Control of Na+-K+-ATPase beta 1-subunit expression: role of 3'-untranslated region

Using in vitro translation and cell transfection assays, we previously demonstrated that the Na⁺-K⁺-ATPase ₁ mRNA species containing its longest 3'-untranslated region (UTR) exhibited the lowest translational efficiency. Here, employing deletions and in vivo expression assays, using direct injection of plasmids into rat ventricular myocardium, we identified a 143-nt segment located in the distal 3'-UTR of ₁ mRNA that was associated with decreased luciferase expression; interestingly, this segment contains three AUUUA motifs. Using RNA-protein binding assays and UV cross-linking of cRNA with cytosolic proteins of rat heart, we identified an 38-kDa protein that specifically bound to the cRNA encoding the 143-nt segment of ₁ mRNA 3'-UTR. Mutation of three nucleotides located in the middle region of the 143-nt segment, which was predicted to greatly disrupt a putative stem-loop structure of the cRNA in this region, was associated with reduced binding of the mutated cRNA to the protein migrating at 38 kDa. The cRNA encoding a segment of cyclooxygenase-2 mRNA 3'-UTR containing six AUUUA sequences did not bind the protein migrating at 38 kDa and did not compete with the binding of the wild-type 143-nt ₁ cRNA to the protein. The above results suggest that the 143-nt segment in the distal segment of the 3'-UTR of ₁ mRNA may play an important role in the control of ₁-subunit expression. RNA-protein binding; AUUUA sequence; plasmid expression in heart; direct myocardial injection; cardiac expression     

Thyroid hormone regulation of cardiac bioenergetics: role of intracellular creatine

The effect of thyroid hormone (T(3)) on the content of myocardial creatine (Cr), Cr phosphate (CrP), and high-energy adenine nucleotides and on cardiac function was examined. In the hearts of control and T(3)-treated rats perfused in vitro, while "low" and "high" contractile work was performed, T(3) treatment resulted in a approximately 50% reduction in CrP, Cr, total Cr content (Cr + CrP), and in the CrP-to-Cr ratio. In addition, there was a slight fall in myocardial content of ATP and a large rise in calculated free ADP (fADP), resulting in a significant decrease in the ATP-to-fADP ratio in the hearts of hyperthyroid compared with euthyroid rats. Moreover, there was a substantial decrease in the level of ATP in hearts of T(3)-treated rats under high work conditions. Importantly, the ratio of cardiac work to oxygen consumption was not altered by thyroid status. Treatment with T(3) also resulted in an almost threefold reduction in the content of Na(+)/Cr transporter mRNA in the ventricular myocardium and skeletal muscle but not in the brain. We conclude with the following: 1) changes in the expression of the Na(+)/Cr transporter mRNA correlate with Cr + CrP in the myocardium; 2) hearts of hyperthyroid rats contain lower levels of ATP and higher levels of fADP under both low and high work conditions but no reduction in efficiency of work output; and 3) the reduction in Cr and ATP in hearts of hyperthyroid rats may be the basis for the reduced maximal work capacity of the hyperthyroid heart.     

Detection of Low-Level Mixed-Population Drug Resistance in Mycobacterium tuberculosis Using High Fidelity Amplicon Sequencing

Undetected and untreated, low-levels of drug resistant (DR) subpopulations in clinical Mycobacterium tuberculosis (Mtb) infections may lead to development of DR-tuberculosis, potentially resulting in treatment failure. Current phenotypic DR susceptibility testing has a theoretical potential for 1% sensitivity, is not quantitative, and requires several weeks to complete. The use of “single molecule-overlapping reads” (SMOR) analysis with next generation DNA sequencing for determination of ultra-rare target alleles in complex mixtures provides increased sensitivity over standard DNA sequencing. Ligation free amplicon sequencing with SMOR analysis enables the detection of resistant allele subpopulations at ≥0.1% of the total Mtb population in near real-time analysis. We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay’s performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples. SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations. This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.     

