Current and future implications of basic and translational research on amyloid-ß peptide production and removal pathways.

Inherited variants in multiple different genes are associated with increased risk for Alzheimer's disease (AD). In many of these genes, the inherited variants alter some aspect of the production or clearance of the neurotoxic amyloid ß-peptide (Aß). Thus missense, splice site or duplication mutants in the presenilin 1 (PS1), presenilin 2 (PS2) or the amyloid precursor protein (APP) genes, which alter the levels or shift the balance of Aß produced, are associated with rare, highly penetrant autosomal dominant forms of Familial Alzheimer's Disease (FAD). Similarly, the more prevalent late-onset forms of AD are associated with both coding and non-coding variants in genes such as SORL1, PICALM and ABCA7 that affect the production and clearance of Aß. This review summarises some of the recent molecular and structural work on the role of these genes and the proteins coded by them in the biology of Aß. We also briefly outline how the emerging knowledge about the pathways involved in Aß generation and clearance can be potentially targeted therapeutically. This article is part of Special Issue entitled "Neuronal Protein".

Antibody response and risk factors for seropositivity in backyard poultry following mass vaccination against highly pathogenic avian influenza and Newcastle disease in Indonesia.

A large-scale mass vaccination campaign was carried out in Java, Indonesia in an attempt to control outbreaks of highly pathogenic avian influenza (HPAI) in backyard flocks and commercial smallholder poultry. Sero-monitoring was conducted in mass vaccination and control areas to assess the proportion of the target population with antibodies against HPAI and Newcastle disease (ND). There were four rounds of vaccination, and samples were collected after each round resulting in a total of 27 293 samples. Sampling was performed irrespective of vaccination status. In the mass vaccination areas, 20-45% of poultry sampled had a positive titre to H5 after each round of vaccination, compared to 2-3% in the control group. In the HPAI + ND vaccination group, 12-25% of the population had positive ND titres, compared to 5-13% in the areas without ND vaccination. The level of seropositivity varied by district, age of the bird, and species (ducks vs. chickens).

Cardiac toxoplasmosis after heart transplantation diagnosed by endomyocardial biopsy.

We describe a case of cardiac toxoplasmosis diagnosed by routine endomyocardial biopsy in a patient with trimethoprim-sulfamethoxazole (TMP-SMX) intolerance on atovaquone prophylaxis. Data are not available on the efficacy of atovaquone as Toxoplasma gondii prophylaxis after heart transplantation. In heart transplant patients in whom TMP-SMX is not an option, other strategies may be considered, including the addition of pyrimethamine to atovaquone.

Determination of hormoligosis of organophosphate insecticides against Phenacoccus solenopsis.

Cotton mealybug is a highly invasive pest of agricultural crops worldwide. Major agriculturists most rely on the use of insecticides for the control of pesticides. So, the indiscriminate use of insecticides leads to resistance development in recent years. For this purpose, an experiment was conducted using different concentrations of the three insecticides (profenfos chlorpyrifos and triazophos) to check the hormoligosis effects against cotton mealybug (CMB) in laboratory conditions. Investigation of variations for % mortality of adults of CMB after three days revealed that all treatments had statistically significant (P ˂ 0.05). The highest mortality was observed at the highest concentrations of profenofos 2.4% (38.55%). After 7 days, all the treatments were significant with difference in means (P ˂ 0.05). The highest mortality was recorded at the highest dilution of pesticide profenofos 2.4% (77.11%). The values of fecundity and longevity exposed a valid difference among treatments (P ˂ 0.05). Maximum fecundity was observed at the concentration 2.4% (181.41%) and longevity showed (38.46%). The highest mortality was observed at a concentration of triazophos 4% (27.98%). For chlorpyriphos the highest mortality was examined at concentration 4% (24.79%). The fecundity showed a statistically significant difference for different concentrations of triazophos and chlorpyriphos (P ˂ 0.05). The results of the recent study provide valuable information regarding the selection of insecticides and hormoligosis effects. The study can be helpful in the implications of integrated pest management of P. solenopsis.

Characterization of the species of genus Physa on the basis of typological species concept from Central Punjab.

