Functional analysis, using in vitro mutagenesis, of amino acids located in the phenylalanine hydroxylase active site

The 3-dimensionaI structure determination of rat phenylalanine hydroxylase (PAH) has identified potentially important amino acids lining the active site cleft with the majority of these having hydrophobic side-chains including several with aromatic side chains. Here we have analyzed the effect on rat PAH enzyme kinetics of in vitro mutagenesis of a number of these amino acids lining the PAH active site. Mutation of F299, Y324, F331, and Y343 caused a significant decrease in enzyme activity but no change in the K-m for substrate or cofactor. me conclude that these aromatic residues are essential for activity but are not significantly involved in binding of the substrate or cofactor. in contrast the PAH mutant, S349T, showed an 18-fold increase in K-m for phenylalanine, showing the first functional evidence that this residue was binding at or near the phenylalanine binding site. This confirms the recently published model for the binding of phenylalanine to the PAH active site that postulated S349 interacts with the amino group on the main chain of the phenylalanine molecule. This result differs with that found for the equivalent mutation (S395T), in the closely related tyrosine hydroxylase, which had no effect on substrate K-m, showing that while the architecture of the two active sites are very similar the amino acids that bind to the respective substrates are different. (C) 2000 Academic Press.

A method for setting the size of plant conservation target areas

Realistic time frames in which management decisions are made often preclude the completion of the detailed analyses necessary for conservation planning. Under these circumstances, efficient alternatives may assist in approximating the results of more thorough studies that require extensive resources and time. We outline a set of concepts and formulas that may be used in lieu of detailed population viability analyses and habitat modeling exercises to estimate the protected areas required to provide desirable conservation outcomes for a suite of threatened plant species. We used expert judgment of parameters and assessment of a population size that results in a specified quasiextinction risk based on simple dynamic models The area required to support a population of this size is adjusted to take into account deterministic and stochastic human influences, including small-scale disturbance deterministic trends such as habitat loss, and changes in population density through processes such as predation and competition. We set targets for different disturbance regimes and geographic regions. We applied our methods to Banksia cuneata, Boronia keysii, and Parsonsia dorrigoensis, resulting in target areas for conservation of 1102, 733, and 1084 ha, respectively. These results provide guidance on target areas and priorities for conservation strategies.

Constitutively Active Mutant D816VKit Induces Megakayocyte and Mast Cell Differentiation of Early Haemopoietic Cells from Murine Foetal Liver

Mutations of Kit at position D816 have been implicated in mastocytosis, acute myeloid leukaemia and germ cell tumours. Expression of this mutant Kit in cell lines results in factor-independent growth, differentiation and increased survival in vitro and tumourigenicity in vivo. Mutant D816VKit and wild-type Kit were expressed in murine primary haemopoietic cells and grown in stem cell factor (SCF) or the absence of factors. Expression of D816VKit did not lead to transformation as assessed by a colony assay, but resulted in enhanced differentiation of cells when compared to control cells. D816VKit induced an increase in the number of cells differentiating along the megakaryocyte lineage in the absence of factors. SCF had an added effect with an increase in differentiation of mast cells. Expression of wild-type Kit in the presence of SCF also failed to cause transformation and induced differentiation of mast cells and megakaryocytes. We conclude that constitutive expression of D816VKit in primary haemopoietic cells is not a sufficient transforming stimulus but leads to the survival and maturation of cells whose phenotype is influenced by the presence of SCF. (C) 2003 Elsevier Science Ltd. All rights reserved.

Active and passive smoking and the risk of subarachnoid hemorrhage - An international population-based case-control study

Background and Purpose - This study was undertaken to better clarify the risks associated with cigarette smoking and subarachnoid hemorrhage (SAH). Methods - The study included 432 incident cases of SAH frequency matched to 473 community SAH-free controls to determine dose-dependent associations of active and passive smoking ( at home) and smoking cessation with SAH. Results - Compared with never smokers not exposed to passive smoking, the adjusted odds ratio for SAH among current smokers was 5.0 (95% confidence interval [CI], 3.1 to 8.1); for past smokers, 1.2 ( 95% CI, 0.8 to 2.0); and for passive smokers, 0.9 ( 95% CI, 0.6 to 1.5). Current and lifetime exposures showed a clear dose-dependent effect, and risks appeared more prominent in women and for aneurysmal SAH. Approximately 1 in 3 cases of SAH could be attributed to current smoking, but risks decline quickly after smoking cessation, even among heavy smokers. Conclusions - A strong positive association was found between cigarette smoking and SAH, especially for aneurysmal SAH and women, which is virtually eliminated within a few years of smoking cessation. Large opportunities exist for preventing SAH through smoking avoidance and cessation programs.