Metabolic programming: fetal origins of obesity and metabolic syndrome in the adult

Exposure of the fetus to the intrauterine milieu can have profound effects on the health of the offspring in adulthood. Results of a series of studies demonstrate the powerful influence of the mother's metabolic state on whether the emerging adult develops obesity and hyperinsulinemia. Importantly, these attributes can be passed on to the next generation nongenetically and can be reversed and prevented.     

Heritability of tolerance to the Cry1Ab toxin of Bacillus thuringiensis in Chilo suppressalis (Lepidoptera: Crambidae)

Heritability of Chilo suppressalis (Walker) tolerance to the Cry1Ab toxin of Bacillus thuringiensis Berliner was estimated using a half-sibling design. Artificial diet with and without Cry1Ab was infested with progenies of 20 males, each mated with 2 females, and mortality was scored 5 d after infestation. The progeny of each female was reared and scored separately. Mean mortality of the 20 families on the Cry1Ab diet was 46.5%. The effects of both male parent and of female parent within male parent were significant. Heritability was estimated to be 0.52, suggesting that a high proportion of phenotypic variation was because of genetic differences. Mortality on the Cry1Ab diet was not correlated with mortality on control diet, indicating that differences among families in tolerance to Cry1Ab were not attributable to differences in general fitness. Our results indicate that "high dose" Bt rice plants may be particularly important for Cry1Ab resistance management in C. suppressalis populations.     

Induction of GLUT1 mRNA in response to azide and inhibition of protein synthesis

The biochemical behaviour of four commercial strains of Saccharomyces cerevisiae was studied in the presence of calcium ions, acrylamide and bisacrylamide. Calcium ions at a concentration of 300 µM induced an increase of NAD(P)⁺ reduction in commercial Turkish and American strains, while in Chilean and Brazilian commercial strains, it diminished NAD(P)⁺ reduction. On the other hand, polyacrylamide monomers (acrylamide and bisacrylamide) induced a decrease of NAD(P)⁺ reduction in all strains studied in this paper. When membrane potential () and oxygen consumption were measured in the presence of polyacrylamide monomers, a decrease of both was observed in all strains studied.     

Simultaneous Application of Rhizobium Strain and Melatonin Improves the Photosynthetic Capacity and Induces Antioxidant Defense System in Common Bean (Phaseolus vulgaris L.) Under Salinity Stress

Melatonin (N-acetyl-5-methoxytryptamine) as a natural biostimulating substance provides a number of benefits in stimulating plant growth in stress situations due to its natural antioxidant capacity. Rhizobia also play crucial roles in supporting plant growth under environmental stress conditions. The overall goal of this research is to study the possible positive effects of melatonin and rhizobacterium in enhancing the growth and salinity tolerance of common bean. To accomplish this objective, we conducted in vitro experiment to select the optimal melatonin concentration and treatment time of seed priming for the best germination. Also, a greenhouse experiment was performed to investigate the effect of melatonin pre-treatment applied before rhizobial inoculation to improve the fitness of common bean under salinity stress. The experiment was conducted using a completely randomized factorial design with six replications and three treatments: priming treatments (melatonin priming (PM100), hydro priming (PH) and dry (PD)), salinity (0, 4, 8, 10 and 16 dS m^?1) and Rhizobium strain (inoculated (RS?+) and uninoculated (RS?)). Our results showed that melatonin priming promoted bacterial colony size in Petri-dishes. The interactive effects of melatonin and RS?+?was found to alleviate reactive oxygen species (ROS) burst, and hence protect common bean chlorophylls a, b and carotenoid and photosynthetic activity and decrease malondialdehyde content through activation of antioxidant enzymes (superoxide dismutase, peroxidase, catalase and ascorbate peroxidase), facilitation of soluble protein synthesis, maintenance of Na⁺ and K⁺ homeostasis, and finally increase shoot dry weight (33.2, 39.5 and 31.5%) and seed yield (78.6, 91 and 54.2%) compared to the combination of PD and RS- treatments under 0, 4 and 8 dS m^?1 salinity levels, respectively. Thus, our findings suggest that seed priming with melatonin, especially 100 µM melatonin is an effective strategy that can be used to enhance salt tolerance in common bean.

