Calorimetric studies of oxyhemoglobin dissociation. II. Erythrocytic oxygen depletion by sodium dithionite

Dithionite causes the depletion of dioxygen from suspensions of erythrocytes by reduction of the external dioxygen and not by diffusion into the cell. The molar enthalpy for the reduction shows a small difference with respect to the values found for free hemoglobin; and the normal stoichiometry of 2 moles dithionite/mole dioxygen found there is not observed with erythrocytes. At low hematocrit, the stoichiometry is 2.6:1 and decreases to 1.5:1 at high hematocrit. The change is not due to differences in the hemoglobin saturation or to an inability of dithionite to reduce all dioxygen present at the higher hematocrit. Neither catalase nor peroxidase added to the extracellular volume significantly alters the stoichiometry or the enthalpy of dioxygen reduction by dithionite. Addition of superoxide dismutase, however, restores the normal stoichiometry at high hematocrit and further increases the stoichiometry at low hematocrit. The calorimetrical signal of hydrogen peroxide, clearly seen with free dioxygen, is not present with erythrocytes. In all these cases the total heat evolved is the same.     

8-Azaguanine versus 6-thioguanine: influence on frequency and expression time of induced HGPRT- mutations in Chinese hamster V79 cells

Chinese hamster V79 cells were mutagenized with ethyl methanesulfonate at various concentrations. Clones resistant to 8-azaguanine (20 and 80 micrograms/ml) or 6-thioguanine (4 micrograms/ml) were selected at different times after the treatments. The total yield of induced mutations was only slightly affected by the kind and concentration of purine analog used in the selection. However, full phenotypic expression of the mutants selected with 8-azaguanine was achieved earlier than that of mutants resistant to 6-thioguanine. This result seems to be best explained by the reported lower affinity of 8-azaguanine for the wild-type HGPRT enzyme, thus providing evidence that, in this gene-mutation assay, the phenotypic expression time has a physiological component.     

Cannabinoid receptor 1 (CNR1) gene variant moderates neural index of cognitive disruption during nicotine withdrawal

Nicotine withdrawal‐related disruption of cognitive control may contribute to the reinforcement of tobacco use. Identification of gene variants that predict this withdrawal phenotype may lead to tailored pharmacotherapy for smoking cessation. Variation on the cannabinoid receptor 1 gene (CNR1) has been related to nicotine dependence, and CNR1 antagonists may increase attention and memory functioning. We targeted CNR1 variants as moderators of a validated neural marker of nicotine withdrawal‐related cognitive disruption. CNR1 polymorphisms comprising the ‘TAG’ haplotype (rs806379, rs1535255 and rs2023239) were tested independently, as no participants in this sample possessed this haplotype. Nicotine withdrawal‐related cognitive disruption was indexed as increased resting electroencephalogram (EEG) alpha‐1 power density across 17 electrodes. Seventy‐three Caucasian Non‐Hispanic smokers (≥15 cigarettes per day) visited the laboratory on two occasions following overnight smoking/nicotine deprivation. Either two nicotine or two placebo cigarettes were smoked prior to collecting EEG data at each session. Analyses showed that rs806379 moderated the effects of nicotine deprivation increasing slow wave EEG (P = 0.004). Smokers homozygous for the major allele exhibited greater nicotine withdrawal‐related cognitive disruption. The current findings suggest potential efficacy of cannabinoid receptor antagonism as a pharmacotherapy approach for smoking cessation among individuals who exhibit greater nicotine withdrawal‐related cognitive disruption.     

Distinct genomic sequence of the CNR/Pcdhalpha genes in chicken

CNR/Pcdhalpha family proteins have been first identified as a receptor family that corporate with Fyn, a family of the Src family of tyrosine kinase, and known as synaptic cadherins. Here we report the complete genomic sequence and organization of the chicken (Gallus gallus) CNR/Pcdhalpha The total length of chicken CNR/Pcdhalpha is 177kb. The chicken CNR/Pcdhalpha cluster encodes 12 variable and 3 constant exons. The genomic organizations of the chicken, rat, mouse, and human CNR/Pcdhalpha are basically orthologous. The constant-region exons (CP1, CP2, and CP3) are highly conserved between chicken and mammals, with percent identities of 90.9%, 90.7%, and 91.8% at the amino-acid level for chicken versus rat, mouse, and human, respectively. In contrast, the percent identities of the variable-region exons between chicken and mammals were lower: 51.8%, 51.3%, and 52.7%, on average, for chicken versus rat, mouse, and human, respectively, at the amino-acid level. Moreover, the chicken variable-region exons (from v1 to v12) are highly conserved paralogously (91.4%: nucleic acid, 92.4%: amino acid) in comparison with those of mammals. The CG content of each variable exon in the chicken (v1 to v12) is 74% on average and the CpG dinucleotide frequency in each variable-region exon is twice that of mammals. Due to the high CG content, chicken variable exons (from v1 to v12) encode 3 to 4 frame-shifted open reading frames, which span 1.5-3.0kb, in both the sense and anti-sense orientations.     