Physids belong to Class Gastropoda; Phylum Mollusca have important position in food web and act as bio indicators, pests and intermediate host. Being resistant these are called cockroaches of malacology. Physid snails were collected from different water bodies of Faisalabad (Punjab) and were identified up to species using morphological markers. The morphometry of the specimens was carried out with the help of a digital Vernier caliper in millimeters (mm) using linear measurement of shell characters. Linear regression analysis of the AL/SW ratio vs AL and SL/SW ratio vs AL indicated that allometric growth exists only in Physa acuta when compared with P.gyrina and P. fontinalis. This study will lead to assess the status of the Physid species in Central Punjab. The Principal component analysis shows that the Component 1 (Shell Length) and component 2 (Shell Width) are the most prolific components and nearly 80 percent of the identification. The distance between P. acuta and P. fontinalis is 5.4699, P. acuta and P. gyrina is 7.6411, P. fontinalis and P. gyrina is 16.6080 showing that P. acuta resembles with P. fontinalis, and both these specimens donot resemble with P. gyrina. P.acuta is an invasive species and shows bioactivity making it a potent candidate for bioactive substances.

Molecular Phylogenetics of Physa acuta (Pulmonata: Basommatophora): an Invasive species in Central Punjab Pakistan.

Physids belong to Class Gastropoda; belong to Phylum Mollusca and being bioindicators, intermediate hosts of parasites and pests hold a key position in the ecosystem. There are three species of Genus Physa i.e. P. fontinalis, Physa acuta and P. gyrina water bodies of Central Punjab and were characterized on the basis of molecular markers High level of genetic diversity was revealed by polymorphic RAPD, however SSR markers were not amplified. The multivariate analysis revealed polymorphism ranging from 9.09 percent to 50 percent among the three Physid species. Total number of 79 loci were observed for the three species under study and 24 loci were observed to be polymorphic. These RAPD fragment(s) can be developed into co dominant markers (SCAR) by cloning and can be further sequenced for the development of the Physa species specific markers to identify the introduced and native species in Pakistan.

Quantitative T1ρ MRI of the Head and Neck Discriminates Carcinoma and Benign Hyperplasia in the Nasopharynx.

BACKGROUND AND PURPOSE: T1ρ imaging is a new quantitative MR imaging pulse sequence with the potential to discriminate between malignant and benign tissue. In this study, we evaluated the capability of T1ρ imaging to characterize tissue by applying T1ρ imaging to malignant and benign tissue in the nasopharynx and to normal tissue in the head and neck. MATERIALS AND METHODS: Participants with undifferentiated nasopharyngeal carcinoma and benign hyperplasia of the nasopharynx prospectively underwent T1ρ imaging. T1ρ measurements obtained from the histogram analysis for nasopharyngeal carcinoma in 43 participants were compared with those for benign hyperplasia and for normal tissue (brain, muscle, and parotid glands) in 41 participants using the Mann-Whitney U test. The area under the curve of significant T1ρ measurements was calculated and compared using receiver operating characteristic analysis and the Delong test, respectively. A P < . 05 indicated statistical significance. RESULTS: There were significant differences in T1ρ measurements between nasopharyngeal carcinoma and benign hyperplasia and between nasopharyngeal carcinoma and normal tissue (all, P < . 05). Compared with benign hyperplasia, nasopharyngeal carcinoma showed a lower T1ρ mean (62.14 versus 65.45 × ms), SD (12.60 versus 17.73 × ms), and skewness (0.61 versus 0.76) (all P < .05), but no difference in kurtosis (P = . 18). The T1ρ SD showed the highest area under the curve of 0.95 compared with the T1ρ mean (area under the curve = 0.72) and T1ρ skewness (area under the curve = 0.72) for discriminating nasopharyngeal carcinoma and benign hyperplasia (all, P < .05). CONCLUSIONS: Quantitative T1ρ imaging has the potential to discriminate malignant from benign and normal tissue in the head and neck.