Proteasomal degradation of N-acetyltransferase 1 is prevented by acetylation of the active site cysteine - A mechanism for the slow acetylator phenotype and substrate-dependent down-regulation

Many drugs and chemicals found in the environment are either detoxified by N-acetyltransferase 1 (NAT1, EC 2.3.1.5) and eliminated from the body or bioactivated to metabolites that have the potential to cause toxicity and/or cancer. NAT1 activity in the body is regulated by genetic polymorphisms as well as environmental factors such as substrate-dependent down-regulation and oxidative stress. Here we report the molecular mechanism for the low protein expression from mutant NAT1 alleles that gives rise to the slow acetylator phenotype and show that a similar process accounts for enzyme down-regulation by NAT1 substrates. NAT1 allozymes NAT1 14, NAT1 15, NAT1 17, and NAT1 22 are devoid of enzyme activity and have short intracellular half-lives (similar to4 h) compared with wild-type NAT1 4 and the active allozyme NAT1 24. The inactive allozymes are unable to be acetylated by cofactor, resulting in ubiquitination and rapid degradation by the 26 S proteasome. This was confirmed by site-directed mutagenesis of the active site cysteine 68. The NAT1 substrate p-aminobenzoic acid induced ubiquitination of the usually stable NAT1 4, leading to its rapid degradation. From this study, we conclude that NAT1 exists in the cell in either a stable acetylated state or an unstable non-acetylated state and that mutations in the NAT1 gene that prevent protein acetylation produce a slow acetylator phenotype.

Physico-chemical characterization and synthesis of neuronally active alpha-conotoxins

The high speciFIcity of alpha-conotoxins for different neuronal nicotinic acetylcholine receptors makes them important probes for dissecting receptor subtype selectivity. New sequences continue to expand the diversity and utility of the pool of available alpha-conotoxins. Their identification and characterization depend on a suite of techniques with increasing emphasis on mass spectrometry and microscale chromatography, which have benefited from recent advances in resolution and capability. Rigorous physicochemical analysis together with synthetic peptide chemistry is a prerequisite for detailed conformational analysis and to provide sufficient quantities of alpha-conotoxins for activity assessment and structure-activity relationship studies.

Bioprocess-centered molecular design (BMD) for the efficient production of an interfacially active peptide

The efficient expression and purification of an interfacially active peptide (mLac21) was achieved by using bioprocess-centered molecular design (BMD), wherein key bioprocess considerations are addressed during the initial molecular biology work. The 21 amino acid mLac21 peptide sequence is derived from the lac repressor protein and is shown to have high affinity for the oil-water interface, causing a substantial reduction in interfacial tension following adsorption. The DNA coding for the peptide sequence was cloned into a modified pET-31(b) vector to permit the expression of mLac21 as a fusion to ketosteroid isomerase (KSI). Rational iterative molecular design, taking into account the need for a scaleable bioprocess flowsheet, led to a simple and efficient bioprocess yielding mLac21 at 86% purity following ion exchange chromatography (and >98% following chromatographic polishing). This case study demonstrates that it is possible to produce acceptably pure peptide for potential commodity applications using common scaleable bioprocess unit operations. Moreover, it is shown that BMD is a powerful strategy that can be deployed to reduce bioseparation complexity. (C) 2004 Wiley Periodicals, Inc.

Synthesis of homoallylic oxygenated alpha-methylene-gamma-butyrolactones: a model for preparing biologically active natural lactones

In this Letter we describe a 12% overall yield synthesis of a model for homoallylic oxygenated alpha-methylene-gamma-butyrolactones with relative stereochemistry defined by selective hydrogenation with Rh/Al(2)O(3). The synthesis was realized in 9 steps involving simple reactions. (C) 2008 Elsevier Ltd. All rights reserved.

Dissolved organic carbon in rainwater from areas heavily impacted by sugar cane burning

This work reports on rainwater dissolved organic carbon (DOC) from Ribeirao Preto (RP) and Araraquara over a period of 3 years. The economies of these two cities, located in Sao Paulo state (Brazil), are based on agriculture and related industries, and the region is strongly impacted by the burning of sugar cane foliage before harvesting. Highest DOC concentrations were obtained when air masses traversed sugar cane fields burned on the same day as the rain event. Significant increases in the DOC volume weighted means (VWM) during the harvest period, for both sites, and a good linear correlation (r=0.83) between DOC and K (a biomass burning marker) suggest that regional scale organic carbon emissions prevail over long-range transport. The DOC VWMs and standard deviations were 272 +/- 22 mu mol L-1 (n=193) and 338 +/- 40 mu mol L-1 (n=80) for RP and Araraquara, respectively, values which are at least two times higher than those reported for other regions influenced by biomass burning, such as the Amazon. These high DOC levels are discussed in terms of agricultural activities, particularly the large usage of biogenic fuels in Brazil, as well as the analytical method used in this work, which includes volatile organic carbon when reporting DOC values. Taking into account rainfall volume, estimated annual rainwater DOC fluxes for RP (4.8 g C m(-2) yr(-1)) and Araraquara (5.4 g C m(-2) yr(-1)) were close to that previously found for the Amazon region (4.8 g C m(-2) yr(-1)). This work also discusses whether previous calculations of the global rainwater carbon flux may have been underestimated, since they did not consider large inputs from biomass combustion sources, and suffered from a possible analytical bias. (c) 2008 Elsevier Ltd. All rights reserved.

