An in vitro demonstration of peroxisome proliferation and increase in peroxisomal β-oxidation system mRNAs in cultured rat hepatocytes treated with ciprofibrate

Using the normal adult rat hepatocytes, plated on rat tail collagen-coated dishes and fed a chemically defined medium, we demonstrate here that ciprofibrate at 0.1 mM concentration, increases significantly the mRNA levels of fatty acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and thiolase (the three enzymes of the β-oxidation system), and causes peroxisome proliferation. Increase in mRNA levels of these genes was evident within 1 h and was maximal 24 h after the addition of ciprofibrate. In hepatocytes cultured in the absence of ciprofibrate, the basal levels of these enzymes were low and further declined with time. Concomitant treatment of hepatocytes with cycloheximide did not inhibit or superinduce the mRNA levels, indicating that this induction may represent a primary (direct) effect of this compound on the expression of these genes and does not apparently involve short-lived repressor protein(s).     

Exploiting the chemical synthesis of RNA.

A number of nucleotide phosphoramidites are now available that permit the chemical synthesis of RNA, modified RNA and RNA-DNA chimeric oligonucleotides. Since the chemical strategy allows the introduction of a particular modification at any given site in a nucleotide polymer, very subtle and specific questions regarding structure-function relationships in RNA may be addressed.     

Prevention of guanine modification and chain cleavage during the solid phase synthesis of oligonucleotides using phosphoramidite derivatives.

Phosphoramidite reagents can phosphitylate guanine bases at the O6-position during solid phase synthesis and serious chain cleavage occurs if the base phosphitylation is not eliminated before the iodine/water oxidation step. This can be accomplished by blocking the O6-position with a 2-cyanoethyl protecting group for deoxyribonucleotides or with a p-nitrophenylethyl group for ribonucleotides, regenerating the guanine base with water or acetate ions, or using N-methylanilinium trifluoroacetate (TAMA) as the phosphoramidite activator. The effectiveness of these methods was demonstrated by both 31P NMR studies and by the synthesis of d(Gp)23G, (Gp)14G, and d-(Gp)13rG sequences.     

Occurrence of hemoglobin in the nitrogen-fixing root nodules of Alnus glutinosa

A true hemoglobin (Hb) was shown to be present in the root nodules of Alnus glutinosa L. After purification by gel filtration and ion exchange, the Hb formed a stable complex with oxygen. This oxygen complex could then be converted to carboxyhemoglobin by treatment with CO. Optical absorption spectra typical of Hb were observed. The molecular weight was estimated to be 15 100 by gel filtration, and 18 300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Hb was largely insoluble when the initial homogenization was done in the absence of a detergent. Under these conditions much of the Hb appears to be associated with clusters of Frankia , the nitrogen-fixing actinomycete that infects plant cells within the nodules. The exact localization of the Hb in vivo is uncertain. The relatively low average concentration of Hb in Alnus nodules suggests that it is either confined to a relatively small fraction of total nodule volume, or has a function other than facilitation of O₂ transport.     

Relationship between 2'-hydroxyls and magnesium binding in the hammerhead RNA domain: a model for ribozyme catalysis.

The use of deoxyribonucleotide substitution in RNA (mixed RNA/DNA polymers) permits an evaluation of the role of 2'-hydroxyl groups in ribozyme catalysis. Specific deoxyribonucleotide substitution at G9 and A13 of the ribozyme decreases the catalytic activity (kcat) of the ribozyme by factors of 14 and 20, respectively. The reduction of the reaction rate concomitant with the absence of these 2'-OHs or the 2'-OH of the substrate U7 position can be partially compensated by increasing the Mg2+ concentration above 10 mM. The KMg of the all-RNA ribozyme is 5.3 mM, and the lack of either of the three influential 2'-OHs increases this value by a factor of approximately 3. These and other reaction constants for the ribozyme and the deoxy-substituted analogues have been determined by assuming a three-step mechanism. The data presented here provide the basis for the formulation of a molecular model of ribozyme activity.     