Identification of a novel duplication CFTRdup2 and functional impact of large rearrangements identified in the CFTR gene

The aim of this study was to investigate associations of two candidate gene SNPs of the endocannabinoid receptor type 1 gene (CNR1) with overweight, obesity and obesity-related traits in Chinese retired women. The study subjects were a subsample of the Taizhou Retiree Women Cohort, consisting of 2812 retired women aged 50-64 years recruited from Taizhou, Jiangsu, China. Neither rs2023239 nor rs806381 polymorphism was significantly associated with body mass index-defined overweight and obesity or waist-to-hip-ratio-defined obesity. For obesity-related traits, rs2023239 was significantly associated with glutamate pyruvate transaminase (GPT) (median, 18.00 vs 17.00 for TT and TC genotypes, respectively, P=0.043). The rs806381 also showed significant association with triglyceride (TG) (mean±SD, 1.46±0.20 vs 1.53±0.20 for GA and GG+AA genotypes, respectively, P=0.013) under the dominant genetic model. In conclusion, the rs2023239 and rs806381 polymorphisms of CNR1 were not associated with increased overweight and obesity risk. But the rs2023239 polymorphism was significantly associated with GPT, and the rs806381 polymorphism was significantly associated with TG.     

Performance in working memory and attentional control is associated with the rs2180619 SNP in the CNR1 gene

Individual differences in cognitive performance are partly dependent, on genetic polymporhisms. One of the single‐nucleotide polymorphisms (SNP) of the CNR1 gene, which codes for cannabinoid receptor 1 (CB1R), is the rs2180619, located in a regulatory region of this gene (6q14–q15). The alleles of the rs2180619 are A > G; the G allele has been associated with addiction and high levels of anxiety (when the G allele interacts with the SS genotype of the 5‐HTTLPR gene). However, GG genotype is observed also in healthy subjects. Considering G allele as risk for ‘psychopathological conditions’, it is possible that GG healthy subjects do not be addicted or anxious, but would have reduced performance, compared to AA subjects, in attentional control and working memory processing. One hundred and sixty‐four healthy young Mexican‐Mestizo subjects (100 women and 64, men; mean age: 22.86 years, SD=2.72) participated in this study, solving a task where attentional control and working memory were required. GG subjects, compared to AA subjects showed: (1) a general lower performance in the task (P = 0.02); (2) lower performance only when a high load of information was held in working memory (P = 0.02); and (3) a higher vulnerability to distractors (P = 0.03). Our results suggest that, although the performance of GG subjects was at normal levels, a lower efficiency of the endocannabinoid system, probably due to a lowered expression of CB1R, produced a reduction in the performance of these subjects when attentional control and working memory processing is challenged .     

Molecular evolution of cadherin-related neuronal receptor/protocadherin(alpha) (CNR/Pcdh(alpha)) gene cluster in Mus musculus subspecies

The mouse cadherin-related neuronal receptor/protocadherin (CNR/Pcdh) gene clusters are located on chromosome 18. We sequenced single-nucleotide polymorphisms (SNPs) of the CNR/Pcdh(alpha)-coding region among 12 wild-derived and four laboratory strains; these included the four major subspecies groups of Mus musculus: domesticus, musculus, castaneus, and bactrianus. We detected 883 coding SNPs (cSNPs) in the CNR/Pcdh(alpha) variable exons and three in the constant exons. Among all the cSNPs, 586 synonymous (silent) and 297 nonsynonymous (amino acid exchanged) substitutions were found; therefore, the K(a)/K(s) ratio (nonsynonymous substitutions per synonymous substitution) was 0.51. The synonymous cSNPs were relatively concentrated in the first and fifth extracellular cadherin domain-encoding regions (ECs) of CNR/Pcdh(alpha). These regions have high nucleotide homology among the CNR/Pcdh(alpha) paralogs, suggesting that gene conversion events in synonymous and homologous regions of the CNR/Pcdh(alpha) cluster are related to the generation of cSNPs. A phylogenetic analysis revealed gene conversion events in the EC1 and EC5 regions. Assuming that the common sequences between rat and mouse are ancestral, the GC content of the third codon position has increased in the EC1 and EC5 regions, although biased substitutions from GC to AT were detected in all the codon positions. In addition, nonsynonymous substitutions were extremely high (11 of 13, K(a)/K(s) ratio 5.5) in the laboratory mouse strains. The artificial environment of laboratory mice may allow positive selection for nonsynonymous amino acid variations in CNR/Pcdh(alpha) during inbreeding. In this study, we analyzed the direction of cSNP generation, and concluded that subspecies-specific nucleotide substitutions and region-restricted gene conversion events may have contributed to the generation of genetic variations in the CNR/Pcdh genes within and between species.     

