Dual role of MutS glutamate 38 in DNA mismatch discrimination and in the authorization of repair

MutS plays a critical role in DNA mismatch repair in Escherichia coli by binding to mismatches and initiating repair in an ATP-dependent manner. Mutational analysis of a highly conserved glutamate, Glu38, has revealed its role in mismatch recognition by enabling MutS to discriminate between homoduplex and mismatched DNA. Crystal structures of MutS have shown that Glu38 forms a hydrogen bond to one of the mismatched bases. In this study, we have analyzed the crystal structures, DNA binding and the response to ATP binding of three Glu38 mutants. While confirming the role of the negative charge in initial discrimination, we show that in vivo mismatch repair can proceed even when discrimination is low. We demonstrate that the formation of a hydrogen bond by residue 38 to the mismatched base authorizes repair by inducing intramolecular signaling, which results in the inhibition of rapid hydrolysis of distally bound ATP. This allows formation of the stable MutS-ATP-DNA clamp, a key intermediate in triggering downstream repair events.     

Dietary habits and folate status in women of childbearing age in Croatia

Folic acid and folates have an important role in prevention of neural tube defect that appears in the first weeks of pregnancy, when women are still not aware of their pregnancy, especially when pregnancy is not planned. Since ensuring sufficient quantities of folates and folic acid in this period is essential, dietary habits of childbearing age women are very important. In line with that the intake of folates and folic acid in nutrition of women age group 20-30 years is examined, as well as the frequency of consumption of foodstuffs rich in vitamins and folic acid supplements. Values of folates in serum are presented, dependent on their nutritional habits. Obtained results indicate that in spite of inadequate intake of folates and folic acid from foodstuffs, clinical deficit is not recorded, which is the result of frequent consumption of dietary supplements. In accordance with these dietary habits, differences in the folates status of examinees were observed.     

Cephalometric indicators of the vertical dimension of occlusion

The aim of this investigation was to establish precise indicators of the vertical dimension of occlusion (VDO) which could be used as objective parameters in prosthodontic treatment providing exact control of the reconstructed vertical dimension of occlusion, early detection of errors and correction of the vertical dimension of occlusion during complete denture manufacturing. A total of 60 lateral cephalometric radiographs of subjects with natural dentition and class I skeletal jaw relationship, of Serbian nationality from the region of Vojvodina, were included in the investigation. Thirty subjects were males, and thirty females, their age range was 20 to 29 years. Cephalometric analysis was performed by using "Dr. Ceph" computer software (FYI Technologies, GA, USA). By evaluation of the craniofacial complex in subjects with natural dentition, horizontal dentofacial developmental growth was established in the examined sample and it was based on the relationship between the posterior and anterior total facial height (S-Go:N-Me, female X=68.96%, male X=72.8%) and statistically significant differences were found between the sexes in almost all linear dimensions and in the relation between the posterior and the anterior total facial height. Highly significant differences (p<0.001) between the sexes were found in regard to anterior total facial height (N-Me, male X=120.75 mm, female X=112.72 mm) and the anterior lower facial height (ANS-Me, male X=67.87 mm, female X=61.50 mm) (p<0.05). The proportions of the upper and lower anterior facial heights (N-ANS:ANS-Me, male X=79.36%, female X=80.63%) were within normal values in both sexes. Facial harmony existed both in men and women. Cephalometric study of the subjects with complete natural dentition included in this study provided important and specific parameters for optimal reconstruction of vertical dimension of occlusion in the treatment of edentulous patients.     

Linear vertical jaw resorption potential in elderly complete denture wearers: a five-year follow-up study

Human bones decrease in quality and increase in porosity beginning at about the third decade of life. The aim of this study was to establish an equation to predict the maxillary and mandibular linear vertical resorption potential for elderly edentulous patients on the basis of the analysis of the cervical vertebrae in a single cephalometric radiograph. The morphology of the bodies of the third and fourth cervical vertebrae and measurements of linear vertical resorption in the frontal region of the jaws were analyzed in two consecutive cephalometric observations of 26 elderly edentulous patients over the five-year period of wearing complete dentures. An equation was determined to obtain maxillary and mandibular linear vertical resorption on the basis of measurements in the third and fourth cervical vertebral bodies and the average errors between the predicted and the actual values were 0.14 mm. The cervical vertebrae exhibited significant decrease in the height and width, and residual alveolar ridges exhibited significant decrease in the height over the 5-year period of wearing dentures (p < 0.01). These results suggest that using cervical vertebral measurements might allow predicting the maxillary and mandibular resorption for edentulous elderly patients wearing complete dentures.     

Bovine platelet-activating factor acetylhydrolase (PAF-AH) activity related to fertility

Plasma platelet-activating factor acetylhydrolase (PAF-AH), the enzyme characterized by the association with plasma lipoproteins, degrades platelet-activating factor (PAF) as well as PAF-like oxidatively fragmented phospholipids produced during oxidative stress. Apart from pro-inflammatory properties, PAF is also related to reproductive processes and successful fertility. In order to get a better insight into the involvement of PAF-AH in the fertility of cows, the aim of the study was to determine the PAF-AH activity as well as the C-reactive protein, cholesterol and high density lipoprotein-cholesterol (HDL-C) in the serum of dairy cows throughout the pregnancy and lactation, as well as in infertile cows. The results showed that serum PAF-AH activity changes throughout pregnancy and lactation with a lower level during periparturient period. It is also found higher PAF-AH activity in lactating cows with reproductive disorders compared to high lactating cows without reproductive disorders. Strong correlation between PAF-AH activity and HDL-C concentration indicates that HDL could have considerable influence on PAF-AH activity in bovine plasma. CRP concentration was also lower during transition period suggesting that lactation might stimulate CRP synthesis in bovine. A higher CRP concentration in cows with reproductive disorders compared to fertile cows at the peak of lactation, demonstrates that milk production is not the only factor influencing CRP in cows. A significant correlation between PAF-AH activity and CRP level shows that both parameters could be influenced by reproductive status of dairy cows.     

