Using 2-aminopurine fluorescence to detect bacteriophage T4 DNA polymerase-DNA complexes that are important for primer extension and proofreading reactions

The fluorescence of the base analogue 2-aminopurine (2AP) was used to probe bacteriophage T4 DNA polymerase-induced conformational changes in the template strand produced during the nucleotide incorporation and proofreading reactions. 2AP fluorescence in DNA is quenched by 2AP interactions with neighboring bases, but T4 DNA polymerase binding to DNA substrates labeled with 2AP in the templating position produces large increases in fluorescence intensity. Fluorescence lifetime studies were performed to characterize the fluorescent complexes. Three fluorescence lifetime components were observed for unbound DNA substrates as reported previously, but T4 DNA polymerase binding modulated the amplitudes of these components and created a new, highly fluorescent 10.5 ns component. Experimental evidence for correlation of fluorescence lifetimes with functionally distinct complexes was obtained by forming complexes under different reaction conditions. T4 DNA polymerase complexes were formed with DNA substrates with matched and mismatched primer ends and with A+T- or G+C-rich primer-terminal regions. dTTP was added to binary complexes to form ternary DNA polymerase-DNA-nucleotide complexes. The effect of temperature on complex formation was studied, and complexes were formed with proofreading-defective T4 DNA polymerases. Complexes characterized by the 10.5 ns lifetime were demonstrated to be formed at the crossroads of the primer-extension and proofreading pathways.     

Mutations in the Drosophila orthologs of the F-actin capping protein alpha- and beta-subunits cause actin accumulation and subsequent retinal degeneration

The progression of several human neurodegenerative diseases is characterized by the appearance of intracellular inclusions or cytoskeletal abnormalities. An important question is whether these abnormalities actually contribute to the degenerative process or whether they are merely manifestations of cells that are already destined for degeneration. We have conducted a large screen in Drosophila for mutations that alter the growth or differentiation of cells during eye development. We have used mitotic recombination to generate patches of homozygous mutant cells. In our entire screen, mutations in only two different loci, burned (bnd) and scorched (scrd), resulted in eyes in which the mutant patches appeared black and the mutant tissue appeared to have undergone degeneration. In larval imaginal discs, growth and cell fate specification occur normally in mutant cells, but there is an accumulation of F-actin. Mutant cells degenerate much later during the pupal phase of development. burned mutations are allelic to mutations in the previously described cpb locus that encodes the beta-subunit of the F-actin capping protein, while scorched mutations disrupt the gene encoding its alpha-subunit (cpa). The alpha/beta-heterodimer caps the barbed ends of an actin filament and restricts its growth. In its absence, cells progressively accumulate actin filaments and eventually die. A possible role for their human orthologs in neurodegenerative disease merits further investigation.     

Replication-independent chromatin loading of Dnmt1 during G2 and M phases

The major DNA methyltransferase, Dnmt1, associates with DNA replication sites in S phase maintaining the methylation pattern in the newly synthesized strand. In view of the slow kinetics of Dnmt1 in vitro versus the fast progression of the replication fork, we have tested whether Dnmt1 associates with chromatin beyond S phase. Using time-lapse microscopy of mammalian cells expressing green-fluorescent-protein-tagged Dnmt1 and DsRed-tagged DNA Ligase I as a cell cycle progression marker, we have found that Dnmt1 associates with chromatin during G2 and M. This association is mediated by a specific targeting sequence, shows strong preference for constitutive but not facultative heterochromatin and is independent of heterochromatin-specific histone H3 Lys 9 trimethylation, SUV39H and HP1. Moreover, photobleaching analyses showed that Dnmt1 is continuously loaded onto chromatin throughout G2 and M, indicating a replication-independent role of Dnmt1 that could represent a novel and separate pathway to maintain DNA methylation.     

Novel glycine transporter type-2 reuptake inhibitors. Part 2: beta- and gamma-amino acid derivatives

Several beta- and gamma-amino acid derivatives were prepared as glycine transport inhibitors and their ability to block the uptake of [(14)C]-glycine in COS7 cells transfected with human glycine transporter-2 (hGlyT-2) were evaluated. A range of lipophilic side chains were tolerated in the beta-amino acid series (i.e., Ph, CH(2)Ph, CH(CH(3))(2), and CH(2)CH(CH(3))(2)). In the gamma-amino acid series, minimal differences in potency were observed between the alpha,beta-unsaturated analogs and the corresponding saturated derivatives. In both series, a 4-biphenyl or 4-phenoxyphenyl substituent appended to the urea or cyanogunaidine moiety was necessary for in vitro activity.     