Characterization of the species of genus Physa on the basis of typological species concept from Central Punjab / Caracterização das espécies do gênero Physa com base no conceito tipológico de espécie do Punjab Central

Physids belong to Class Gastropoda; Phylum Mollusca have important position in food web and act as bio indicators, pests and intermediate host. Being resistant these are called cockroaches of malacology. Physid snails were collected from different water bodies of Faisalabad (Punjab) and were identified up to species using morphological markers. The morphometry of the specimens was carried out with the help of a digital Vernier caliper in millimeters (mm) using linear measurement of shell characters. Linear regression analysis of the AL/SW ratio vs AL and SL/SW ratiovs AL indicated that allometric growth exists only in Physa acuta when compared with P.gyrina and P. fontinalis. This study will lead to assess the status of the Physid species in Central Punjab. The Principal component analysis shows that the Component 1 (Shell Length) and component 2 (Shell Width) are the most prolific components and nearly 80 percent of the identification. The distance between P. acuta and P. fontinalis is 5.4699, P. acuta and P. gyrina is 7.6411, P. fontinalis and P. gyrina is 16.6080 showing that P. acuta resembles with P. fontinalis, and both these specimens donot resemble with P. gyrina. P.acuta is an invasive species and shows bioactivity making it a potent candidate for bioactive substances.

Os físidos pertencem à classe Gastropoda; o filo Mollusca possui importante posição na teia alimentar e atua como bioindicador, praga e hospedeiro intermediário. Por serem resistentes, são chamadas baratas de malacologia. Os caramujos físidos foram coletados em diferentes corpos d’água de Faisalabad (Punjab) e identificados até as espécies por meio de marcadores morfológicos. A morfometria dos corpos de prova foi realizada com auxílio de paquímetro digital Vernier em milímetros (mm) por meio de medida linear dos caracteres da casca. A análise de regressão linear da razão AL / SW vs. AL e razão SL / SW vs. AL indicou que o crescimento alométrico existe apenas em Physa acuta quando comparado com P. gyrina e P. fontinalis. Este estudo levará a avaliar a situação das espécies de físido no Punjab Central. A análise do componente principal mostra que o componente 1 (comprimento da casca) e o componente 2 (largura da casca) são os componentes mais prolíficos e quase 80% da identificação. A distância entre P. acuta e P. fontinalis é 5,4699, P. acuta e P. gyrina é 7,6411, P. fontinalis e P. gyrina é 16,6080, mostrando que P. acuta se assemelha a P. fontinalis, e ambos os espécimes não se parecem com P. gyrina. P. acuta é uma espécie invasora e apresenta bioatividade, tornando-se uma candidata potente para substâncias bioativas.

Molecular Phylogenetics of Physa acuta (Pulmonata: Basommatophora): an invasive species in Central Punjab Pakistan / Molecular Phylogenetics of Physa acuta (Pulmonata: Basommatophora): uma espécie invasora no centro de Punjab, Paquistão

Physids belong to Class Gastropoda; belong to Phylum Mollusca and being bioindicators, intermediate hosts of parasites and pests hold a key position in the ecosystem. There are three species of Genus Physa i.e. P. fontinalis, Physa acuta and P. gyrina water bodies of Central Punjab and were characterized on the basis of molecular markers High level of genetic diversity was revealed by polymorphic RAPD, however SSR markers were not amplified. The multivariate analysis revealed polymorphism ranging from 9.09 percent to 50 percent among the three Physid species. Total number of 79 loci were observed for the three species under study and 24 loci were observed to be polymorphic. These RAPD fragment(s) can be developed into co dominant markers (SCAR) by cloning and can be further sequenced for the development of the Physa species specific markers to identify the introduced and native species in Pakistan.

Os físidos pertencem à classe Gastropoda; pertencem ao filo Mollusca e, sendo bioindicadores, hospedeiros intermediários de parasitas e pragas, ocupam uma posição-chave no ecossistema. Existem três espécies do gênero Physa, ou seja, P. fontinalis, Physa acuta e P. gyrina em corpos d’água do Punjab Central e foram caracterizadas com base em marcadores moleculares. Alto nível de diversidade genética foi revelado por RAPD polimórfico, no entanto os marcadores SSR não foram amplificados. A análise multivariada revelou polimorfismo variando de 9,09% a 50% entre as três espécies de Physid. Um número total de 79 loci foi observado para as três espécies em estudo e 24 loci foram observados como polimórficos. Esses fragmentos RAPD podem ser desenvolvidos em marcadores codominantes (SCAR) por clonagem e podem ser posteriormente sequenciados para o desenvolvimento de marcadores específicos da espécie Physa para identificar as espécies introduzidas e nativas no Paquistão.

