Familial porphyria cutanea tarda: hybridization analysis of the uroporphyrinogen decarboxylase locus

Familial porphyria cutanea tarda (PCT) results from a deficiency of uroporphyrinogen decarboxylase (URO-D) activity. Hybridization analysis of genomic DNA from unrelated normal individuals and PCT pedigree members failed to detect any major deletions, rearrangements or restriction fragment length polymorphisms at the URO-D locus.     

Synthesis and anticonvulsant activity of 3-aryl-5H-2,3-benzodiazephine AMPA antagonists.

A novel series of 3-aryl-5H-2,3-benzodiazepines with N-3 aromatic substituents has been synthesized. Good in vivo anticonvulsant activity of the new compounds has been demonstrated employing the maximal electroshock seizure test in mice. Evaluation of a subset of the compounds in the cortical wedge assay confirmed the new structures to be AMPA antagonists.     

Dietary induction of hepatic glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme in lean and obese female Zucker rats

Responses of the hepatic lipogenic enzymes, glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and malic enzyme (ME) to starvation refeeding and diet shifting were determined in lean and obese female Zucker rats. Rats were either fed nonpurified diet, starved 48 hr, and then refed nonpurified diet or one of the refined carbohydrate diets containing either glucose, fructose, cornstarch, or sucrose for 72 hr, or shifted from nonpurified diet directly to one of the refined carbohydrate diets for 72 hr. Initial activities were greater in obese than lean rats for all three enzymes studied. Similar to other strains of female rats, lean Zucker rats failed to demonstrate a starve-refeed response when refed nonpurified diet. Obese female littermates showed a statistically significant increase in enzymes when refed a nonpurified diet. Both lean and obese female Zucker rats demonstrated increases in enzyme activities above controls when starved and refed any of the refined carbohydrate diets. The greatest responses were observed when female rats were starved and refed sucrose; activities increased 2.6- to 3.5-fold in lean and 3.0- to 4.3-fold in obese Zuckers. In lean females 50-70% of the starve-refeed response observed with G6PDH and ME can be accounted for by simply shifting from a nonpurified diet to the respective refined carbohydrate diet, whereas in obese females only 33-55% of the increase could be attributed to diet shifting. Plasma testosterone/estrogen ratios were consistently 1.5 times higher in obese than in lean female rats. This phenotypic difference may potentiate the heightened starve-refeed overshoot response observed in obese rats.     

Satellite 2 methylation patterns in normal and ICF syndrome cells and association of hypomethylation with advanced replication

Mutation in the DNMT3B DNA methyltransferase gene is a common cause of ICF (immunodeficiency, centromeric heterochromatin, facial anomalies) immunodeficiency syndrome and leads to hypomethylation of satellites 2 and 3 in pericentric heterochromatin. This hypomethylation is associated with centromeric decondensation and chromosomal rearrangements, suggesting that these satellite repeats have an important structural role. In addition, the satellite regions may have functional roles in modifying gene expression. The extent of satellite hypomethylation in ICF cells is unknown because methylation status has only been determined with restriction enzymes that cut infrequently at these loci. We have therefore developed a bisulfite conversion-based method to determine the detailed cytosine methylation patterns at satellite 2 sequences in a quantitative manner for normal and ICF samples. From our sequence analysis of unmodified DNA, the internal repeat region analyzed for methylation contains an average of 17 CpG sites. The average level of methylation in normal lymphoblasts and fibroblasts is 69% compared with 20% in such cells from ICF patients with DNMT3B mutations and 29% in normal sperm. Although the mean satellite 2 methylation values for these groups do not overlap, there is considerable overlap at the level of individual DNA strands. Our analysis has also revealed a pattern of methylation specificity, suggesting that some CpGs in the repeat are more prone to methylation than other sites. Variation in satellite 2 methylation among lymphoblasts from different ICF patients has prompted us to determine the frequency of cytogenetic abnormalities in these cells. Although our data suggest that some degree of hypomethylation is necessary for pericentromeric decondensation, factors other than DNA methylation appear to play a major role in this phenomenon. Another such factor may be altered replication timing because we have discovered that the hypomethylation of satellite 2 in ICF cultures is associated with advanced replication.     

Glycerol and dihydroxyacetone dissimilation in Desulfovibrio strains

During growth on glycerol two marine Desulfovibrio strains that can grow on an unusually broad range of substrates contained high activities of glycerol kinase, NAD(P)-independent glycerol 3-phosphate dehydrogenase and the other enzymes necessary for the conversion of dihydroxyacetone phosphate to pyruvate. Glycerol dehydrogenase and a specific dihydroxyacetone kinase were absent. During growth on dihydroxyacetone, glycerol kinase is involved in the initial conversion of this compound to dihydroxyacetone phosphate which is then further metabolized. Some kinetic properties of the partially purified glycerol kinase were determined. The role of NAD as electron carrier in the energy metabolism during growth of these strains on glycerol and dihydroxyacetone is discussed.Glycerol also supported growth of three out of four classical Desulfovibrio strains tested. D. vulgaris strain Hildenborough grew slowly on glycerol and contained glycerol kinase, glycerol 3-phosphate dehydrogenase and enzymes for the dissimilation of dihydroxyacetone phosphate. In D. gigas which did not grow on glycerol the enzymes glycerol kinase and glycerol 3-phosphate dehydrogenase were absent in lactate-grown cells.Abbreviations DHA dihydroxyacetone - DHAP dihydroxyacetone phosphate - G3P glycerol 3-phosphate - GAP glyceraldehyde 3-phosphate - 3-PGA 3-phosphoglycerate - 2-PGA 2-phosphoglycerate - 2,3-DPGA 2,3-diphosphoglycerate - PEP phosphoenolpyruvate - DH dehydrogenase - GK glycerol kinase - DHAK dihydroxyacetone kinase - TIM triosephosphate isomerase - PGK 3-phosphoglycerate kinase - PK pyruvate kinase - LDH lactate dehydrogenase - DTT dithiotreitol - HEPES 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid - PIPES piperazine-1,1-bis(2-ethane sulfonic acid) - BV²⁺/BV⁺ oxidized/reduced benzylviologen - PMS phenazine methosulfate - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide     