Electrophysiological studies with repeated episodes of ischaemia on isolated rat heart

In order to know the beneficial effect of preconditioning electrocardiography recording were used as tool to assess myocardial malfunction and for this perfusion apparatus was setup. Electrophysiological changes for each heart were recorded during perfusion at 1, 2, 3, 5, 10, 20, 30 and 60 min of global ischaemia and also during the equal period of reperfusion. Recordings dembnstrate that the normal rate was about 240 beats/min with an "R" amplitude of 4mV. During the first ischaemic episode of 1min the rate was 180 +/- 15 beats/min (counted as per 'R' wave deflection), at 2 mins it was 60 +/- 6 beats/min, at 3 min the rate was 40 +/- 2 beats/min, at 5 mins of ischaemia it was 90 +/- 6 beats/min, at 10 min 20 +/- 2 beats/min, at 20 min the rate was 60 +/- 4 beats/min, and at 30 mins there were nil beats/min. The recovery during all the periods of reperfusion was restored to between 120 and 180 beats/min in all episodes. Further after a 60 min of ischaemia the heart stopped to elicit any mechanical response. It is concluded that short term ischaemia can induce a resilient effect on the beating of the heart after a few episodes as seen subsequent to 1 and 2 min of ischaemia. Further, preconditioning was beneficial up to 30 min, beyond which the heart showed signs of fatigue and irreversible injury.     

Regulation of aflatoxin production by Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation

To elucidate Ca(2+)-mediated regulation of aflatoxin production, the status of Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation was investigated employing toxigenic and non-toxigenic strains of Aspergillus parasiticus. Incubation of cytoplasmic extracts with [gamma-(32)P]ATP followed by SDS-PAGE and autoradiography revealed total absence of protein phosphorylation during periods corresponding to aflatoxin production in the toxigenic strain (NRRL 2999). In contrast, protein phosphorylation was unaffected in the non-toxigenic strain (SRRC 255). Aflatoxin production in the toxigenic strain was also accompanied by enhanced (26-fold) activity of calcineurin (calmodulin-dependent protein phosphatase 2B) concomitant with a lowered (6-fold) activity of calmodulin-dependent protein kinase. In addition, the in vitro activity of Ca(2+)/calmodulin-dependent protein kinase was susceptible to dose-dependent inhibition by aflatoxin. Since calcineurin remains active in the absence of phosphorylation by calmodulin-dependent protein kinase, it is suggested that calcineurin-mediated dephosphorylation of regulatory enzymes ensures continued production of aflatoxins.     

Polypurine.polypyrimidine sequences in complete bacterial genomes: preference for polypurines in protein-coding regions

The genomes of Methanococcus jannaschii, Mycoplasma genitalium, Haemophilus influenzae, Archaeoglobus fulgidus, Helicobacter pylori, Treponema pallidum, Borrelia burgdorferri, Rickettsia prowazekeii, Mycobacterium tuberculosis, Methanobacterium thermoautotrophicum, Synechocystis sp. PCC6803, Bacillus subtilis, Chlamydia trachomatis, Pyrococcus horikoshii, Aquifex aeolicus, Mycoplasma pneumoniae and Escherichia coli have been analysed for the presence of polypurine.polypyrimidine tracts, in order to understand their distribution in these genomes. We observed a variation in abundance of such sequences in these bacteria, with the archaeal genomes forming a high-abundance group and the canonical eubacteria forming a low-abundance group. The genomes of M. tuberculosis and A. aeolicus are unique among the organisms analysed here in the abnormal underrepresentation and overrepresentation of polypurine.polypyrimidine, respectively. We also observe a strand bias, i.e., a preferential occurrence of polypurines in coding strands. It varies widely among the bacteria, from the very high bias in M. jannaschii to the slightly inverse bias in the parasitic genomes of T. pallidum and C. trachomatis. The extent of strand bias, however, cannot be explained on the basis of the GC-content of the genome, use of all-purine codons or an excess in the amino acids that are encoded by such codons. The probable causes and effects of this phenomenon are discussed.     

Sympathovagal Imbalance Contributes to Prehypertension Status and Cardiovascular Risks Attributed by Insulin Resistance,Inflammation, Dyslipidemia and Oxidative Stress in First Degree Relatives of Type 2 Diabetics

Background

Though cardiovascular (CV) risks are reported in first-degree relatives (FDR) of type 2 diabetics, the pathophysiological mechanisms contributing to these risks are not known. We investigated the association of sympathovagal imbalance (SVI) with CV risks in these subjects.

Subjects and Methods

Body mass index (BMI), basal heart rate (BHR), blood pressure (BP), rate-pressure product (RPP), spectral indices of heart rate variability (HRV), autonomic function tests, insulin resistance (HOMA-IR), lipid profile, inflammatory markers, oxidative stress (OS) marker, rennin, thyroid profile and serum electrolytes were measured and analyzed in subjects of study group (FDR of type 2 diabetics, n = 72) and control group (subjects with no family history of diabetes, n = 104).