Characterization of the species of genus Physa on the basis of typological species concept from Central Punjab

Abstract Physids belong to Class Gastropoda; Phylum Mollusca have important position in food web and act as bio indicators, pests and intermediate host. Being resistant these are called cockroaches of malacology. Physid snails were collected from different water bodies of Faisalabad (Punjab) and were identified up to species using morphological markers. The morphometry of the specimens was carried out with the help of a digital Vernier caliper in millimeters (mm) using linear measurement of shell characters. Linear regression analysis of the AL/SW ratio vs AL and SL/SW ratio vs AL indicated that allometric growth exists only in Physa acuta when compared with P.gyrina and P. fontinalis. This study will lead to assess the status of the Physid species in Central Punjab. The Principal component analysis shows that the Component 1 (Shell Length) and component 2 (Shell Width) are the most prolific components and nearly 80 percent of the identification. The distance between P. acuta and P. fontinalis is 5.4699, P. acuta and P. gyrina is 7.6411, P. fontinalis and P. gyrina is 16.6080 showing that P. acuta resembles with P. fontinalis, and both these specimens donot resemble with P. gyrina. P.acuta is an invasive species and shows bioactivity making it a potent candidate for bioactive substances.

Resumo Os físidos pertencem à classe Gastropoda; o filo Mollusca possui importante posição na teia alimentar e atua como bioindicador, praga e hospedeiro intermediário. Por serem resistentes, são chamadas baratas de malacologia. Os caramujos físidos foram coletados em diferentes corpos dágua de Faisalabad (Punjab) e identificados até as espécies por meio de marcadores morfológicos. A morfometria dos corpos de prova foi realizada com auxílio de paquímetro digital Vernier em milímetros (mm) por meio de medida linear dos caracteres da casca. A análise de regressão linear da razão AL / SW vs. AL e razão SL / SW vs. AL indicou que o crescimento alométrico existe apenas em Physa acuta quando comparado com P. gyrina e P. fontinalis. Este estudo levará a avaliar a situação das espécies de físido no Punjab Central. A análise do componente principal mostra que o componente 1 (comprimento da casca) e o componente 2 (largura da casca) são os componentes mais prolíficos e quase 80% da identificação. A distância entre P. acuta e P. fontinalis é 5,4699, P. acuta e P. gyrina é 7,6411, P. fontinalis e P. gyrina é 16,6080, mostrando que P. acuta se assemelha a P. fontinalis, e ambos os espécimes não se parecem com P. gyrina. P. acuta é uma espécie invasora e apresenta bioatividade, tornando-se uma candidata potente para substâncias bioativas.

Molecular Phylogenetics of Physa acuta (Pulmonata: Basommatophora): an Invasive species in Central Punjab Pakistan

Abstract Physids belong to Class Gastropoda; belong to Phylum Mollusca and being bioindicators, intermediate hosts of parasites and pests hold a key position in the ecosystem. There are three species of Genus Physa i.e. P. fontinalis, Physa acuta and P. gyrina water bodies of Central Punjab and were characterized on the basis of molecular markers High level of genetic diversity was revealed by polymorphic RAPD, however SSR markers were not amplified. The multivariate analysis revealed polymorphism ranging from 9.09 percent to 50 percent among the three Physid species. Total number of 79 loci were observed for the three species under study and 24 loci were observed to be polymorphic. These RAPD fragment(s) can be developed into co dominant markers (SCAR) by cloning and can be further sequenced for the development of the Physa species specific markers to identify the introduced and native species in Pakistan.

Resumo Os físidos pertencem à classe Gastropoda; pertencem ao filo Mollusca e, sendo bioindicadores, hospedeiros intermediários de parasitas e pragas, ocupam uma posição-chave no ecossistema. Existem três espécies do gênero Physa, ou seja, P. fontinalis, Physa acuta e P. gyrina em corpos dágua do Punjab Central e foram caracterizadas com base em marcadores moleculares. Alto nível de diversidade genética foi revelado por RAPD polimórfico, no entanto os marcadores SSR não foram amplificados. A análise multivariada revelou polimorfismo variando de 9,09% a 50% entre as três espécies de Physid. Um número total de 79 loci foi observado para as três espécies em estudo e 24 loci foram observados como polimórficos. Esses fragmentos RAPD podem ser desenvolvidos em marcadores codominantes (SCAR) por clonagem e podem ser posteriormente sequenciados para o desenvolvimento de marcadores específicos da espécie Physa para identificar as espécies introduzidas e nativas no Paquistão.

