Structural Basis of Drug Resistance by Genetic Variants of HIV Type 1 Clade C Protease from India

Using computer modeling of three-dimensional structures and structural information available on the crystal structures of HIV-1 protease, we investigated the structural effects of mutations, in treatment-naive and treatment-exposed individuals from India and postulated mechanisms of resistance in clade C variants. A large number of models (14) have been generated by computational mutation of the available crystal structures of drug bound proteases. Localized energy minimization was carried out in and around the sites of mutation in order to optimize the geometry of interactions present. Most of the mutations result in structural differences at the flap that favors the semiopen state of the enzyme. Some of the mutations were also found to confer resistance by affecting the geometry of the active site. The E35D mutation affects the flap structure in clade B strains and E35N and E35K mutation, seen in our modeled strains, have a more profound effect. Common polymorphisms at positions 36 and 63 in clade C also affected flap structure. Apart from a few other residues Gln-58, Asn-83, Asn-88, and Gln-92 and their interactions are important for the transition from the closed to the open state. Development of protease inhibitors by structure-based design requires investigation of mechanisms operative for clade C to improve the efficacy of therapy.

Inverse Correlation between Vitamin D and C-Reactive Protein in Newborns

Some studies suggested that adequate vitamin D might reduce inflammation in adults. However, little is known about this association in early life. We aimed to determine the relationship between cord blood 25-hydroxyvitamin D (25(OH)D) and C-reactive protein (CRP) in neonates. Cord blood levels of 25(OH)D and CRP were measured in 1491 neonates in Hefei, China. Potential confounders including maternal sociodemographic characteristics, perinatal health status, lifestyle, and birth outcomes were prospectively collected. The average values of cord blood 25(OH)D and CRP were 39.43 nmol/L (SD = 20.35) and 6.71 mg/L (SD = 3.07), respectively. Stratified by 25(OH)D levels, per 10 nmol/L increase in 25(OH)D, CRP decreased by 1.42 mg/L (95% CI: 0.90, 1.95) among neonates with 25(OH)D <25.0 nmol/L, and decreased by 0.49 mg/L (95% CI: 0.17, 0.80) among neonates with 25(OH)D between 25.0 nmol/L and 49.9 nmol/L, after adjusting for potential confounders. However, no significant association between 25(OH)D and CRP was observed among neonates with 25(OH)D ≥50 nmol/L. Cord blood 25(OH)D and CRP levels showed a significant seasonal trend with lower 25(OH)D and higher CRP during winter-spring than summer-autumn. Stratified by season, a significant linear association of 25(OH)D with CRP was observed in neonates born in winter-spring (adjusted β = −0.11, 95% CI: −0.13, −0.10), but not summer-autumn. Among neonates born in winter-spring, neonates with 25(OH)D <25 nmol/L had higher risk of CRP ≥10 mg/L (adjusted OR = 3.06, 95% CI: 2.00, 4.69), compared to neonates with 25(OH)D ≥25 nmol/L. Neonates with vitamin D deficiency had higher risk of exposure to elevated inflammation at birth.

Metabolism of 1,8-Cineole in Rat: Its Effects on Liver and Lung Microsomal Cytochrome P-450 Systems

The monoterpene cyclic ether, cineole (l,8-cineole, I) also known as eucalyptol, is a component of many essential oils and is widely distributed in nature. It is extensively used in pharmaceutical preparations for external application and also as a nasal spray. It was reported earlier that cineole when administered to sheep may be largely oxidized in the system (Scheline 1978). However the mode of metabolism of cineole is not known. Hence the present study was undertaken to investigate the metabolic fate of this ubiquitous terpenoid following its administration to rats by gastric intubation.

New microsatellite loci for Carcharhinid sharks (Carcharhinus tilstoni and C. sorrah) and their cross-amplification in other shark species

Twelve microsatellite DNA markers were isolated in the spot-tail shark (Carcharhinus sorrah) and nine were isolated in Australian black-tip shark (Carcharhinus tilstoni). These loci plus 18 others developed for sharks from the genera Negaprion, Ginglymostoma, Carcharodon and Isurus were tested for amplification success on four species of Carcharhinus (including C. sorrah and C. tilstoni) and four other species representing three diverse families. Cross-amplification was most common within families. Five loci were subsequently tested for polymorphism on 50 C. sorrah and 60 C. tilstoni. The number of alleles per locus was two to 24 and the average heterozygosity was 0.54 (range 0.16-0.87) for C. sorrah and 0.64 (range 0.44-0.78) for C. tilstoni. These loci may be useful tools for genetic analyses of the Carcharhinidae.

