Comparison of membranes and glass slides for 16S rDNA array analyses of microbial communities by sequence-specific labeling of DNA probes

Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for a DNA array based analyzes of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions, and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Arrays prepared on positively charged nylon membranes and coated glass slides were compared. The advantage of using membranes is that chromogenic signal amplification can be used for the detection. Furthermore, the chromogenic detection does not require any sophisticated equipment. The advantage of the glass slides is that multiple fluorescence colors can be detected simultaneously, and that internal controls can be used for normalization. This approach is also suited for high throughput screenings.     

Two modes of gating during late Na+ channel currents in frog sartorius muscle

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.     

Expression of the Escherichia coli pntA and pntB genes, encoding nicotinamide nucleotide transhydrogenase, in Saccharomyces cerevisiae and its effect on product formation during anaerobic glucose fermentation.

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD+ in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD+ by NADPH and by NADH in the presence of NADP+, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD+, NADPH, and NADP+ were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD+.     

Stability of nucleosome placement in newly repaired regions of DNA

Rearrangements of chromatin structure during excision repair of UV-damaged DNA appear to involve unfolding of nucleosomal DNA while repair is taking place, followed by refolding of this DNA into a native nucleosome structure. Recently, we found that repair patches are not distributed uniformly along the DNA in nucleosome core particles immediately following their refolding into nucleosomes (Lan, S. Y., and Smerdon, M. J. (1985) Biochemistry, 24,7771). Therefore, the distribution of repair patches in nucleosome core DNA was used to monitor the stability of nucleosome placement in these regions. Our results indicate that in nondividing human cells undergoing excision repair there is a slow change in the positioning of nucleosomes in newly repaired regions of chromatin, resulting in the eventual randomization of repair patches in nucleosome core DNA. Furthermore, the nonrandom placement of nucleosomes observed just after the refolding event is not re-established during DNA replication. Possible mechanisms for this change in nucleosome placement along the DNA are discussed.     

Modulatory and catalytic modes of ATP binding by the calcium pump

We present crystal structures of the calcium-free E2 state of the sarcoplasmic reticulum Ca2+ -ATPase, stabilized by the inhibitor thapsigargin and the ATP analog AMPPCP. The structures allow us to describe the ATP binding site in a modulatory mode uncoupled from the Asp351 phosphorylation site. The Glu439 side chain interacts with AMPPCP via an Mg2+ ion in accordance with previous Fe2+ -cleavage studies implicating this residue in the ATPase cycle and in magnesium binding. Functional data on Ca2+ mediated activation indicate that the crystallized state represents an initial stage of ATP modulated deprotonation of E2, preceding the binding of Ca2+ ions in the membrane from the cytoplasmic side. We propose a mechanism of Ca2+ activation of phosphorylation leading directly from the compact E2-ATP form to the Ca2E1-ATP state. In addition, a role of Glu439 in ATP modulation of other steps of the functional cycle is suggested.     

Follicular Development and Ovulation in Sows: Effect of hCG and GnRH Treatment

Follicular growth, chronology of ovulation and embryo morphology were compared in sows ovulating spontaneously and sows, in which the ovulation was attempted induced by hCG or GnRH. Indwelling catheters were placed on day 1 (weaning = day 0) in the ear veins of 18 sows, which were then randomly divided into 3 groups: a control group (N = 6), a group (N = 6) given 750 iu hCG (Physex®) im 76h after weaning (hCG group) and a group (N = 6) given 500 µg GnRH (Fertagyl®) im 76h (N = 3) or 100h after weaning (N = 3) (GnRH group). Follicular diameter and time of ovulation were monitored by ultrasonography every 4h from day 3 until ovulation or development of cysts by means of a sector scanner fitted with a 5.0/7.5 MHz multiangle probe. Heat detection was performed every 8h from day 3 until ovulation. On day 13, the sows were slaughtered, the number of corpora luteae (CL) was counted, and embryos were flushed from the uteri. The control group showed clear heat symptoms, and on day 3, the follicles were typically 3–7 mm and grew up to 7–10 mm over 2 days, where they remained for approximately 24h until ovulation took place 41h ± 9h after first sign of standing heat. The hCG group exhibited no signs of heat, and the follicles only reached 5–8 mm in diameter at time of ovulation, which occurred 40h ± lh after hCG-injection. The GnRH group exhibited inconsistent signs of heat, and the follicles reached a maximum size of 7–12 mm in diameter where they remained for more than 24h. Only 2 sows in this group ovulated within 84–92h after the GnRH injection, and development of bursa cysts and cystic follicles was a common finding. The average number of CL was 18.2 ±5.7 per sow (N = 16, range: 3–27) with no significant difference between the groups. Total embryo recovery was 79 ± 13 % with no significant difference between groups. The embryo diversity calculated as standard deviation of the maximum diameter was higher in the hCG group as compared with the control group. It is concluded that (1) transrectal ultrasonography can be used in sows for accurate assessment of follicular growth and ovulation; (2) the use of hCG results in lack of heat symptoms and reduced follicle size at the time of ovulation when injected 76h after weaning; (3) administration of a single injection of GnRH, if given before the first signs of heat, results in inconsistent heat symptoms and no or late ovulations.     

