Start codon FokI and intron 8 BsmI variants in the vitamin D receptor gene and susceptibility to colorectal cancer

Epidemiological evidence suggests the protective effect of vitamin D against colorectal cancer (CRC) and the polymorphisms in vitamin D receptor (VDR) gene may influence the development of CRC. In this study the possible association of VDR FokI and BsmI gene polymorphisms with CRC risk was examined. A total of 904 subjects, including 452 cases with CRC and 452 controls were enrolled in this study. All 904 subjects were genotyped for VDR FokI and BsmI gene polymorphisms by PCR-RFLP method. We observed no significant difference in genotype and allele frequencies between the cases with CRC and controls for the both FokI and BsmI polymorphisms either before or after adjustment for confounding factors including age, BMI, sex, and smoking status. Furthermore, no evidence for effect modification of the association VDR gene FokI and BsmI variants and CRC by BMI, sex, or tumor site was observed. In addition, there was no significant difference in genotype and allele frequencies between the normal weight (BMI <25 kg/m²) cases with CRC and overweight/obese (BMI ≥25 kg/m²) cases with CRC for the two SNPs. Our results do not lend support to the hypothesis that VDR gene FokI and BsmI polymorphisms are associated with the risk of CRC. However, further studies are required to confirm this finding.     

Microbial enzyme activity at the watershed scale: response to chronic nitrogen deposition and acute phosphorus enrichment

Microbial enzymes play a critical role in organic matter decomposition and enzyme activity can dynamically respond to shifts in inorganic nutrient and substrate availability, reflecting the nutrient and energy limitation of the microbial community. We characterized microbial enzyme response to shifting nitrogen (N) and phosphorus (P) availability across terrestrial and aquatic environments at the Bear Brook Watershed in Maine, the site of a whole-watershed N enrichment experiment. We compared activity of β-1,4-glucosidase (BG); β-1,4-N-acetylglucosaminidase (NAG); acid phosphatase (AP) in soil, leaf litter in terrestrial and stream habitats and stream biofilms in a reference and N enriched watershed, representing whole-ecosystem response to chronic N enrichment. In addition, we used shorter, experimental P enrichments to address potential P limitation under ambient and elevated N availability. We found that BG and NAG activity were not affected by the long-term N enrichment in either habitat. Enhanced P limitation due to N enrichment was evident only in the aquatic habitats with 5- and 8-fold higher treated watershed AP activity in stream biofilms and stream litter, respectively. Acute P additions reduced AP activity and increased BG activity and these effects were also most pronounced in the streams. The stoichiometry of enzyme activity was constrained across ecosystem compartments with regression slopes for lnBG:lnNAG, lnBG:lnAP, and lnNAG:lnAP close to 1, ranging 1.142–1.241. We found that microbial enzyme response to shifting N and P availability varied among watershed compartments, typically with stronger effects in aquatic habitats. This suggests that understanding the response of ecosystem function to disturbance at the watershed scale requires simultaneous consideration of all compartments.     

Heterogeneous effects of cholecystokinin on neuronal response properties in deep layers of rat barrel cortex

Abstract

Cholecystokinin (CCK) is one of the most studied neuropeptides in the brain. In this study, we investigated the effects of CCK-8s and LY225910 (CCK₂ receptor antagonist) on properties of neuronal response to natural stimuli (whisker deflection) in deep layers of rat barrel cortex. This study was done on 20 male Wistar rats, weighing 230–260?g. CCK-8s (300?nmol/rat) and LY225910 (1?µmol/rat) were administered intracerebroventricularly (ICV). Neuronal responses to deflection of principal (PW) and adjacent (AW) whiskers were recorded in the barrel cortex using tungsten microelectrodes. Computer controlled mechanical displacement was used to deflect whiskers individually or in combination at 30?ms inter-stimulus intervals. ON and OFF responses for PW and AW deflections were measured. A condition-test ratio (CTR) was computed to quantify neuronal responses to whisker interaction. ICV administration of CCK-8s and LY225910 had heterogeneous effects on neuronal spontaneous activity, ON and OFF responses to PW and/or AW deflections, and CTR for both ON and OFF responses. The results of this study demonstrated that CCK-8s can modulate neuronal response properties in deep layers of rat barrel cortex probably via CCK₂ receptors.     

