under control of the gentamycin resistance gene promoter resulting in high levels of expression in Escherichia coli and A. brasilense. The constructed plasmids were stably maintained in A. brasilense strains even in the absence of selective pressure. The colonization of wheat plants grown under controlled conditions in sterilized vermiculite by A. brasilense strain FP2 (a Sp7-derivative) transconjugants containing these plasmids was monitored. Bacteria expressing GFP were easily observed in fresh plant material by fluorescence microscopy. Cell aggregates and single bacteria were visualized on the surfaces of young root zones, such as roots hairs and lateral roots. Large cellular clumps were observed at the points of lateral root emergence or at intercellular spaces of root epidermal cells 30 days after inoculation. Although we failed to detected bacteria in internal cortical and xylem tissues of wheat roots, the initial stage of endophytic colonization by A. brasilense may involve the sites detected in this work.     

Molecular pharmacological dissection of short- and long-term memory

1. It has been discussed for over 100 years whether short-term memory (STM) is separate from, or just an early phase of, long-term memory (LTM). The only way to solve this dilemma is to find out at least one treatment that blocks STM while keeping LTM intact for the same task in the same animal.2. The effect of a large number of treatments infused into the hippocampus, amygdala, and entorhinal, posterior parietal or prefrontal cortex on STM and LTM of a one-trial step-down inhibitory avoidance task was studied. The animals were tested at 1.5 h for STM, and again at 24 h for LTM. The treatments were given after training.3. Eleven different treatments blocked STM without affecting LTM. Eighteen treatments affected the two memory types differentially, either blocking or enhancing LTM alone. Thus, STM is separate from, and parallel to the first hours of processing of, LTM of that task.4. The mechanisms of STM are different from those of LTM. The former do not include gene expression or protein synthesis; the latter include a double peak of cAMP-dependent protein kinase activity, accompanied by the phosphorylation of CREB, and both gene expression and protein synthesis.5. Possible cellular and molecular events that do not require mRNA or protein synthesis should account for STM. These might include a hyperactivation of glutamate AMPA receptors, ribosome changes, or the exocytosis of glycoproteins that participate in cell addition.