A Pilot Study of Demographic and Dopaminergic Genetic Contributions to Weight Change in Kidney Transplant Recipients

Kidney transplant recipients often experience a significant amount of weight gain in the first year post-transplantation. While demographic factors such as age, race, and sex have been associated with weight gain in this population, these factors do not explain all of the variability seen. A number of studies have suggested that genetics also plays a critical role in weight changes. Recently, alterations in the activity of the neurotransmitter dopamine have been associated with weight change, and gene expression studies in kidney transplant recipients have supported this association. The purpose of this pilot study is to first examine the feasibility and methodology, and then to examine the associations of age, race, sex, and genotype for 13 SNPs and 3 VNTRs in 9 dopaminergic pathway genes (ANKK1, DRD2, DRD3, DRD4, SLC6A3/DAT1, MAOA, MAOB, COMT, CPE) for associations with percent weight change at 12 months post-transplantation. Seventy kidney transplant recipients had demographic and clinical data collected as a part of a larger observational study. DNA was extracted from repository buffy coat samples taken at the time of transplant, and genotyped using Taqman and PCR based methods. Three SNPs were independently associated with percent weight change: ANKK1 rs1800497 (r = -0.28, p = 0.05), SLC6A3/DAT1 rs6347 (p = 0.046), and CPE rs1946816 (p = 0.028). Stepwise regression modelling confirmed the combined associations of age (p = 0.0021), DRD4 VNTR 4/5 genotype (p = 0.0074), and SLC6A3/DAT1 rs6347 CC genotype (p = 0.0009) and TT genotype (p = 0.0004) with percent weight change in a smaller sample (n = 35) of these kidney transplant recipients that had complete genotyping. These associations indicate that there may be a genetic, and an age component to weight changes post transplantation.     

Gas chromatographic method using photoionization detection for the determination of breath pentane

Lipid peroxidation is thought to be an important event in the pathogenesis of atherosclerosis. It has been suggested that pentane, which can be formed during the oxidation of ω-6 fatty acids, is a marker of lipid peroxidation. Previous studies have reported elevated breath pentane and serum markers of lipid peroxidation in smokers. However, chromatographic separation of pentane from isoprene in virtually all of these studies was incomplete and the methods used did not resolve pentane into its isomers, n-pentane and isopentane. Additionally, most current methods are complicated, requiring trapping and concentrating steps to obtain adequate sensitivity prior to hydrocarbon analysis. The purpose of the current study was to develop a gas chromatographic system to analyze breath pentane, that addressses the above technical problems and that would provide a simple in vivo method for measuring lipid. n-Pentane and isopentane standards were easily separated from isoprene with a Al₂O₃/KCl capillary column contained in a portable gas chromatograph equipped with a photoionization detector. The analysis of repeated measures showed a low coefficient of variation for measurements of n-pentane (10%) and isopentane (9%). We measured breath pentane in 27 subjects (15 smokers, 12 non-smokers). There were no significant difference between the baseline and 4 week interval measurements of n-pentane for smokers both before and after cigarette smoking. The within-subject variability data showed that the assay is highly reproducible for both low and high pentane levels in smokers. Smokers were found to have higher levels of both n-pentane and isopentane than non-smokers (P<0.001). In addition, smokers had further significant elevation of pentane levels 10 min after smoking (P<0.001), which returned to baseline by 1 h. These studies demonstrate that measurement of breath pentane, using a gas chromatograph with a photoionization detector, is simple and reproducible. Additionally, these results suggest that pentane elevation associated with smoking is secondary to the oxidant effects of cigarette smoke and an important temporal relationship exists between cigarette smoking and breath sample analysis.     

A graphical method for detecting recombination in phylogenetic data sets

Current phylogenetic tree reconstruction methods assume that there is a single underlying tree topology for all sites along the sequence. The presence of mosaic sequences due to recombination violates this assumption and will cause phylogenetic methods to give misleading results due to the imposition of a single tree topology on all sites. The detection of mosaic sequences caused by recombination is therefore an important first step in phylogenetic analysis. A graphical method for the detection of recombination, based on the least squares method of phylogenetic estimation, is presented here. This method locates putative recombination breakpoints by moving a window along the sequence. The performance of the method is assessed by simulation and by its application to a real data set.      

bilbo, a non-LTR retrotransposon of Drosophila subobscura: a clue to the evolution of LINE-like elements in Drosophila

We used the repetitive character of transposable elements to isolate a non-LTR retrotransposon in Drosophila subobscura. bilbo, as we have called it, has homology to TRIM and LOA elements. Sequence analysis showed a 5' untranslated region (UTR), an open reading frame (ORF) with no RNA-binding domains, a downstream ORF that had structural homology to that of the I factor, and, finally, a 3' UTR which ended in several 5-nt repeats. The results of our phylogenetic and structural analyses shed light on the evolution of Drosophila non-LTR retrotransposons and support the hypothesis that an ancestor of these elements was structurally complex.      

Variation of physico-chemical parameters along a river transect through the Okavango Delta,Botswana

The Okavango Delta depends on water quantity and quality to sustain its ecosystem services. Whereas many studies have been carried out on its hydrology, few have been done on water quality in the delta. Water pH, electrical conductivity (EC), dissolved oxygen (DO), turbidity, total suspended solids (TSS) and dissolved organic carbon (DOC) were monitored at 10 sites along the Okavango–Boro–Thamalakane–Lake Ngami system almost fortnightly from June 2008 to June 2010. Water quality in the delta was generally good, despite high evapotranspiration rates which would normally produce very saline waters. Electrical conductivity and water temperature increased with distance from Mohembo to Lake Ngami, the former most likely due to evapoconcentration. In contrast, pH, DO, turbidity and TSS decreased with distance from Mohembo to Boro at the lower end of the seasonal floodplain, before increasing again to Lake Ngami. Dissolved oxygen and TSS most likely declined due to biological uptake and particle sedimentation, respectively. Strong and significant relationships were observed between TSS and turbidity and between DOC and EC, indicating that turbidity and EC could be useful proxies for routine estimations of TSS and DOC, respectively, in the delta.     