     

The effect of 8 weeks of high-intensity interval training and moderate-intensity continuous training on cardiac angiogenesis factor in diabetic male rats

Journal of Physiology and Biochemistry - The balance of pro-angiogenic and anti-angiogenic factors has a significant role in the development of diabetic cardiomyopathy. The purpose of this study...     

Performance Comparison of Three Rapid Tests for the Diagnosis of Drug-Resistant Tuberculosis

Background

The aim of this study was to compare the performance of several recently developed assays for the detection of multi- and extensively drug-resistant tuberculosis (M/XDR-TB) in a large, multinational field trial.

Methods

Samples from 1,128 M/XDR-TB suspects were examined by Line Probe Assay (LPA), Pyrosequencing (PSQ), and Microscopic Observation of Drug Susceptibility (MODS) and compared to the BACTEC MGIT960 reference standard to detect M/XDR-TB directly from patient sputum samples collected at TB clinics in India, Moldova, and South Africa.

Results

Specificity for all three assays was excellent: 97–100% for isoniazid (INH), rifampin (RIF), moxifloxacin (MOX) and ofloxacin (OFX) and 99–100% for amikacin (AMK), capreomycin (CAP) and kanamycin (KAN) resistance. Sensitivities were lower, but still very good: 94–100% for INH, RIF, MOX and OFX, and 84–90% for AMK and CAP, but only 48–62% for KAN. In terms of agreement, statistically significant differences were only found for detection of RIF (MODS outperformed PSQ) and KAN (MODS outperformed LPA and PSQ) resistance. Mean time-to-result was 1.1 days for LPA and PSQ, 14.3 days for MODS, and 24.7 days for MGIT.

Conclusions

All three rapid assays evaluated provide clinicians with timely detection of resistance to the drugs tested; with molecular results available one day following laboratory receipt of samples. In particular, the very high specificity seen for detection of drug resistance means that clinicians can use the results of these rapid tests to avoid the use of toxic drugs to which the infecting organism is resistant and develop treatment regiments that have a higher likelihood of yielding a successful outcome.     

Intrinsic Up-Regulation of 2-AG Favors an Area Specific Neuronal Survival in Different In Vitro Models of Neuronal Damage

Background

The endocannabinoid 2-arachidonoyl glycerol (2-AG) acts as a retrograde messenger and modulates synaptic signaling e. g. in the hippocampus. 2-AG also exerts neuroprotective effects under pathological situations. To better understand the mechanism beyond physiological signaling we used Organotypic Entorhino-Hippocampal Slice Cultures (OHSC) and investigated the temporal regulation of 2-AG in different cell subsets during excitotoxic lesion and dendritic lesion of long range projections in the enthorhinal cortex (EC), dentate gyrus (DG) and the cornu ammonis region 1 (CA1).

Results

2-AG levels were elevated 24 h after excitotoxic lesion in CA1 and DG (but not EC) and 24 h after perforant pathway transection (PPT) in the DG only. After PPT diacylglycerol lipase alpha (DAGL) protein, the synthesizing enzyme of 2-AG was decreased when Dagl mRNA expression and 2-AG levels were enhanced. In contrast to DAGL, the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MAGL) showed no alterations in total protein and mRNA expression after PPT in OHSC. MAGL immunoreaction underwent a redistribution after PPT and excitotoxic lesion since MAGL IR disappeared in astrocytes of lesioned OHSC. DAGL and MAGL immunoreactions were not detectable in microglia at all investigated time points. Thus, induction of the neuroprotective endocannabinoid 2-AG might be generally accomplished by down-regulation of MAGL in astrocytes after neuronal lesions.

Conclusion

Increase in 2-AG levels during secondary neuronal damage reflects a general neuroprotective mechanism since it occurred independently in both different lesion models. This intrinsic up-regulation of 2-AG is synergistically controlled by DAGL and MAGL in neurons and astrocytes and thus represents a protective system for neurons that is involved in dendritic reorganisation.