Results

BMI, BP, BHR, HOMA-IR, lipid profile, inflammatory and OS markers, renin, LF-HF (ratio of low-frequency to high-frequency power of HRV, a sensitive marker of SVI) were significantly increased (p<0.0001) in study group compared to the control group. SVI in study group was due to concomitant sympathetic activation and vagal inhibition. There was significant correlation and independent contribution of markers of insulin resistance, dyslipidemia, inflammation and OS to LF-HF ratio. Multiple-regression analysis demonstrated an independent contribution of LF-HF ratio to prehypertension status (standardized beta 0.415, p<0.001) and bivariate logistic-regression showed significant prediction (OR 2.40, CI 1.128–5.326, p = 0.002) of LF-HF ratio of HRV to increased RPP, the marker of CV risk, in study group.

Conclusion

SVI in FDR of type 2 diabetics occurs due to sympathetic activation and vagal withdrawal. The SVI contributes to prehypertension status and CV risks caused by insulin resistance, dyslipidemia, inflammation and oxidative stress in FDR of type 2 diabetics.     

Changes in Morphology,Gene Expression and Protein Content in Chondrocytes Cultured on a Random Positioning Machine

Tissue engineering of chondrocytes on a Random Positioning Machine (RPM) is a new strategy for cartilage regeneration. Using a three-dimensional RPM, a device designed to simulate microgravity on Earth, we investigated the early effects of RPM exposure on human chondrocytes of six different donors after 30 min, 2 h, 4 h, 16 h, and 24 h and compared the results with the corresponding static controls cultured under normal gravity conditions. As little as 30 min of RPM exposure resulted in increased expression of several genes responsible for cell motility, structure and integrity (beta-actin); control of cell growth, cell proliferation, cell differentiation and apoptosis (TGF-β₁, osteopontin); and cytoskeletal components such as microtubules (beta-tubulin) and intermediate filaments (vimentin). After 4 hours of RPM exposure disruptions in the vimentin network were detected. These changes were less dramatic after 16 hours on the RPM, when human chondrocytes appeared to reorganize their cytoskeleton. However, the gene expression and protein content of TGF-β₁ was enhanced during RPM culture for 24 h. Taking these results together, we suggest that chondrocytes exposed to the RPM seem to change their extracellular matrix production behaviour while they rearrange their cytoskeletal proteins prior to forming three-dimensional aggregates.     

Discovery of P1736, a Novel Antidiabetic Compound That Improves Peripheral Insulin Sensitivity in Mice Models

Insulin resistance is a characteristic feature of Type 2 diabetes. Insulin resistance has also been implicated in the pathogenesis of cardiovascular disease. Currently used thiazolidinedione (TZD) insulin sensitizers although effective, have adverse side effects of weight gain, fluid retention and heart failure. Using fat cell-based phenotypic drug discovery approach we identified P1736, a novel antidiabetic molecule that has completed Phase II clinical trials. The present study evaluated the in vitro and in vivo pharmacological properties of P1736. P1736 is a non-TZD and it did not activate human PPAR(Peroxisome Proliferator Activated Receptor Gamma )receptors. P1736 caused dose dependent increase in glucose uptake (EC₅₀-400nM) in the insulin resistant 3T3 adipocytes. The compound (10µM) induced translocation of GLUT-4 (Glucose Transporter type 4) transporters in these adipocytes while metformin (1.0mM) was inactive. In diabetic db/db mice, P1736 (150mg/kg) was more efficacious than metformin in lowering plasma glucose (35% vs 25%) and triglyceride levels (38% vs 31%). P1736 tested at 5mg/kg, twice daily doses, reduced glucose by 41% and triglycerides by 32%, in db/db mice. These effects were not associated with adverse effects on body weight or liver function. Rosiglitazone (5mg/kg, twice daily) caused 60% and 40 % decreases in glucose and triglyceride levels, respectively. However, rosiglitazone induced 13% weight gain (p<0.05) in db/db mice. P1736 was also efficacious in ob/ob mice wherein 30-35% decrease in glucose and significant improvement in hyperinsulinemia were observed. Administration of P1736 to ob/ob mice resulted in 70% increase in glucose uptake in soleus muscles while metformin caused 38% increase. P1736 exhibited excellent safety profile and was weight neutral in all preclinical models of diabetes. Thus, P1736 with its unique pharmacology coupled with PPAR- independent mode of action could represent an alternative option in the management of insulin resistant Type 2 diabetic patients.