The Inhibition Effect of Lactobacilli Against Growth and Biofilm Formation of <Emphasis Type="BoldItalic">Pseudomonas aeruginosa</Emphasis>

The emergence of antibiotic-resistant and food-spoilage microorganisms has renewed efforts to identify safe and natural alternative agents of antibiotics such as probiotics. The aim of this study was the isolation of lactobacilli as potential probiotics from local dairy products with broad antibacterial and anti-biofilm activities against antibiotic-resistant strains of Pseudomonas aeruginosa and determination of their inhibition mechanism. Antibiotic susceptibility and classification of acquired resistance profiles of 80 P. aeruginosa strains were determined based on Centers for Disease Control and Prevention (CDC) new definition as multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) followed by antibacterial assessment of lactobacilli against them by different methods. Among the 80 P. aeruginosa strains, 1 (1.3%), 50 (62.5%), and 78 (97.5%) were PDR, XDR, and MDR, respectively, and effective antibiotics against them were fosfomycin and polymyxins. Among 57 isolated lactobacillus strains, two strains which were identified as Lactobacillus fermentum using biochemical and 16S rDNA methods showed broad inhibition/killing and anti-biofilm effects against all P. aeruginosa strains. They formed strong biofilms and had bile salts and low pH tolerance. Although investigation of inhibition mechanism of these strains showed no bacteriocin production, results obtained by high-performance liquid chromatography (HPLC) analysis indicated that their inhibitory effect was the result of production of three main organic acids including lactic acid, acetic acid, and formic acid. Considering the broad activity of these two L. fermentum strains, they can potentially be used in bio-control of drug-resistant strains of P. aeruginosa.     

The closed and compact domain organization of the 70-kDa human cytochrome P450 reductase in its oxidized state as revealed by NMR

The NADPH cytochrome P450 reductase (CPR), a diflavin enzyme, catalyzes the electron transfer (ET) from NADPH to the substrate P450. The crystal structures of mammalian and yeast CPRs show a compact organization for the two domains containing FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), with a short interflavin distance consistent with fast ET from the NADPH-reduced FAD to the second flavin FMN. This conformation, referred as "closed", contrasts with the alternative opened or extended domain arrangements recently described for partially reduced or mutant CPR. Internal domain flexibility in this enzyme is indeed necessary to account for the apparently conflicting requirements of having FMN flavin accessible to both the FAD and the substrate P450 at the same interface. However, how interdomain dynamics influence internal and external ETs in CPR is still largely unknown. Here, we used NMR techniques to explore the global, domain-specific and residue-specific structural and dynamic properties of the nucleotide-free human CPR in solution in its oxidized state. Based on the backbone resonance assignment of this 70-kDa protein, we collected residue-specific (15)N relaxation and (1)H-(15)N residual dipolar couplings. Surprisingly and in contrast with previous studies, the analysis of these NMR data revealed that the CPR exists in a unique and predominant conformation that highly resembles the closed conformation observed in the crystalline state. Based on our findings and the previous observations of conformational equilibria of the CPR in partially reduced states, we propose that the large-scale conformational transitions of the CPR during the catalytic cycle are tightly controlled to ensure optimal electron delivery.     

On the substrate specificity of DNA methyltransferases. adenine-N6 DNA methyltransferases also modify cytosine residues at position N4

Methylation of DNA is important in many organisms and essential in mammals. Nucleobases can be methylated at the adenine-N6, cytosine-N4, or cytosine-C5 atoms by specific DNA methyltransferases. We show here that the M.EcoRV, M.EcoRI, and Escherichia coli dam methyltransferases as well as the N- and C-terminal domains of the M. FokI enzyme, which were formerly all classified as adenine-N6 DNA methyltransferases, also methylate cytosine residues at position N4. Kinetic analyses demonstrate that the rate of methylation of cytosine residues by M.EcoRV and the M.FokI enzymes is reduced by only 1-2 orders of magnitude in relation to methylation of adenines. This result shows that although these enzymes methylate DNA in a sequence specific manner, they have a low substrate specificity with respect to the target base. This unexpected finding has implications on the mechanism of adenine-N6 DNA methyltransferases. Sequence comparisons suggest that adenine-N6 and cytosine-N4 methyltransferases have changed their reaction specificity at least twice during evolution, a model that becomes much more likely given the partial functional overlap of both enzyme types. In contrast, methylation of adenine residues by the cytosine-N4 methyltransferase M.BamHI was not detectable. On the basis of our results, we suggest that adenine-N6 and cytosine-N4 methyltransferases should be grouped into one enzyme family.     

Levels of Bcl-2 and P53 Are Altered in Superior Frontal and Cerebellar Cortices of Autistic Subjects

