Positron emission tomographic studies of cerebral benzodiazepine-receptor binding in chronic alcoholics

Positron emission tomography was used with [¹¹C] flumazenil (FMZ) and [¹⁸F] fluorodeoxyglucose to study GABA type A/benzodiazepine (GABA-A/BDZ) receptors and cerebral metabolic rates for glucose (1CMRglc) in 17 male patients with severe chronic alcoholism (ALC), 8 with (ACD) and 9 without alcoholic cerebellar degeneration (non-ACD). In comparison with male normal controls of similar ages, the ALC group had significantly reduced FMZ ligand influx (K₁), FMZ distribution volume (DV), and 1CMRglc bilaterally in the medial frontal lobes, including superior frontal gyrus and rostral cingulate gyrus; the ACD group had significant reductions of K₁, DV, and ICMRglc bilaterally in the same distribution, and also in the superior cerebellar vermis; and the non-ACD group had significant reductions of K₁, DV, and ICMRglc bilaterally in the same regions of the frontal lobes but not in the superior cerebellar vermis. When compared with the non-ACD group, the ACD group had significant reductions of K₁ and DV bilaterally in the superior cerebellar vermis. The results suggest that severe chronic alcoholism damages neurons containing GABA-A/BDZ receptors in the superior medial apsects of the frontal lobes, and in patients with clinical signs of ACD, neurons containing GABA-A/BDZ receptors in the superior cerebellar vermis.     

Immunochemical detection of DNA in alkaline sucrose gradient fractions

A procedure for the detection of non-radioactive DNA in alkaline sucrose gradients is described. This method consists of the following steps: (1) fractionation of the DNA-containing gradients into microtiter plates, neutralization and overnight adsorption; (2) covalent labelling of the guanine bases of the adsorbed DNA by reaction with N-acetoxy-2-acetylaminofluorene; and (3) determination of the gradient profile by means of an enzyme-linked immunosorbent assay using antibodies with high affinity for dG-AAF. The method has been found suitable for the rapid and sensitive determination of gamma-ray-induced single-strand breaks and UV-induced pyrimidine dimers in mammalian cells in vitro. In addition, it has been shown that the method can be used for the determination of pyrimidine dimers induced in skin cells in vivo.     

Combination of real-time PCR and sequencing to detect multiple clinically relevant genetic variations in the lactase gene

Background: Lactase persistence is an autosomal dominant trait commonly distributed in Europe as well as some parts of east Africa and the Arabian Peninsula. Using real-time PCR to detect the ?13910C?>?T variant common in the European population is a reliable analysis although other variants in the probe-binding site may cause errors in analysis. The aim of this study was to determine the prevalence of the variants in a Danish cohort examined for lactose intolerance as well as to improve the real-time PCR analysis for detection of the different variants.

Methods: We genotyped 3395 routine samples using real-time PCR for the ?13910C?>?T-variant. All consecutive samples identified as ?13910CC were sequenced using Sanger Sequencing. Using the SDS software we examined various quality value settings to improve on the genetic analysis.

Results: Using real-time PCR resulted in 100% successful genotyping of the ?13910C?>?T variant. By using a quality value of 99% and sequencing the undetermined samples we improved the ability of the assay to identify variants other than ?13910C?>?T. This resulted in a reduction of the diagnostic error rate by a factor of 2.4 while increasing the expenses only 3%.

Conclusions: We conclude that using a quality value of 99% in the SDS software significantly improves the diagnostic efficiency of the real-time PCR assay for detecting variants associated to lactase persistence.     

Awareness of Medicare Regulation Changes: Occupational Therapists' Perceptions and Implications for Practice

Aims: Occupational therapists who work with Medicare beneficiaries should keep abreast about Medicare regulations relevant to practice. This study identified how occupational therapists employed by contractual companies working in skilled nursing facilities (SNFs) learn and interpret changes about Medicare regulations impacting practice. Methods: Researchers analyzed qualitative data from 11 occupational therapists employed in a Midwest metropolitan area through an open-ended questionnaire, focus group, and phone interview. Results: Major themes identified included primary reliance on company resources to learn about Medicare regulation changes, reasons therapists were for or against utilization of noncompany resources for enhanced awareness, barriers to, and suggestions for deepening understanding of Medicare regulation changes. Conclusions: Findings have important implications for the occupational therapy field by contributing to better understanding of how current communication and interpretation regarding Medicare regulations occurs with therapists employed in contracted SNFs and conceivable ways for occupational therapists to gain deeper understanding of Medicare regulations impacting practice.     