MaGIC: a program to generate targeted marker sets for genome-wide association studies

High-throughput genotyping technologies such as DNA pooling and DNA microarrays mean that whole-genome screens are now practical for complex disease gene discovery using association studies. Because it is currently impractical to use all available markers, a subset is typically selected on the basis of required saturation density. Restricting markers to those within annotated genomic features of interest (e.g., genes or exons) or within feature-rich regions, reduces workload and cost while retaining much information. We have designed a program (MaGIC) that exploits genome assembly data to create lists of markers correlated with other genomic features. Marker lists are generated at a user-defined spacing and can target features with a user-defined density. Maps are in base pairs or linkage disequilibrium units (LDUs) as derived from the International HapMap data, which is useful for association studies and fine-mapping. Markers may be selected on the basis of heterozygosity and source database, and single nucleotide polymorphism (SNP) markers may additionally be selected on the basis of validation status. The import function means the method can be used for any genomic features such as housekeeping genes, long interspersed elements (LINES), or Alu repeats in humans, and is also functional for other species with equivalent data. The program and source code is freely available at http://cogent.iop.kcl.ac.uk/MaGIC.cogx.     

GABAA Modulation of S100B Secretion in Acute Hippocampal Slices and Astrocyte Cultures

Astrocytes are the major glial cells in brain tissue and are involved, among many functions, ionic and metabolic homeostasis maintenance of synapses. These cells express receptors and transporters for neurotransmitters, including GABA. GABA signaling is reportedly able to affect astroglial response to injury, as evaluated by specific astrocyte markers such as glial fibrillary acid protein and the calcium-binding protein, S100B. Herein, we investigated the modulatory effects of the GABA_A receptor on astrocyte S100B secretion in acute hippocampal slices and astrocyte cultures, using the agonist, muscimol, and the antagonists pentylenetetrazol (PTZ) and bicuculline. These effects were analyzed in the presence of tetrodotoxin (TTX), fluorocitrate (FLC), cobalt and barium. PTZ positively modify S100B secretion in hippocampal slices and astrocyte cultures; in contrast, bicuculline inhibited S100B secretion only in hippocampal slices. Muscimol, per se, did not change S100B secretion, but prevented the effects of PTZ and bicuculline. Moreover, PTZ-induced S100B secretion was prevented by TTX, FLC, cobalt and barium indicating a complex GABA_A communication between astrocytes and neurons. The effects of two putative agonists of GABA_A, β-hydroxybutyrate and methylglyoxal, on S100B secretion were also evaluated. In view of the neurotrophic role of extracellular S100B under conditions of injury, our data reinforce the idea that GABA_A receptors act directly on astrocytes, and indirectly on neurons, to modulate astroglial response.

     

Altered temporal characteristics of the rectoanal inhibitory reflex in patients with abdominal distension

The rectoanal inhibitory reflex (RAIR) is important in gas and stool evacuation. We examined RAIR features in patients with chronic constipation who exhibited bloating with and without abdominal distension, to determine whether alterations in RAIR may be a factor in the pathogenesis of abdominal distension. Seventy-five female patients with chronic constipation with or without abdominal distension were included in the study. The presence or absence of abdominal distension was assessed according to the Rome II questionnaire. All patients underwent both RAIR and rectal sensitivity testing, and specific RAIR parameters were analyzed. Patients were divided into two groups: abdominal bloating with distension (D, n = 55) and abdominal bloating without distension (ND, n = 20). D had a longer time to the onset of anal sphincter inhibition (latency of inhibition) (P = 0.03) compared with ND. In logistic regression analysis, a combination of age, latency of inhibition and the time measured from onset of inhibition to the point of maximum inhibition predicted abdominal distension (P = 0.002). There were no differences between groups for the time from point of maximum inhibition to recovery and for the percentage of internal anal sphincter relaxation. This is the first study to examine the role of RAIR in patients with abdominal distension. Female patients with constipation and abdominal distension exhibited differences in the temporal characteristics of, but not in the degree of, anal sphincter relaxation compared with patients without distension. Since this study was uncontrolled, further studies are necessary to determine the contribution of altered anorectal reflexes to abdominal distension.     

Spatio-temporal distribution and production of calanoid copepods in the central Baltic Sea

The aim of our study was the exploration of species-specificdistribution and production patterns of dominant copepods inthe Central Baltic Sea (Bornholm Basin). Spatio-temporal distribution,egg and secondary production were studied by means of net-samplingand egg production experiments from April to August 1999. Verticaland horizontal distribution patterns appeared to be linked witheach other and were primarily species-dependent. Both Acartiaspp. and Temora longicornis preferentially inhabited the upper30 m of the water column and the shallower marginal regionsof the Bornholm Basin. In contrast, the C4–5 and C6 ofPseudocalanus spp. preferentially inhabited the halocline region(50–70 m) and the deep central part of the area. Observeddifferences in horizontal distribution among these three copepodsappear to result from different depth preferences and depth-dependentwater circulation patterns. Egg production rates (EPR) clearlyexhibited seasonal differences with maximum EPR in May and minimumEPR in July. Mean EPR was significantly different between species,being highest in Acartia spp. and lowest in Pseudocalanus spp.The variability in EPR was related to differences in hydrographyand modelled food environment.     

Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 10⁵ FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm² within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.