1. Autistic disease (AD) is a severe neuropsychiatric disorder affecting 2-4 children per 10,000. We have recently shown reduction of Bcl-2 and increase in P53, two important markers of apoptosis, in parietal cortex of autistic subjects. 2. We hypothesized that brain levels of Bcl-2 and P53 would also be altered in superior frontal cortex and cerebellum of age-, sex, and postmortem-interval (PMI)-matched autistic subjects (N = 5 autistic, N = 4 controls). 3. Brain extracts were prepared from superior frontal cortex and cerebellum and subjected to Western blotting. 4. Results showed that levels of Bcl-2 decreased by 38% and 36% in autistic superior frontal and cerebellar cortices, respectively when compared to control tissues. By the same token, levels of P53 increased by 67.5% and 38% in the same brain areas in autistic subjects vs. controls respectively. Calculations of ratios of Bcl-2/P53 values also decreased by 75% and 43% in autistic frontal and cerebellar cortices vs. controls respectively. The autistic cerebellar values were significantly reduced (p < 0.08) vs. control only. There were no significant differences in levels of beta-actin between the two groups. Additionally, there were no correlations between Bcl-2, P53, and beta-actin concentrations vs. age or PMI in either group. 5. These results confirm and extend previous data that levels of Bcl-2 and P53 are altered in three important brain tissues, i.e. frontal, parietal, and cerebellar cortices of autistic subjects, alluding to deranged apoptotic mechanisms in autism.     

Copper(I) interaction with model peptides of WD6 and TM6 domains of Wilson ATPase: regulatory and mechanistic implications

With the aim to investigate the mechanism of Cu(I) transport by Wilson ATPase (ATP7B), we have studied the interaction of the peptides 2K10p (CH(3)CO-Lys-Gly-Met-Thr-Cys-Ala-Ser-Cys-Val-His-Asn-Lys-CONH(2)), and 2K8p (CH(3)CO-Lys-Leu-Cys-Ile-Ala-Cys-Pro-Cys-Ser-Lys-CONH(2)), part of the sixth metal binding domain (WD6) and the sixth transmembrane segment (TM6) of Wilson ATPase, respectively, by means of CD, NMR spectroscopy and homology modeling. In addition, the interaction of Cu(I) with the 2K8p mutants 1s (CH(3)CO-Lys-Leu-Ser-Ile-Ala-Cys-Pro-Cys-Ser-Lys-CONH(2)), 2s (CH(3)CO-Lys-Leu-Cys-Ile-Ala-Ser-Pro-Cys-Ser-Lys-CONH(2)) and 3s (CH(3)CO-Lys-Leu-Cys-Ile-Ala-Cys-Pro-Ser-Ser-Lys-CONH(2)), containing two cysteines in various positions, have been studied with the same methods, in order to understand the role of each cysteine in copper binding. Our studies show that the three cysteine thiolates present in the 2K8p peptide sequence act mainly as bridging ligands for Cu(I) binding, and dithiothreitol acts as an important ligand in Cu(I) ligation by 2K10p and the 2K8p mutants. Formation of oligomeric species has been evidenced for all peptides except 2s. Shift of the equilibrium between the various oligomeric species has been accomplished by reducing the Cu(I):peptide ratio. Significant shifts of proline protons upon interaction with Cu(I) have been observed for all proline containing peptides implying a possible role of proline in facilitating Cu(I) binding. These findings have been further discussed with respect to the molecular basis of copper trafficking and intermolecular interactions.     

Efficacy of plant essential oils on post-harvest control of rot caused by Botrytis cinerea on kiwi fruits

Antifungal activity of the essential oils of Carum carvi and Pimpinella anisum against Botrytis cinerea fruit rot of key kiwi fruit was studied. In vitro experiments, antifungal activities of essential oils were tested on potato dextrose agar media. Results of an in vitro experiment showed that these essential oils, at all applied concentrations, inhibited grey mould growth. Black caraway essential oil at concentrations of 600 and 800?μL?L^?1 inhibited germination spores of grey mould. Then, the fruits were artificially inoculated with a suspension at 1?×?10⁵?conidia/ml and then treated with different concentrations of these essential oils. The results of in vivo conditions showed that black caraway and anise essential oils applied at all concentrations were increasing the shelf life and inhibited the grey mould growth on kiwi fruits completely in comparison to control. The result showed that black caraway and anise oils at a concentration of 800?μL?L^?1 had higher total soluble solids, ascorbic acid, titrable acidity and antioxidant content compared to untreated fruits.     

Inhibition of green mould in blood orange (Citrus sinensis var. Moro) with salicylic acid treatment

The aim of this study was to find an alternative to synthetic fungicides currently used in the control of devastating fungal pathogen Penicillium digitatum Sacc, the causal agent of green mould disease of blood orange. Antifungal activities of salicylic acid (SA) were investigated against P. digitatum. Treatments consisted of five concentrations (0, 1, 2, 4 and 5?mM). The SA application significantly decreased weight loss percentage and increased life storage of fruits. Also, SA positively affected on post-harvest quality factors including total soluble solids (TSS), titrable acidity, anthocyanin, antioxidant content and pH value. It was observed that treated fruits at a concentration of 5?mM had the highest TSS, titrable acidity, anthocyanin and antioxidant content, and it had the lowest decay and acidity. Thus, these results showed that SA has strong impact on post-harvest decay and fruit quality of blood orange.