Carcinogen-induced loss of heterozygosity at the Aprt locus in somatic cells of the mouse

Genetic events leading to the loss of heterozygosity (LOH) have been shown to play a crucial role in the development of cancer. However, LOH events do not occur only in genetically unstable cancer cells but also have been detected in normal somatic cells of mouse and man. Mice, in which one of the alleles for adenine phosphoribosyltransferase (Aprt) has been disrupted by gene targeting, were used to investigate the potency of carcinogens to induce LOH in vivo. After 7,12-dimethyl-1,2-benz[a]anthracene (DMBA) exposure, a 3-fold stronger mutagenic response was detected at the autosomal Aprt gene than at the X chromosomal hypoxantine-guanine phosphoribosyltransferase (Hprt) gene in splenic T-lymphocytes. Allele-specific PCR analysis showed that the normal, nontargeted Aprt allele was lost in 70% of the DMBA-induced Aprt mutants. Fluorescence in situ hybridization analysis demonstrated that the targeted allele had become duplicated in almost all DMBA-induced mutants that displayed LOH at Aprt. These results indicate that the main mechanisms by which DMBA caused LOH were mitotic recombination or chromosome loss andduplication but not deletion. However, after treatment with the alkylating agent N-ethyl-N-nitrosourea, Aprt had a similar mutagenic response to Hprt while the majority (90%) of N-ethyl-N-nitrosourea-induced Aprt mutants had retained both alleles. Unexpectedly, irradiation with x-rays, which induce primarily large deletions, resulted in a significant increase of the mutant frequency at Hprt but not at Aprt. This in vivo study clearly indicates that, in normal somatic cells, carcinogen exposure can result in the induction of LOH events that are compatible with cell survival andmay represent an initiating event in tumorigenesis.     

Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation

Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1-166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47- phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates assembly of the p47 and p67 oxidase components on the cytosolic face of the plasma membrane via cytoskeletal reorganization, whereas Rap1a functions downstream as the final activation switch involving direct physical interaction with the transmembrane flavocytochrome component of the NADPH oxidase.     

Lower levels of maternal capital in early life predict offspring obesity in adulthood

Background: As of 2013, 65% of the world’s population lived in countries where overweight/obesity kills more people than being underweight. Evolutionary perspectives provide a holistic understanding of both how and why obesity develops and its long-term implications.

Aim: To test whether the maternal capital hypothesis, an evolutionary perspective, is viable for explaining the development of obesity in adulthood.

Subjects and methods: Restricted-use data from the National Longitudinal Study of Adolescent Health (Add Health; n?=?11?403) was analysed using logistic regressions. The sample included adolescents and their biological mothers.

Results: The odds of obesity in adulthood increased by 22% for every standard deviation increase in lack of maternal capital (Exp (B)?=?1.22, p?Conclusion: The maternal capital perspective is useful for explaining how and why early life characteristics (including maternal resources) predict obesity in adulthood. Implications of the findings are discussed.     

Evaluation of Portability and Cost of a Fluorescent PCR Ribotyping Protocol for Clostridium difficile Epidemiology

Clostridium difficile is the most commonly identified pathogen among health care-associated infections in the United States. There is a need for accurate and low-cost typing tools that produce comparable data across studies (i.e., portable data) to help characterize isolates during epidemiologic investigations of C. difficile outbreaks and sporadic cases of disease. The most popular C. difficile-typing technique is PCR ribotyping, and we previously developed methods using fluorescent PCR primers and amplicon sizing on a Sanger-style sequencer to generate fluorescent PCR ribotyping data. This technique has been used to characterize tens of thousands of C. difficile isolates from cases of disease. Here, we present validation of a protocol for the cost-effective generation of fluorescent PCR ribotyping data. A key component of this protocol is the ability to accurately identify PCR ribotypes against an online database (http://walklab.rcg.montana.edu) at no cost. We present results from a blinded multicenter study to address data portability across four different laboratories and three different sequencing centers. Our standardized protocol and centralized database for typing of C. difficile pathogens will increase comparability between studies so that important epidemiologic linkages between cases of disease and patterns of emergence can be rapidly identified.