Nucleotide sequence of the Acinetobacter calcoaceticus trpGDC gene cluster

A plasmid library of Acinetobacter calcoaceticus HindIII fragments was constructed, and clones that complemented an Escherichia coli pabA mutant were selected. Plasmids containing a 3.9-kb fragment of A. calcoaceticus DNA that also complemented E. coli trpD and trpC-(trpF+) mutants were obtained. We infer that complementation of E. coli pabA mutants was the result of the expression of the amphibolic anthranilate- synthase/p-aminobenzoate-synthase glutamine-amidotransferase gene and that the plasmid insert carried the entire trpGDC gene cluster. In E. coli minicells, the plasmid insert directed the synthesis of polypeptides of 44,000, 33,000, and 20,000 daltons, molecular masses that are consistent with the reported molecular masses of phosphoribosylanthranilate transferase, indoleglycerol-phosphate synthase, and anthranilate-synthase component II, respectively. A 3,105- bp nucleotide sequence was determined. Comparison of the A. calcoaceticus trpGDC sequences with other known trp gene sequences has allowed insight into (1) the evolution of the amphibolic trpG gene, (2) varied strategies for coordinate expression of trp genes, and (3) mechanisms of gene fusions in the trp operon.      

Only one independent genetic association with rheumatoid arthritis within the KIAA1109-TENR-IL2-IL21 locus in Caucasian sample sets: confirmation of association of rs6822844 with rheumatoid arthritis at a genome-wide level of significance

Introduction

To investigate whether accelerated hand bone mineral density (BMD) loss is associated with progressive joint damage in hands and feet in the first year of rheumatoid arthritis (RA) and whether it is an independent predictor of subsequent progressive total joint damage after 4 years.

Methods

In 256 recent-onset RA patients, baseline and 1-year hand BMD was measured in metacarpals 2-4 by digital X-ray radiogrammetry. Joint damage in hands and feet were scored in random order according to the Sharp-van der Heijde method at baseline and yearly up to 4 years.

Results

68% of the patients had accelerated hand BMD loss (>-0.003 g/cm²) in the first year of RA. Hand BMD loss was associated with progressive joint damage after 1 year both in hands and feet with odds ratios (OR) (95% confidence intervals [CI]) of 5.3 (1.3-20.9) and 3.1 (1.0-9.7). In univariate analysis, hand BMD loss in the first year was a predictor of subsequent progressive total joint damage after 4 years with an OR (95% CI) of 3.1 (1.3-7.6). Multivariate analysis showed that only progressive joint damage in the first year and anti-citrullinated protein antibody positivity were independent predictors of long-term progressive joint damage.

Conclusions

In the first year of RA, accelerated hand BMD loss is associated with progressive joint damage in both hands and feet. Hand BMD loss in the first year of recent-onset RA predicts subsequent progressive total joint damage, however not independent of progressive joint damage in the first year.     

Large Scale Identification and Categorization of Protein Sequences Using Structured Logistic Regression

Background

Structured Logistic Regression (SLR) is a newly developed machine learning tool first proposed in the context of text categorization. Current availability of extensive protein sequence databases calls for an automated method to reliably classify sequences and SLR seems well-suited for this task. The classification of P-type ATPases, a large family of ATP-driven membrane pumps transporting essential cations, was selected as a test-case that would generate important biological information as well as provide a proof-of-concept for the application of SLR to a large scale bioinformatics problem.

Results

Using SLR, we have built classifiers to identify and automatically categorize P-type ATPases into one of 11 pre-defined classes. The SLR-classifiers are compared to a Hidden Markov Model approach and shown to be highly accurate and scalable. Representing the bulk of currently known sequences, we analysed 9.3 million sequences in the UniProtKB and attempted to classify a large number of P-type ATPases. To examine the distribution of pumps on organisms, we also applied SLR to 1,123 complete genomes from the Entrez genome database. Finally, we analysed the predicted membrane topology of the identified P-type ATPases.

Conclusions

Using the SLR-based classification tool we are able to run a large scale study of P-type ATPases. This study provides proof-of-concept for the application of SLR to a bioinformatics problem and the analysis of P-type ATPases pinpoints new and interesting targets for further biochemical characterization and structural analysis.