The introduction and release of Chiasmia inconspicua and C. assimilis (Lepidoptera: Geometridae) for the biological control of Acacia nilotica in Australia

Two geometrid moths Chiasmia inconspicua and Chiasmia assimilis, identified as potential biological control agents for prickly acacia Acacia nilotica subsp. indica, were collected in Kenya and imported into quarantine facilities in Australia where laboratory cultures were established. Aspects of the biologies of both insects were studied and CLIMEX® models indicating the climatically favourable areas of Australia were developed. Host range tests were conducted using an approved test list of 74 plant species and no-choice tests of neonate larvae placed on both cut foliage and potted plants. C. inconspicua developed through to adult on prickly acacia and, in small numbers, Acacia pulchella. C. assimilis developed through to adult on prickly acacia and also in very small numbers on A. pulchella, A. deanei, A. decurrens, and A. mearnsii. In all experiments, the response on prickly acacia could be clearly differentiated from the responses on the non-target species. Both insects were approved for release in Australia. Over a three-year period releases were made at multiple sites in north Queensland, almost all in inland areas. There was no evidence of either insect's establishment and both colonies were terminated. A new colony of C. assimilis was subsequently established from insects collected in South Africa and releases of C. assimilis from this new colony were made into coastal and inland infestations of prickly acacia. Establishment was rapid at one coastal site and the insect quickly spread to other infestations. Establishment at one inland area was also confirmed in early 2006. The establishment in coastal areas supported a CLIMEX model that indicated that the climate of coastal areas was more suitable than inland areas.

Expression of Cytochrome P-450 and Albumin Genes in Rat Liver: Effect of Xenobioticst

Thioacetamide, a hepatocarcinogen and an inhibitor of heme synthesis, blocks the phenobarbitone- mediated increase in the transcription of cytochrome P-450b+e messenger RNA in rat liver. This property is also shared by CoCl, and 3-amino-l,2,4-triazole, two other inhibitors of heme synthesis. Thus, it appears feasible that heme may serve as a positive regulator of cytochrome P-450b+e gene transcription. Thioacetamide enhances albumin messenger RNA concentration, whereas phenobarbitone decreases the same. However, these changes in albumin messenger RNA concentration are not accompanied by corresponding changes in the transcription rates. Therefore, drug-mediated changes in albumin messenger RNA concentration are due to posttranscriptional regulation. The property of thioacetamide to enhance the albumin messenger RNA concentration is not shared by CoC1, and 3-amino- 1,2,4-triazole. Therefore, heme does not appear to be a regulatory molecule mediating the reciprocal changes brought about in the concentrations of cytochrome P-450b+e and albumin messenger RNAs.

Generation of H2O2 in biomembranes

Knowledge of the generation of H202 in cellular oxidations has existed for many years. It has been assumed that H202 is tOxiC tO cells and the presence of catalase is indicative of a detoxication mechanism. Other radicals of oxygen were recently recognized to be more potent destructive agents of biological material than H202. Also catalase and other peroxidases utilize H202 in some cellular oxidation processes leading to several important metabolites. Thus, the generation of H202 in cellular processes seems to be purposeful and H202 can not be dismissed as a mere undesirable byproduct. Biological formation of H202 is not limited to the previously known flavoproteins and some copper enzymes, but other redox systems, particularly heme and non-heme iron proteins, are now found to undergo auto-oxidation yielding H202. The capacity for generation of H202 is now found to be widespread in a variety of organisms and in the organdies of the cells. The reduction of oxygen to H20 by mitochondrial cytochrome oxidase being the predominant oxygen-utilizing reaction had over-shadowed the importance of the quantitatively minor pathways. Under aerobic conditions generation of H202 by a Variety of biomembranes has now been found to be a physiological event interlinked with phenomena such as phagocytosis, transport processes and thermogenesis in some as yet unidentified way. The underlying mechanisms of these processes seem to involve generation and utilization of H202 in mitochondria, microsomes, peroxisomes or plasma membranes. This review gives an account of the potential of biomembranes to generate H202 and its implication in the cellular processes.

Retrogressed eclogite (20 kbar, 1020 °C) from the Neoproterozoic Palghat–Cauvery suture zone, southern India

The Palghat–Cauvery suture zone in southern India separates Archaean crustal blocks to the north and the Proterozoic Madurai block to the south. Here we present the first detailed study of a partially retrogressed eclogite (from within the Sittampundi anorthositic complex in the suture zone) that occurs as a 20-cm wide layer in a garnet gabbro layer in anorthosite. The eclogite largely consists of an assemblage of coexisting porphyroblasts of almandine–pyrope garnet and augitic clinopyroxene. However, a few garnets contain inclusions of omphacite. Rims and symplectites composed of Na–Ca amphibole and plagioclase form a retrograde assemblage. Petrographic analysis and calculated phase equilibria indicate that garnet–omphacite–rutile–melt was the peak metamorphic assemblage and that it formed at ca. 20 kbar and above 1000 °C. The eclogite was exhumed on a very tight hairpin-type, anticlockwise P–T path, which we relate to subduction and exhumation in the Palghat–Cauvery suture zone. The REE composition of the minerals suggests a basaltic oceanic crustal protolith metamorphosed in a subduction regime. Geological–structural relations combined with geophysical data from the Palghat–Cauvery suture zone suggest that the eclogite facies metamorphism was related to formation of the suture zone. Closure of the Mozambique Ocean led to development of the suture zone and to its western extension in the Betsimisaraka suture of Madagascar.