Synthesis of 6-Aza- & 6-Methyl-pyrimidine Ribonucleoside Phosphoramidites and Their Incorporation in Hammerhead Ribozymes

Abstract

The synthesis of phosphoramidites of 6-modified pyrimidine ribonucleosides and their incorporation into hammerhead ribozymes and influence on nuclease stability and catalytic activity is described.     

Foliar Application of Phosphorus Enhances Photosynthesis and Biochemical Characteristics of Maize under Drought Stress

Water is essential for the growth period of crops; however, water unavailability badly affects the growth and physiological attributes of crops, which considerably reduced the yield and yield components in crops. Therefore, a pot experiment was conducted to investigate the effect of foliar phosphorus (P) on morphological, gas exchange, biochemical traits, and phosphorus use efficiency (PUE) of maize (Zea mays L.) hybrids grown under normal as well as water deficit situations at the Department of Agronomy, University of Agriculture Faisalabad, Pakistan in 2014. Two different treatments (control and P @ 8 kg ha⁻¹ ) and four hybrids (Hycorn, 31P41, 65625, and 32B33) of maize were tested by using a randomized complete block design (RCBD) with three replications. Results showed that the water stress caused a remarkable decline in total soluble protein (9.7%), photosynthetic rate (9.4%) and transpiration rate (13.4%), stomatal conductance (10.2%), and internal CO₂ rate (20.4%) comparative to well-watered control. An increase of 37.1%, 36.8%, and 24.5% were recorded for proline, total soluble sugar, and total free amino acid, respectively. However, foliar P application minimized the negative impact of drought by improving plant growth, physio-biochemical attributes, and PUE in maize plants under water stress conditions. Among the hybrids tested, the hybrid 6525 performed better both under stress and non-stress conditions. These outcomes confirmed that the exogenous application of P improved drought stress tolerance by modulating growth, physio-biochemical attributes, and PUE of maize hybrids.     

In-Silico Analyses of Nonsynonymous Variants in the BRCA1 Gene

BReast CAncer gene 1 (BRCA1)—a tumor suppressor gene plays an important role in the DNA repair mechanism. Several BRCA1 variants perturb its structure and function, including synonymous and nonsynonymous single nucleotide polymorphisms (SNPs). In the present study, we performed in-silico analyses of nonsynonymous SNPs (nsSNPs) of the BRCA1 gene. In total, 122 nsSNPs were retrieved from the NCBI SNP database and in-silico analyses were performed using computational prediction tools: SIFT, PROVEAN, Mutation Taster, PolyPhen-2, MutPred, and ConSurf. Of these tools, SIFT, PROVEAN, and Mutation Taster predicted 61 out of 122 nsSNPs as “damaging”, based on structural homology analysis. PolyPhen-2 classified 22 nsSNPs as “probably damaging”. These nsSNPs were further analyzed by MutPred to predict basic molecular mechanisms of amino acid alteration. ConSurf analysis predicted eleven conserved amino acid residues with structural and functional consequences. We identified five amino acid residues in the RING finger domain (L22, C39, H41, C44, and C47) and two in the BRCT domain (P1771 and I1707) with the potential to deter the BRCA1 protein function. This study provides insights into the effect of nsSNPs and amino acid substitutions in BRCA1.

     

Perspectives of pluripotent stem cells in livestock

The recent progress in derivation of pluripotent stem cells(PSCs)from farm animals opens new approaches not only for reproduction,genetic engineering,treatment and conservation of these species,but also for screening novel drugs for their efficacy and toxicity,and modelling of human diseases.Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages,or lost their cellular potency;indicating that the protocols which allowed the derivation of murine or human embryonic stem(ES)cells were not sufficient to support the maintenance of ES cells from farm animals.This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support na?ve pluripotency in ES cells from livestock species.However,the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging,and requires further refinements.Here,we review the current achievements in the derivation of PSCs from farm animals,and discuss the potential application areas.