Characterization of ribonuclease P isolated from rat liver cytosol.

Rat liver ribonuclease P was isolated from a cytosolic fraction and shown to have optimal activity in the presence of 1 mM MgCl2 and 150-200 mM KCl using Escherchia coli pre-tRNA(Tyr) as substrate. In cesium sulfate isopycnic density gradients, the enzyme had a buoyant density of 1.36 g/ml, indicating that it is a ribonucleoprotein complex. Analysis of the RNAs in the enzyme sample purified through two successive Cs2SO4 density gradient steps revealed the copurification of two major species of RNA (RRP1 and RRP2) along with several less abundant RNAs. Rat liver ribonuclease P activity was insensitive to micrococcal nuclease pretreatment. However, the nuclease-treated preparations contained several incompletely degraded RNA species that may have been sufficient to support the ribonuclease P activity. When RNase A was substituted for micrococcal nuclease, the ribonuclease P activity was diminished by greater than 90%, suggesting the requirement for an RNA subunit for activity.     

Nuclear magnetic resonance studies of 5-aminolevulinate demonstrate multiple forms in aqueous solution

5-Aminolevulinic acid (ALA), the common precursor of heme and chlorophyll, can exist in a variety of forms at neutral pH. ¹³C NMR studies of [3-¹³C]ALA, [4-¹³C]ALA, and [5-¹³C]ALA have been used to demonstrate that the predominant species in solution under physiologic conditions is the ketone. The mole fraction of the hydrate is about 0.6%. To further substantiate the existence of the hydrate, ¹³C NMR was used to monitor ¹⁸O exchange at C₄ of [4-¹³C]ALA with H₂, ¹⁸O. Confirmation of the existence of the hydrate was achieved through direct observation by ¹H NMR. The mole fractions of the enol forms of ALA are each below 0.3%. Although direct observation of the enol forms of ALA has not been achieved, enol formation has been indirectly demonstrated by monitoring hydrogen exchange at the C₃ and C₅ methylene groups by ¹H NMR in D₂O. In neutral phosphate buffer, hydrogen exchange occurs readily at both C₃ and C₅ at a ratio of rates of 1:4. In N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid-KOH buffer the hydrogen exchange rates are more than an order of magnitude slower than in phosphate buffer, but the ratio of the exchange rates remains unchanged. The results suggest that phosphate catalyzes enolization at both C₃ and C₅. To evaluate the role of the C₅ substituent in the proton exchange reactions, levulinate and 5-chlorolevulinate (5-CLA) were also monitored for proton exchange at C₃ and C₅. For levulinate, the hydrogen exchange rates in phosphate buffer are two to three orders of magnitude slower than for ALA, and the rate of hydrogen exchange at C₅ is three times slower than hydrogen exchange at C₃. The enolization rate at C₅ of 5-CLA is identical to ALA while enolization at C₃ is about threefold slower for 5-CLA than ALA. These NMR and kinetic studies suggest that under physiologic conditions, ALA rapidly equilibrates between the ketone, the hydrate at C₄, and two or more different enols (C₃---C₄ and C₄---C₅). The alternative forms of ALA may be biologically significant as active site structures for ALA synthase, glutamate semialdehyde transaminase, or porphobilinogen synthase. These NMR studies have also elucidated the structures of condensation products of ALA which can be formed under physiologic conditions. The alternative forms of ALA, as well as the autocondensation products, may serve as the active toxin in porphyrias characterized by elevated ALA levels (e.g., lead poisoning).     

Pathways of carbohydrate metabolism in mycobacterium tuberculosis H37Rv1.

Radiorespirometric studies using glucose labelled at 1, 2, 3-4, and 6 positions and enzymatic studies were conducted to determine the primary pathways of glucose dissimilation in Mycobacterium tuberculosis H37Rv. The pattern of 14CO2 recovery was C3-4 greater than C1 greater than C6 = C2. The Embden-Meyerhof pathway was found to be the predominant pathway for glucose oxidation, operative to the extent of 94%. The pentose phosphate pathway accounted for the remaining 6%. Maximum incorporation of 14C into cellular components was from C2 and C6 labelled glucose.     