Macroinvertebrates as bioindicators of water quality in the Mkondoa River,Tanzania, in an agricultural area

The suitability of using macroinvertebrates as bioindicators of stream water quality was tested in the Mkondoa River in an agricultural area at Kilosa, using the rapid bioassessment protocol. The family biotic index (FBI) showed marked variation in water quality along the stream from values ranging from 4.1 to 5.0 in the upstream reaches, indicating good water quality, 5.3 to 5.5 in the mid-reaches and 6.0 to 6.5 in the lower reaches. The water quality index (WQI) indicated that water quality was fair (77 ± 0.98) in the upstream reach of the Mkondoa, marginal (55 ± 0.86) in the midstream reach and poor (33 ± 0.45) in the downstream reach. There were significant relationships between biological oxygen demand and dissolved oxygen and the occurrence of specific taxa, mainly Chironomus and Caenis. Significant changes in macroinvertebrate abundance were mostly related to changes in water quality. As in other parts of the world, macroinvertebrate communities proved to be good biological indicators of water quality and they should be used as bioindicators in long-term monitoring of this river.     

Analysis of cell surface galactosyltransferase activity during mouse trophectodermal differentiation

The ectoplacental cone (EPC) of the Day 7.5 mouse embryo consists of a core of adhesive, proliferating trophoblast cells which transform to invasive trophoblast giant cells during implantation. Adhesive trophoblast cell types express monoclonally defined lactosaminoglycans (LAGs) at the cell surface; transformation to giant cells results in a loss of LAG cell surface expression (H. J. Hathaway and B. S. Babiarz, 1988, Cell Differ. 24, 55-66). LAGs can serve as substrates for cell surface galactosyltransferase (GalTase), providing an adhesive mechanism between a number of different cell types (B. D. Shur, 1984, Mol. Cell. Biochem. 61, 143-158). It was hypothesized that the LAGs in the EPC represented a substrate for a similar GalTase-mediated cell:cell adhesion system. Cell surface GalTase activity was demonstrated on EPC trophoblast on Day 7.5 of development by the incorporation of galactose from exogenous radiolabeled substrate. In 24- to 48-hr EPC trophoblast cultures the enzyme was localized by immunofluorescence to areas of cell:cell contact. Monolayers of differentiated trophoblast giant cells lacked this labeling pattern. The cell surface glycopeptide substrate for GalTase eluted as a single peak with an apparent molecular mass of 15,000 Da. A portion of this material was sensitive to endo-beta-galactosidase digestion, indicating that it contained a LAG structure. Perturbation of the enzyme:substrate complex in 24- to 48-hr EPC outgrowths, with alpha-lactalbumin, uridine 5'-diphosphogalactose, or anti-GalTase antibody, resulted in the disruption of cell:cell contacts. Differentiation to trophoblast giant cells resulted in a loss of sensitivity to surface GalTase perturbation. The results suggest that adhesive EPC trophoblast cells possess a GalTase-mediated cell:cell adhesion system which is downregulated upon differentiation to invasive trophoblast giant cells.     

The effects of caldesmon on smooth muscle heavy actomeromyosin ATPase activity and binding of heavy meromyosin to actin

Caldesmon was purified to homogeneity from both chicken gizzard and bovine aortic smooth muscles. Caldesmon purified from bovine aorta was slightly larger than caldesmon purified from chicken gizzards (Mr = 140,000) when the two were compared electrophoretically. Caldesmon bound tightly to actin saturating at a molar ratio of 1 caldesmon monomer per 6.6 actin monomers. Ca2+-calmodulin appeared to reduce the affinity of caldesmon for actin. Caldesmon was also a potent inhibitor of heavy actomeromyosin ATPase activity producing a maximal effect at a ratio of 1 caldesmon monomer per 7-10 actin monomers. This effect was also antagonized by Ca2+-calmodulin. While caldesmon inhibited heavy actomeromyosin ATPase activity, it greatly enhanced binding of both unphosphorylated and phosphorylated heavy meromyosin to actin in the presence of MgATP, reducing the Kd for binding by a factor of 40 for each form of heavy meromyosin. Although we did identify a Ca2+-calmodulin-stimulated "caldesmon kinase" activity in caldesmon preparations purified under nondenaturing conditions, we observed no effect of phosphorylation (2 mol of PO4/mol of caldesmon) on the capacity to inhibit heavy actomeromyosin ATPase activity. Our results suggest that caldesmon could serve some role in smooth muscle function by enhancing cross-bridge affinity while inhibiting actomyosin ATPase activity.     

The kinetics of the reversible inhibition of heart lactate dehydrogenase through the formation of the enzyme–oxidized nicotinamide–adenine dinucleotide–pyruvate compound

The inhibition of lactate dehydrogenase at high pyruvate concentration was studied in three ways. First, a rapid decrease in the rate of the enzyme reaction was observed; secondly, the rate of formation of a pyruvate-NAD(+) compound was followed by the change in E(325); thirdly, the rate of quenching of the protein fluorescence was measured. The data obtained at pH6.0 at different temperatures and ionic strengths as functions of pyruvate, NAD(+) and enzyme concentrations show that the extent of inhibition can be correlated with the reversible formation of a compound between pyruvate and enzyme-bound NAD(+). It is suggested that the detailed kinetic analysis of the formation of this abortive ternary compound will give pertinent information about properties of the enzyme-NAD(+) compound involved in the normal catalytic process.