Characterization of the species of genus Physa on the basis of typological species concept from Central Punjab / Caracterização das espécies do gênero Physa com base no conceito tipológico de espécie do Punjab Central

Abstract Physids belong to Class Gastropoda; Phylum Mollusca have important position in food web and act as bio indicators, pests and intermediate host. Being resistant these are called cockroaches of malacology. Physid snails were collected from different water bodies of Faisalabad (Punjab) and were identified up to species using morphological markers. The morphometry of the specimens was carried out with the help of a digital Vernier caliper in millimeters (mm) using linear measurement of shell characters. Linear regression analysis of the AL/SW ratio vs AL and SL/SW ratio vs AL indicated that allometric growth exists only in Physa acuta when compared with P.gyrina and P. fontinalis. This study will lead to assess the status of the Physid species in Central Punjab. The Principal component analysis shows that the Component 1 (Shell Length) and component 2 (Shell Width) are the most prolific components and nearly 80 percent of the identification. The distance between P. acuta and P. fontinalis is 5.4699, P. acuta and P. gyrina is 7.6411, P. fontinalis and P. gyrina is 16.6080 showing that P. acuta resembles with P. fontinalis, and both these specimens donot resemble with P. gyrina. P.acuta is an invasive species and shows bioactivity making it a potent candidate for bioactive substances.

Resumo Os físidos pertencem à classe Gastropoda; o filo Mollusca possui importante posição na teia alimentar e atua como bioindicador, praga e hospedeiro intermediário. Por serem resistentes, são chamadas baratas de malacologia. Os caramujos físidos foram coletados em diferentes corpos d'água de Faisalabad (Punjab) e identificados até as espécies por meio de marcadores morfológicos. A morfometria dos corpos de prova foi realizada com auxílio de paquímetro digital Vernier em milímetros (mm) por meio de medida linear dos caracteres da casca. A análise de regressão linear da razão AL / SW vs. AL e razão SL / SW vs. AL indicou que o crescimento alométrico existe apenas em Physa acuta quando comparado com P. gyrina e P. fontinalis. Este estudo levará a avaliar a situação das espécies de físido no Punjab Central. A análise do componente principal mostra que o componente 1 (comprimento da casca) e o componente 2 (largura da casca) são os componentes mais prolíficos e quase 80% da identificação. A distância entre P. acuta e P. fontinalis é 5,4699, P. acuta e P. gyrina é 7,6411, P. fontinalis e P. gyrina é 16,6080, mostrando que P. acuta se assemelha a P. fontinalis, e ambos os espécimes não se parecem com P. gyrina. P. acuta é uma espécie invasora e apresenta bioatividade, tornando-se uma candidata potente para substâncias bioativas.

Molecular Phylogenetics of Physa acuta (Pulmonata: Basommatophora): an Invasive species in Central Punjab Pakistan / Molecular Phylogenetics of Physa acuta (Pulmonata: Basommatophora): uma espécie invasora no centro de Punjab, Paquistão

Abstract Physids belong to Class Gastropoda; belong to Phylum Mollusca and being bioindicators, intermediate hosts of parasites and pests hold a key position in the ecosystem. There are three species of Genus Physa i.e. P. fontinalis, Physa acuta and P. gyrina water bodies of Central Punjab and were characterized on the basis of molecular markers High level of genetic diversity was revealed by polymorphic RAPD, however SSR markers were not amplified. The multivariate analysis revealed polymorphism ranging from 9.09 percent to 50 percent among the three Physid species. Total number of 79 loci were observed for the three species under study and 24 loci were observed to be polymorphic. These RAPD fragment(s) can be developed into co dominant markers (SCAR) by cloning and can be further sequenced for the development of the Physa species specific markers to identify the introduced and native species in Pakistan.

Resumo Os físidos pertencem à classe Gastropoda; pertencem ao filo Mollusca e, sendo bioindicadores, hospedeiros intermediários de parasitas e pragas, ocupam uma posição-chave no ecossistema. Existem três espécies do gênero Physa, ou seja, P. fontinalis, Physa acuta e P. gyrina em corpos d'água do Punjab Central e foram caracterizadas com base em marcadores moleculares. Alto nível de diversidade genética foi revelado por RAPD polimórfico, no entanto os marcadores SSR não foram amplificados. A análise multivariada revelou polimorfismo variando de 9,09% a 50% entre as três espécies de Physid. Um número total de 79 loci foi observado para as três espécies em estudo e 24 loci foram observados como polimórficos. Esses fragmentos RAPD podem ser desenvolvidos em marcadores codominantes (SCAR) por clonagem e podem ser posteriormente sequenciados para o desenvolvimento de marcadores específicos da espécie Physa para identificar as espécies introduzidas e nativas no Paquistão.