Functional analysis of HNPCC-related missense mutations in MSH2

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in the human DNA mismatch repair (MMR) genes, most frequently MSH2 and MLH1. The majority of HNPCC mutations cause truncations and thus loss of function of the affected polypeptide. However, a significant proportion of MMR mutations found in HNPCC patients are single amino acid substitutions and the functional consequences of many of these mutations in DNA repair are unclear. We have examined the consequences of seven MSH2 missense mutations found in HNPCC families by testing the MSH2 mutant proteins in functional assays as well as by generating equivalent missense mutations in Escherichia coli MutS and analyzing the phenotypes of these mutants. Here we show that two mutant proteins, MSH2-P622L and MSH2-C697F confer multiple biochemical defects, namely in mismatch binding, in vivo interaction with MSH6 and EXO1, and in nuclear localization in the cell. Mutation G674R, located in the ATP-binding region of MSH2, appears to confer resistance to ATP-dependent mismatch release. Mutations D167H and H639R show reduced mismatch binding. Results of in vivo experiments in E. coli with MutS mutants show that one additional mutant, equivalent of MSH2-A834T that do not show any defects in MSH2 assays, is repair deficient. In conclusion, all mutant proteins (except for MSH2-A305T) have defects; either in mismatch binding, ATP-release, mismatch repair activity, subcellular localization or protein-protein interactions.     

Different aggregation approaches in the chironomid community and the threshold of acceptable information loss

Due to the problem of identification, Chironomidae larvae, although very abundant, are often avoided or not properly used in bioassessment programs. The aim of this work was to test how different aggregation processes—taxonomic resolution and the random aggregation approach (best practicable aggregation of species—BestAgg) affect the analysis of chironomid communities regarding any information loss. The self-organizing map method, together with classification strength analysis and Spearman’s rank correlation, revealed that the genus-level and BestAgg-abundance matrix most accurately approximated the species-level community pattern. The subfamily-level dataset was ineffective at presenting the chironomid community structure, with a substantially lower concordance with the species-level dataset. The biologic environmental gradients analyses presented the same set of important environmental variables for the species-level, genus-level, and BestAgg-abundance matrix. The indicator values analysis showed that indicator genera provide information very close to that gained from species indicators. According to our results, the numeric relationship between species and higher taxa influences taxonomic scaling, limiting Chironomidae family aggregation, with acceptable information loss only up to genus level. In addition, the BestAgg approach, with the maximum level of aggregation, properly assesses the community structure and consequently describes environmental conditions.     

The effects of zolpidem treatment on GABA(A) receptors in cultured cerebellar granule cells: changes in functional coupling

AimsHypnotic zolpidem is a positive allosteric modulator of γ-aminobutyric acid (GABA) action, with preferential although not exclusive binding for α1 subunit-containing GABA_A receptors. The pharmacological profile of this drug is different from that of classical benzodiazepines, although it acts through benzodiazepine binding sites at GABA_A receptors. The aim of this study was to further explore the molecular mechanisms of GABA_A receptor induction by zolpidem.Main methodsIn the present study, we explored the effects of two-day zolpidem (10 μM) treatment on GABA_A receptors on the membranes of rat cerebellar granule cells (CGCs) using [³H]flunitrazepam binding and semi-quantitative PCR analysis.Key findingsTwo-day zolpidem treatment of CGCs did not significantly affect the maximum number (B_max) of [³H]flunitrazepam binding sites or the expression of α1 subunit mRNA. However, as shown by decreased GABA [³H]flunitrazepam binding, two-day exposure of CGCs to zolpidem caused functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptor complexes.SignificanceIf functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptors is the mechanism responsible for the development of tolerance following long-term administration of classical benzodiazepines, chronic zolpidem treatment may induce tolerance.     

Impact of Oral Typhoid Vaccination on the Human Gut Microbiota and Correlations with S. Typhi-Specific Immunological Responses

The resident microbial consortia of the human gastrointestinal tract play an integral role in modulating immune responses both locally and systemically. However, detailed information regarding the effector immune responses after vaccine administration in relation to the gastrointestinal microbiota is absent. In this study, the licensed oral live-attenuated typhoid vaccine Ty21a was administered in a clinical study to investigate whether oral immunization resulted in alterations of the microbiota and to identify whether a given microbiota composition, or subsets of the community, are associated with defined S. Typhi-specific immunological responses. The fecal microbiota composition and temporal dynamics were characterized using bacterial 16S rRNA pyrosequencing from individuals who were either immunized with the Ty21a typhoid vaccine (n = 13) or served as unvaccinated controls (n = 4). The analysis revealed considerable inter- and intra-individual variability, yet no discernible perturbations of the bacterial assemblage related to vaccine administration were observed. S. Typhi-specific cell mediated immune (CMI) responses were evaluated by measurement of intracellular cytokine production using multiparametric flow cytometry, and humoral responses were evaluated by measurement of serum anti-LPS IgA and IgG titers. Volunteers were categorized according to the kinetics and magnitude of their responses. While differences in microbial composition, diversity, or temporal stability were not observed among individuals able to mount a positive humoral response, individuals displaying multiphasic CMI responses harbored more diverse, complex communities. In line with this preliminary observation, over two hundred operational taxonomic units (OTUs) were found to differentiate multiphasic and late CMI responders, the vast majority of which classified within the order Clostridiales. These results provide an unprecedented view into the dramatic temporal heterogeneity of both the gut microbiota and host immune responses.