Activation of visual areas during the attentional blink

When two targets are presented in rapid succession, identification of the first target is nearly perfect while identification of the second is severely impaired at shorter inter-target lags, and then gradually improves as lag increases. This second-target deficit is known as the attentional blink (AB). Numerous studies have implicated competition for access to higher-order processing mechanisms as the primary cause of the AB. However, relatively few studies have directly examined how the AB modulates activity in specific brain areas. To this end, we used fMRI to measure activation in the occipital and parietal cortices (including V1, V2, and area MT) during an AB task. Participants were presented with an initial target of oriented line segments embedded in a central stream of letter distractors. This central target was followed 100 - 700 ms later by a peripheral ‘X’ presented at one of four locations along with three ‘+’ distractors. All peripheral items were presented in the centre of a small field of moving dots. Participants made non-speeded judgments about line-segment orientation and the location of the second target at the end of a trial and to ignore all other stimuli. The results showed a robust AB characterised by a linear improvement in second-target accuracy as lag increased. This pattern of behavioural results was mirrored by changes in activation patterns across a number of visual areas indicating robust modulation of brain activity by the AB.

Social Wasp Diversity (Vespidae, Polistinae) in a Forest Fragment in the Northeast of Sao Paulo State Sampled with Different Methodologies

Social wasp diversity in Semideciduous Seasonal Forests of the northeast of Sao Paulo State is poorly known, causing a lack of information on the diversity of these wasps from these areas which have been degraded. The objective of this work was to evaluate the social wasp (Vespidae, Polistinae) diversity in a Semideciduous Seasonal Forest of the northeast of Sao Paulo State and to compare three different kinds of sampling methodology. Surveys were conducted from August 2005 to September 2006 in the interior, edge and matrix of a Semideciduous Seasonal Forest fragment in Patrocinio Paulista city, Sao Paulo State. Three methodologies were used: 1. Active collection in flowers, 2. Searching for nests, 3. Active collection with attractive liquid. Thirty species of social wasps were collected in the fragment, but the diversity was highest in the edge. Active collection with attractive liquid was the most efficient methodology. Despite the high levels of deforestation, forest fragments in Sao Paulo State have a high diversity of social wasps, reinforcing the importance of their preservation.

Dyslipidemia in a Cohort of HIV-infected Latin American Children Receiving Highly Active Antiretroviral Therapy*

In order to describe the prevalence of hypercholesterolemia and hypertriglyceridemia in a cohort of HIV-infected children and adolescents in Latin America and to determine associations with highly active antiretroviral therapy (HAART), we performed this cross-sectional analysis within the NICHD International Site Development Initiative pediatric cohort study. Eligible children had to be at least 2 years of age and be on HAART. Among the 477 eligible HIV-infected youth, 98 (20.5%) had hypercholesterolemia and 140 (29.4%) had hypertriglyceridemia. In multivariable analyses, children receiving protease inhibitor (PI)-containing HAART were at increased risk for hypercholesterolemia [adjusted odds ratio (AOR) = 2.7, 95% confidence interval (CI) 1.3-5.6] and hypertriglyceridemia (AOR = 3.5, 95% CI 1.9-6.4) compared with children receiving non-nucleoside reverse transcriptase inhibitor (NNRTI)-containing HAART. In conclusion, HIV-infected youth receiving PI-containing HAART in this Latin American cohort were at increased risk for hypercholesterolemia and hypertriglyceridemia compared with those receiving NNRTI-containing HAART.

Atrial Coronary Arteries in Areas Involved in Atrial Fibrillation Catheter Ablation

Background-The proximity to vascular structures is a limiting factor during radiofrequency ablation. However, little or no attention has been given to the atrial arterial circulation during the development of atrial fibrillation (AF) catheter ablation techniques. Methods and Results-We examined the atrial arterial circulation in areas involved in AF ablation in 24 heart specimens by colored resin injection and careful dissection. The sinus node artery (SNA) arose from the circumflex artery in 42% of case; proximal to the LA appendage in 29%, crossing the left atrium (LA) anterior wall; and after the LA appendage in the remaining 13%, crossing the mitral isthmus and passing close to the left pulmonary veins (PVs), the LA roof, and the right superior PV. In 58%, the SNA arose from the right coronary artery. Major arteries (>= 1 mm in external diameter) were found in the mitral isthmus in 54%, at the LA roof in 54%, and at the LA anterior wall in 29%. Around the left PV ostia, there were areas with major arteries in up to 37% (at the roof and inferior segments) and around the right PV ostia in up to 29% (at the roof segment). Conclusions-Major atrial coronary arteries, including the SNA, were commonly found in the areas involved in AF ablation and could cause difficulties in obtaining transmural lesions and electric isolation or even lead to ischemic sinus node or atrial dysfunction. (Circ Arrhythm Electrophysiol. 2010;3:600-605.)