Clustered protocadherin family

The brain is a complex system composed of enormous numbers of differentiated neurons, and brain structure and function differs among vertebrates. To examine the molecular mechanisms underlying brain structure and function, it is important to identify the molecules involved in generating neural diversity and organization. The clustered protocadherin (Pcdh) family is the largest subgroup of the diverse cadherin superfamily. The clustered Pcdh proteins are predominantly expressed in the brain and their gene structures in vertebrates are diversified. In mammals, the clustered Pcdh family consists of three gene clusters: Pcdh -α, Pcdh -β, and Pcdh -γ. During brain development, this family is upregulated by neuronal differentiation, and Pcdh-α is then dramatically downregulated by myelination. Clustered Pcdh expression continues in the olfactory bulb, hippocampus, and cerebellum until adulthood. Structural analysis of the first cadherin domain of the Pcdh-α protein revealed it lacks the features that classical cadherins require for homophilic adhesiveness, but it contains Pcdh-specific loop structures. In Pcdh-α, an RGD motif on a specific loop structure binds β1-integrin. For gene expression, the gene clusters are regulated by multiple promoters and alternative cis splicing. At the single-cell level, several dozen Pcdh -α and -γ mRNA are regulated monoallelically, resulting in the combinatorial expression of distinct variable exons. The Pcdh-α and Pcdh-γ proteins also form oligomers, further increasing the molecular diversity at the cell surface. Thus, the unique features of the clustered Pcdh family may provide the molecular basis for generating individual cellular diversity and the complex neural circuitry of the brain.     

Sulfonamide inhibition profile of the γ-carbonic anhydrase identified in the genome of the pathogenic bacterium Burkholderia pseudomallei the etiological agent responsible of melioidosis

A new γ-carbonic anhydrase (CA, EC 4.1.1.1) was cloned and characterized kinetically in the genome of the bacterial pathogen Burkholderia pseudomallei, the etiological agent of melioidosis, an endemic disease of tropical and sub-tropical regions of the world. The catalytic activity of this new enzyme, BpsCAγ, is significant with a k_cat of 5.3 × 10⁵ s^?1 and k_cat/K_m of 2.5 × 10⁷ M^?1 × s^?1 for the physiologic CO₂ hydration reaction. The inhibition constant value for this enzyme for 39 sulfonamide inhibitors was obtained. Acetazolamide, benzolamide and metanilamide were the most effective (K_Is of 149–653 nM) inhibitors of BpsCAγ activity, whereas other sulfonamides/sulfamates such as ethoxzolamide, topiramate, sulpiride, indisulam, sulthiame and saccharin were active in the micromolar range (K_Is of 1.27–9.56 μM). As Burkholderia pseudomallei is resistant to many classical antibiotics, identifying compounds that interfere with crucial enzymes in the B. pseudomallei life cycle may lead to antibiotics with novel mechanisms of action.     

Genome-wide identification of cytosine-5 DNA methyltransferases and demethylases in Solanum lycopersicum

Recent studies have reported that decreased level of DNA cytosine methylation in the global genome was closely related to the initiation of tomato (Solanum lycopersicum) fruit ripening. However, genome-scale analysis of cytosine-5 DNA methyltransferases (C5-MTases) and demethylases in tomato has not been engaged. In this study, 7 C5-MTases and 3 demethylases were identified in tomato genome, which probably contributed to DNA cytosine methylation level in tomato. The 7 C5-MTases were categorized into 4 subgroups, and the 3 demethylases were classified into 2 subgroups based on phylogenetic analyses. Comprehensive analysis of their structure and genomic localization was also performed in this paper. According to online RNA-seq data, 4 S. lycopersicum C5-MTase (SlC5-MTase) genes (SlMET, SlDRM1L1, SlDRM5, SlMET3L) were expressed higher than others, and one DNA demethylase gene (SlDML) was significantly changed during tomato fruit development and ripening. Furthermore, all these five gene expressions at breaker (BK) stage changed with 1-methylcyclopropene (1-MCP) treatment, indicating that they were regulated by ethylene directly or indirectly in tomato fruit. In addition, subcellular localization analysis indicated that SlDRM1L1 and SlDRM5 located in the nucleus might have responsibility for RNA-directed DNA methylation (RdDM). Collectively, this paper provided a framework for gene discovery and functional characterization of C5-MTases and DNA demethylases in other Solanaceae species.