The crystal structure of a class II fructose-1,6-bisphosphate aldolase shows a novel binuclear metal-binding active site embedded in a familiar fold.

BACKGROUND: [corrected] Aldolases catalyze a variety of condensation and cleavage reactions, with exquisite control on the stereochemistry. These enzymes, therefore, are attractive catalysts for synthetic chemistry. There are two classes of aldolase: class I aldolases utilize Schiff base formation with an active-site lysine whilst class II enzymes require a divalent metal ion, in particular zinc. Fructose-1,6-bisphosphate aldolase (FBP-aldolase) is used in gluconeogenesis and glycolysis; the enzyme controls the condensation of dihydroxyacetone phosphate with glyceraldehyde-3-phosphate to yield fructose-1,6-bisphosphate. Structures are available for class I FBP-aldolases but there is a paucity of detail on the class II enzymes. Characterization is sought to enable a dissection of structure/activity relationships which may assist the construction of designed aldolases for use as biocatalysts in synthetic chemistry. RESULTS: The structure of the dimeric class II FBP-aldolase from Escherichia coli has been determined using data to 2.5 A resolution. The asymmetric unit is one subunit which presents a familiar fold, the (alpha/beta)8 barrel. The active centre, at the C-terminal end of the barrel, contains a novel bimetallic-binding site with two metal ions 6.2 A apart. One ion, the identity of which is not certain, is buried and may play a structural or activating role. The other metal ion is zinc and is positioned at the surface of the barrel to participate in catalysis. CONCLUSIONS: Comparison of the structure with a class II fuculose aldolase suggests that these enzymes may share a common mechanism. Nevertheless, the class II enzymes should be subdivided into two categories on consideration of subunit size and fold, quaternary structure and metal-ion binding sites.

Involvement of lysine residues of goat serum albumin in high-affinity binding of bilirubin.

Four maleylated derivatives of goat serum albumin having percent modification as 40%, 46%, 84% and 98% were prepared using varying molar ratio of maleic anhydride over protein. These preparations were found to be pure, both with respect to size and charged as judged by gel filtration and polyacrylamide gel electrophoresis. Maleylation caused significant change in protein conformation as revealed by the change in Stokes radius and frictional ratio of serum albumin, from 3.46 nm and 1.28 to 4.96 nm and 1.79, respectively, upon 98% modification. Immunodiffusion results of native and modified albumins with anti-goat serum albumin antiserum also suggested significant conformational changes in serum albumin upon maleylation. About 88% reduction in bilirubin binding was observed after modification of 98% amino groups of serum albumin as studied by visible difference spectroscopy at pH 8.0, and at 0.15 ionic strength. Increase in ionic strength to 1.0 did not lead to any significant reversal in bilirubin binding. These results prove the involvement of lysine residues in bilirubin-albumin interaction.

Conserved residues in the mechanism of the E. coli Class II FBP-aldolase.

The two classes of fructose-1,6-bisphosphate aldolase both catalyse the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The Class I aldolases use Schiff base formation as part of their catalytic mechanism, whereas the Class II enzymes are zinc-containing metalloproteins. The mechanism of the Class II enzymes is less well understood than their Class I counterparts. We have combined sequence alignments of the Class II family of enzymes with examination of the crystal structure of the enzyme to highlight potentially important aspartate and asparagine residues in the enzyme mechanism. Asp109, Asp144, Asp288, Asp290, Asp329 and Asn286 were targeted for site-directed mutagenesis and the resulting proteins purified and characterised by steady-state kinetics using either a coupled assay system to study the overall cleavage reaction or using the hexacyanoferrate (III) oxidation of the enzyme bound intermediate carbanion to investigate partial reactions. The results showed only minor changes in the kinetic parameters for the Asp144, Asp288, Asp290 and Asp329 mutants, suggesting that these residues play only minor or indirect roles in catalysis. By contrast, mutation of Asp109 or Asn286 caused 3000-fold and 8000-fold decreases in the kcat of the reaction, respectively. Coupled with the kinetics measured for the partial reactions the results clearly demonstrate a role for Asn286 in catalysis and in binding the ketonic end of the substrate. Fourier transform infra-red spectroscopy of the wild-type and mutant enzymes has further delineated the role of Asp109 as being critically involved in the polarisation of the carbonyl group of glyceraldehyde 3-phosphate.