Energetic differences between the specific binding of a 40 bp DNA duplex and the lac promoter to lac repressor protein

The energetics of LRP binding to a 104 bp lac promoter determined from ITC measurements were compared to the energetics of binding to a shorter 40 bp DNA duplex with the 21 bp promoter binding site sequence. The promoter binding affinity of 2.47 +/- 0.0 1x 10(7) M(-1) was higher than the DNA binding affinity of 1.81 +/- 0.67 x 10(7) M(-1) while the binding enthalpy of -804 +/- 41 kJ mol(-1) was lower than that of the DNA binding enthalpy of -145 +/- 16 kJ mol(-1) at 298.15 K. Both the promoter and DNA binding reactions were exothermic in phosphate buffer but endothermic in Tris buffer that showed the transfer of four protons to LRP in the former reaction but only two in the latter. A more complicated dependence of these parameters on temperature was observed for promoter binding. These energetic differences are attributable to additional LRP-promoter interactions from wrapping of the promoter around the LRP.     

RNA splicing promotes translation and RNA surveillance

Aberrant mRNAs harboring premature termination codons (PTCs or nonsense codons) are degraded by the nonsense-mediated mRNA decay (NMD) pathway. mRNAs transcribed from genes that naturally acquire PTCs during lymphocyte development are strongly downregulated by PTCs. Here we show that a signal essential for this robust mRNA downregulatory response is efficient RNA splicing. Strong mRNA downregulation can be conferred on a poor NMD substrate by either strengthening its splicing signals or removing its weak introns. Efficient splicing also strongly promotes translation, providing a molecular explanation for enhanced NMD and suggesting that efficient splicing may have evolved to enhance both protein production and RNA surveillance. Our results suggest simple approaches for increasing protein expression from expression vectors and treating human genetic diseases caused by nonsense and frameshift mutations.     

Aluminum concentrations in tissues of rats: effect of soft drink packaging

Aluminum is a commonly occurring trace element for which no nutritional requirements have been set. Some non-conclusive evidence exists suggesting a need of aluminum for growth, reproduction or health of man and animals. There is concern that exposure or consumption of aluminum may be toxic to humans and animals. The objective of the current study was to compare tissue levels of aluminum of rats fed soft drinks packaged in aluminum cans, glass bottles or distilled water. Thirty male weanling rats (Sprague-Dawley) were divided into three treatment groups of 10 rats each. All rats were fed rodent chow ad libitum throughout the study. Three different fluids, i.e. distilled water, diet soft drinks from aluminum cans and diet soft drinks from glass bottles, were fed for a period of 3 weeks. Aluminum contents of tissues were measured by atomic absorption spectrophotometry. Canned soft drink fed rats had significantly higher blood, liver and bone aluminum concentration than rats that were given glass bottled soft drink. There was a 69% higher bone aluminum concentration and 16% lower femur weight in rats fed aluminum canned soft drinks when compared with rats fed with distilled water.     

Pharmacophore feature prediction and molecular docking approach to identify novel anti‐HCV protease inhibitors

Discovering a potential drug for HCV treatment is a challenging task in the field of drug research. This study initiates with computational screening and modeling of promising ligand molecules. The foremost modeling method involves the identification of novel compound and its molecular interaction based on pharmacophore features. A total of 197 HCV compounds for NS3/4A protein target were screened for our study. The pharmacophore models were generated using PHASE module implemented in Schrodinger suite. The pharmacophore features include one hydrogen bond acceptor, one hydrogen bond donor, and three hydrophobic sites. As a result, based on mentioned hypothesis the model ADHHH.159 corresponds to the CID 59533233. Furthermore, docking was performed using maestro for all the 197 compounds. Among these, the CID 59533313 and 59533233 possess the best binding energy of ?11.75 and ?10.40 kcal/mol, respectively. The interactions studies indicated that the CID complexed with the NS3/4A protein possess better binding affinity with the other compounds. Further the compounds were subjected to calculate the ADME properties. Therefore, it can be concluded that these two compounds could be a potential alternative drug for the development of HCV.     

Structure and interactions of RecA: plasticity revealed by molecular dynamics simulations

Eleven independent simulations, each involving three consecutive molecules in the RecA filament, carried out on the protein from Mycobacterium tuberculosis, Mycobacterium smegmatis and Escherichia coli and their Adenosine triphosphate (ATP) complexes, provide valuable information which is complementary to that obtained from crystal structures, in addition to confirming the robust common structural framework within which RecA molecules from different eubacteria function. Functionally important loops, which are largely disordered in crystal structures, appear to adopt in each simulation subsets of conformations from larger ensembles. The simulations indicate the possibility of additional interactions involving the P-loop which remains largely invariant. The phosphate tail of the ATP is firmly anchored on the loop while the nucleoside moiety exhibits substantial structural variability. The most important consequence of ATP binding is the movement of the ‘switch’ residue. The relevant simulations indicate the feasibility of a second nucleotide binding site, but the pathway between adjacent molecules in the filament involving the two nucleotide binding sites appears to be possible only in the mycobacterial proteins.