Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3′ acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.     

Microbial utilization of electrically reduced neutral red as the sole electron donor for growth and metabolite production.

Electrically reduced neutral red (NR) served as the sole source of reducing power for growth and metabolism of pure and mixed cultures of H2-consuming bacteria in a novel electrochemical bioreactor system. NR was continuously reduced by the cathodic potential (-1.5 V) generated from an electric current (0.3 to 1.0 mA), and it was subsequently oxidized by Actinobacillus succinogenes or by mixed methanogenic cultures. The A. succinogenes mutant strain FZ-6 did not grow on fumarate alone unless electrically reduced NR or hydrogen was present as the electron donor for succinate production. The mutant strain, unlike the wild type, lacked pyruvate formate lyase and formate dehydrogenase. Electrically reduced NR also replaced hydrogen as the sole electron donor source for growth and production of methane from CO2. These results show that both pure and mixed cultures can function as electrochemical devices when electrically generated reducing power can be used to drive metabolism. The potential utility of utilizing electrical reducing power in enhancing industrial fermentations or biotransformation processes is discussed.     

An oxidative and salinity stress induced peroxisomal ascorbate peroxidase from Avicennia marina: Molecular and functional characterization

APX (EC, 1.11.1.11) has a key role in scavenging ROS and in protecting cells against their toxic effects in algae and higher plants. A cDNA encoding a peroxisomal ascorbate peroxidase, Am-pAPX1, was isolated from salt stressed leaves of Avicennia marina (Forsk.) Vierh. by EST library screening and its expression in the context of various environmental stresses was investigated. Am-pAPX1 contains an ORF of 286 amino acids coding for a 31.4kDa protein. The C-terminal region of the Am-pAPX1 ORF has a putative transmembrane domain and a peroxisomal targeting signal (RKKMK), suggesting peroxisomal localization. The peroxisomal localization of Am-pAPX1 was confirmed by stable transformation of the GFP-(Ala)(10)-Am-pAPX1 fusion in tobacco. RNA blot analysis revealed that Am-pAPX1 is expressed in response to salinity (NaCl) and oxidative stress (high intensity light, hydrogen peroxide application and excess iron). The isolated genomic clone of Am-pAPX1 was found to contain nine exons. A fragment of 1616bp corresponding to the 5' upstream region of Am-pAPX1 was isolated by TAIL-PCR. In silico analysis of this sequence reveals the presence of putative light and abiotic stress regulatory elements.     

Relationship Between Molecular Structure and Thermo-mechanical Properties of Candelilla Wax and Amides Derived from (<Emphasis Type="Italic">R</Emphasis>)-12-Hydroxystearic Acid as Gelators of Safflower Oil

In this research, we studied the relationship between the molecular structure of (R)-12-hydroxyoctadecanamide, (R)-N-propyl-12-hydroxyoctadecanamide, and (R)-N-octadecyl-12-hydroxyoctadecanamide and the thermo-mechanical properties of their 2% (wt/wt) organogels developed using safflower oil high in oleic acid (HOSFO) as the liquid phase. Candelilla wax (CW), a well-known edible gelling additive whose main component is hentriacontane, also was studied for comparative purposes. The results obtained show that the attractive interactions (i.e., hydrogen bonding and dipolar interactions) between amide groups and between hydroxyl groups present in the amides resulted in organogels with higher melting temperature, heat of melting, and crystallization parameters than those found in the CW organogel. The rheological parameters associated to the strength of the amide or CW-based gels developed in HOSFO (i.e., yield stress and elastic modulus) seem to be associated with the nature of amide groups (i.e., primary or secondary amide) and the increase in the length of the self-assembly molecular unit (i.e., L value determined by X-ray diffraction) and therefore to the extent of London dispersion forces along the hydrocarbon chain. The creep and recovery measurements allowed an evaluation among the internal structures of the different organogels and demonstrated that independent of the hydrogen bonding and dipolar interaction provided by the amide and the hydroxyl groups, the increase in the hydrocarbon chain length results in higher organogel resistance to deformation and higher instant recovery capacity. However, the stabilization of the self-assembly unit through polar groups (i.e., –CONH₂ in HOA) reduces organogel elasticity but provides a higher extended recovery capacity. The results reported in this investigation showed some relationships between gelator structure and the thermo-mechanical properties of low-molecular-mass organic gelator amides. Our long-term objective is to understand the organogelation process to eventually develop trans-free vegetable oil-based food products with novel textures for the consumers.     

Influence of age,weaning, season and body weight on the levels of progesterone,oestradiol-17β and luteinizing hormone in growing buffalo heifers (Bubalus bubalis)

The influence of age, weaning, season of the year and body weight on the peripheral levels of progesterone, oestradiol-17β and luteinizing hormone (LH) were studied during neonatal, perinatal and peripubertal periods in buffalo heifers. The buffalo heifers exhibited oestrus only after 30 months of age and had higher levels of LH and oestradiol-17β and a lower level of progesterone on the day of oestrus. The progesterone concentration was affected significantly (P < 0.01) by different seasons, by weaning (P < 0.05) and varied between pubertal and neonatal periods (P < 0.01), whereas the oestradiol-17β level was affected significantly (P < 0.01) by weaning and varied at different seasons and with body weight. However, the LH concentration was greater during the neonatal period than the pre- and peripubertal periods and changed significantly (P < 0.01) between groups of ages and body weights. The results suggest that increases in the levels of oestradiol-17β and progesterone after 30 months of age are probably indicative of the onset of puberty in buffalo heifers. However, a further increase in oestradiol-17β, LH, and a decrease in progesterone are essential for oestrus and cyclicity to be exhibited in buffalo heifers.     

A small RNA that complements mutants in the RNA processing enzyme ribonuclease P

Four temperature-sensitive RNase P mutants were analyzed for the accumulation of 10 S RNA. In the 10 S region of the polyacrylamide gel two molecules appear, a and b. While the level of 10 Sa seems to be affected in some of the mutants, the 10 Sb molecule was not found in rnpB mutants. A plasmid (pL2), which contains Escherichia coli DNA sequences that complement, at least partially, rnp mutations, directs the synthesis of 10 Sb RNA. The presence of the pL2 plasmid complements the rnpA49, rnpB3187 and the rnpC241 mutations, as revealed by colony formation at “non-permissive” temperatures. However, the complementation of the rnpA49 mutation is much better than that of the other mutations. The complementation can also be measured by the increased level of RNase P activity in extracts. 10 Sa and b RNAs are unique among all RNAs tested thus far, since they are stable during exponential growth at 30 °C and 37 °C. However, at higher temperatures, such as 43 °C, the molecules are somewhat less stable, and they become rather labile when RNA synthesis is blocked by rifampicin. Structural analysis revealed that the 10 Sa and 10 Sb RNA molecules have dissimilar sequences.     

Complementarity between ferritin H mRNA and 28 S ribosomal RNA

We have found an interesting complementarity in sequences of human ferritin H mRNA and 28 S ribosomal RNA. Immediately upstream of the initiating AUG in the ferritin mRNA is a stretch of 67 nucleotides which contains sequences complementary to several regions in 28 S RNA. One such region can form 55 base pairings with the 5' noncoding region of the ferritin H mRNA. Most of the complementarity is due to repeats of CCG in the ferritin mRNA and GGC in the ribosomal RNA. The regions of complementarity in the 28 S RNA appear to be expansion sequences that have arisen in the evolution of eukaryotic ribosomal RNA. We suggest that interaction of ferritin mRNA and 28 S RNA may function to regulate the stability and/or translatability of ferritin mRNA.