Identification of arginine 331 as an important active site residue in the class II fructose-1,6-bisphosphate aldolase of Escherichia coli.

Treatment of the Class II fructose-1,6-bisphosphate aldolase of Escherichia coli with the arginine-specific alpha-dicarbonyl reagents, butanedione or phenylglyoxal, results in inactivation of the enzyme. The enzyme is protected from inactivation by the substrate, fructose 1,6-bisphosphate, or by inorganic phosphate. Modification with [7-14C] phenylglyoxal in the absence of substrate demonstrates that enzyme activity is abolished by the incorporation of approximately 2 moles of reagent per mole of enzyme. Sequence alignment of the eight known Class II FBP-aldolases shows that only one arginine residue is conserved in all the known sequences. This residue, Arg-331, was mutated to either alanine or glutamic acid. The mutant enzymes were much less susceptible to inactivation by phenylglyoxal. Measurement of the steady-state kinetic parameters revealed that mutation of Arg-331 dramatically increased the K(m) for fructose 1,6-bisphosphate. Comparatively small differences in the inhibitor constant Ki for dihydroxyacetone phosphate or its analogue, 2-phosphoglycolate, were found between the wild-type and mutant enzymes. In contrast, the mutation caused large changes in the kinetic parameters when glyceraldehyde 3-phosphate was used as an inhibitor. Kinetic analysis of the oxidation of the carbanionic aldolase-substrate intermediate of the reaction by hexacyanoferrate (III) revealed that the K(m) for dihydroxyacetone phosphate was again unaffected, whereas that for fructose 1,6-bisphosphate was dramatically increased. Taken together, these results show that Arg-331 is critically involved in the binding of fructose bisphosphate by the enzyme and demonstrate that it interacts with the C-6 phosphate group of the substrate.

Probing the determinants of protein solubility with amino acid modification.

Chemical modification was used as a probe to study the effect of structural features of serum albumin (charge, conformation, surface hydrophobicity, etc.) on its solubility behavior in concentrated ammonium sulfate solutions. Four different acetylated derivatives of goat serum albumin namely 18% acetylated, 40% acetylated, 53% acetylated, and 93% acetylated albumins were prepared. The homogeneity of these preparations was established by gel chromatography and polyacrylamide gel electrophoresis. Hydrodynamic data on the Stokes radius of native and acetylated albumins suggested gradual change in conformation on increasing modification. Solubility experiments performed in concentrated ammonium sulfate solutions at pH 7.0 and at 30 degrees C showed a slight decrease in salting-out parameter, Ks, up to 40% modification, whereas a significant decrease was obtained at higher modification. However, the salting-out parameter, beta, decreased monotonously. Similar decrease in these parameters was also observed with different modified albumins at other pH values viz. pH 5.5, 4.5, and 3.6. From these results we conclude that the decrease in solubility of serum albumin on increasing modification was primarily due to change in conformation.

Effect of acetylation on conformation and bilirubin-binding properties of goat serum albumin.

Four acetylated derivatives of goat serum albumin with percentage modifications of 18, 40, 53 and 93% were checked for bilirubin-binding and conformational properties. Acetylation caused marked changes in protein conformation, as evidenced by double immunodiffusion and proteolytic digestion results, as well as a decrease in bilirubin binding. An increase in ionic strength had a dramatic effect on the bilirubin-binding characteristics of modified proteins. The results suggest that the lysine residues of goat serum albumin modified in this study are not involved in bilirubin-albumin interaction.

The bioavailability and pharmacokinetics of mefenamic acid in alloxan-diabetic rabbits.

The bioavailability and pharmacokinetics of mefenamic acid was studied in alloxan-diabetic rabbits. Mefenamic acid in plasma was assayed by high performance liquid chromatography. A paired t-test for normal and alloxan treated rabbits revealed a significant decrease in all the bioavailability and disposition kinetic parameters of mefenamic acid during diabetes was observed in the present study. The altered bioavailability and disposition of mefenamic acid in the diabetic state will require adjustment of the dosage regimen prescribed for diabetics in a clinical setting.