Evidence for H2O2 as the product of reduction of oxygen by alternative oxidase in mitochondria from potato tubers

Oxygen Consumption by alternative oxidase (AOX), present in mitochondria of many angiosperms, is known to be cyanide-resistant in contrast to cytochrome oxidase. Its activity in potato tuber (Solarium tuberosum L.) was induced following chilling treatment at 4 degrees C.About half of the total O-2 consumption of succinate oxidation in such mitochondria was found to be sensitive to SHAM, a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive oxygen consumption by nearly half, and addition at the end of the reaction released nearly half of the consumed oxygen by AOX, both typical of catalase action on H2O2. These findings with catalase suggest that the product of reduction of AOX is H2O2 and not H2O, as previously Surmised. In potatoes Subjected to chill stress (4 degrees C) for periods of 3, 5 and >= 8 days the activity of AOX in mitochondria increased progressively with a corresponding increase in the AOX protein detected by immunoblot of the protein.

Central America: Labor Reports and Child Labor Reports Pursuant to the Trade Act of 2002, Section 2102(c)(8)-(9)

[Excerpt] These comments are in response to the “Request for Information Concerning Labor Rights in Costa Rica, El Salvador, Guatemala, Honduras and Nicaragua and their Laws Governing Exploitative Child Labor” published at 68 Fed. Reg. 19580 (April 21, 2003). This Request for Information was issued pursuant to Section 2102(c)(8) and (9) of the Trade Act of 2002, Pub. L. 107-210, which requires the President, with respect to any proposed trade agreement, to submit to Congress a “meaningful labor rights report” and a “report describing the extent to which the country or countries that are parties to the agreement have in effect laws governing exploitative child labor.”

Sequence-dependent conformation of an A-DNA double helix: The crystal structure of the octamer d(G-G-T-A-T-A-C-C)

The crystal structures of the synthetic self-complementary octamer d(G-G-T-A-T-A-C-C) and its 5-bromouracil-containing analogue have been refined to R values of 20% and 14% at resolutions of 1·8 and 2·25 Å, respectively. The molecules adopt an A-DNA type double-helical conformation, which is minimally affected by crystal forces. A detailed analysis of the structure shows a considerable influence of the nucleotide sequence on the base-pair stacking patterns. In particular, the electrostatic stacking interactions between adjacent guanine and thymine bases produce symmetric bending of the double helix and a major-groove widening. The sugar-phosphate backbone appears to be only slightly affected by the base sequence. The local variations in the base-pair orientation are brought about by correlated adjustments in the backbone torsion angles and the glycosidic orientation. Sequence-dependent conformational variations of the type observed here may contribute to the specificity of certain protein-DNA interactions.

Microsomal 11-Alpha-hydroxylation of progesterone in Aspergillius ochraceus: Part I: Characterization of the hydroxylase system

Microsomes (105,000xg sediment) prepared from induced cells of A.ochraceus was found to hydroxylate progesterone to 11-Alpha-hydroxyprogesterone (11a-OHP) in high yields (85-�90% in 30 min.) in the presence of NADPH and O2. The pH optimum for the hydroxylase was found to be 7.7. However, for the isolation of active microsomes grinding of the mycelium should be carried out at pH 8.3. Metyrapone, carbon monoxide, SKF-525A, p-CMB and N-methyl maleimide inhibited the hydroxylase activity indicating the involvement of cytochrome P-450 system. The inhibition of the hydroxylase by cytochrome Image and the presence of high levels of NADPH-cytochrome Image reductase in induced microsomes suggest that the reductase could be one of the components in the hydroxylase system.

In vitro propagation of Corymbia torelliana x C. citriodora (Myrtaceae) via cytokinin-free node culture

Hybrids between Corymbia torelliana (F.Muell.) K.D.Hill & L.A.S.Johnson and C. citriodora subsp. variegata (F.Muell.) A.R.Bean & M.W.McDonald are used extensively to establish forestry plantations in subtropical Australia. Methods were developed for in vitro seed germination, shoot multiplication and plantlet formation that could be used to establish in vitro and ex vitro clone banks of juvenile Corymbia hybrids. Effects of sodium hypochlorite concentration and exposure time on seed contamination and germination, and effects of cytokinin and auxin concentrations on shoot multiplication and subsequent rooting, were assessed. A two-step surface sterilisation procedure, involving 70% ethanol followed by 1% sodium hypochlorite, provided almost no contamination and at least 88% germination. A novel method of cytokinin-free node culture proved most effective for in vitro propagation. Lateral bud break of primary shoots was difficult to induce by using cytokinin, but primary shoots rooted prolifically, elongated rapidly and produced multiple nodes in the absence of exogenous cytokinin. Further multiplication was obtained either by elongating lateral shoots of nodal explants in cytokinin-free medium or by inducing organogenic callus and axillary shoot proliferation with 2.2 µm benzyladenine. Plantlets were produced using an in vitro soil-less method that provided extensive rooting in sterile propagation mixture. These methods provide a means for simultaneous laboratory storage and field-testing of clones before selection and multiplication of desired genotypes.