Isolation and characterisation of a novel rabbit sulfotransferase isoform belonging to the SULT1A subfamily

Sulfotransferases (SULTs) catalyse the sulfonation of both endogenous and exogenous compounds including hormones, catecholamines. drugs and xenobiotics. While in most occasions, sulfonation is a detoxication pathway. in the case of certain drugs and carcinogens. it leads to metabolic activation. Since, the rabbit has been extensively used for both pharmacological and toxicological studies, the purpose of this study was to further characterise the sulfotransferase system of this animal. In the present study, a novel sulfotransferase isoform (GenBank Accession no. AF360872) was isolated from a rabbit liver cDNA lambdaZAP 11 library. The full-length sequence of the clone was 1138 bp long and contained a coding region of 888 bp encoding a cytosolic protein of 295 amino acids (deduced molecular weight 34,193 Da). The amino acid sequence of this novel SULT isoform showed >70% identity with members of the SULT1A subfamily of sulfotransferases from other species. Upon expression of the encoded rabbit sulfotransferase in Escherchia coli (E. coli), it was shown that the enzyme was capable of sulfonating both p-nitrophenot (K-m and V-max values of 0.15 muM and 897.5 nmol/min/mg protein. respectively) and dopamine (K-m and V-max values of 175.3 muM and 151.1 nmol/min/mg protein, respectively). Based on the sequence data obtained and substrate specificity, this new rabbit sulfotransferase was named rabSULT1A1. Immunoblotting was used to demonstrate that rabSULT1A1 protein is expressed in liver, duodenum, jejunum, ileum, colon and recturm. (C) 2002 Elsevier Science Ltd. All rights reserved.

Phylogeny of the lice (Insecta, Phthiraptera) inferred from small subunit rRNA

There has been much argument about the phylogenetic relationships of the four suborders of lice (Insecta: Phthiraptera). Lyal's study of the morphology of lice indicated that chewing/biting lice (Mallophaga) are paraphyletic with respect to sucking lice (Anoplura). To test this hypothesis we inferred the phylogeny of 33 species of lice from small subunit (SSU) rRNA sequences (18S rRNA). Liposcelis sp. from the Liposcelididae (Psocoptera) was used for outgroup reference. Phylogenetic relationships among the four suborders of lice inferred from these sequences were the same as those inferred from morphology. The Amblycera is apparently the sister-group to all other lice whereas the Rhynchophthirina is apparently sister to the Anoplura; these two suborders are sister to the Ischnocera, i.e. (Amblycera (Ischnocera (Anoplura, Rhynchophthirina))). Thus, the Mallophaga (Amblycera, Ischnocera, Rhynchophthirina) is apparently paraphyletic with respect to the Anoplura. Our analyses also provide evidence that: (i) each of the three suborders of lice that are well represented in our study (the Amblycera, Ischnocera, and Anoplura) are monophyletic; (ii) the Boopiidae is monophyletic; (iii) the genera Heterodoxus and Latumcephalum (Boopiidae) are more closely related to one another than either is to the genus Boopia (also Boopiidae); (iv) the Ricinidae and Laemobothridae may be sister-taxa; (v) the Philopteridae may be paraphyletic with respect to the Trichodectidae; (vi) the genera Pediculus and Pthirus are more closely related to each other than either is to the genus Pedicinus ; and (vii) in contrast to published data for mitochondrial genes, the rates of nucleotide substitution in the SSU rRNA of lice are not higher than those of other insects, nor do substitution rates in the suborders differ substantially from one another.

Biochemical characterization of two mutants of human pyruvate dehydrogenase, F205L and T231A of the E1 alpha subunit

Mutations in the E1alpha subunit of the pyruvate dehydrogenase multienzyme complex may result in congenital lactic acidosis, but little is known about the consequences of these mutations at the enzymatic level. Here we characterize two mutants (F205L and T231A) of human pyruvate dehydrogenase in vitro, using the enzyme expressed in Escherichia coli. Wild-type and mutant proteins were purified successfully and their kinetic parameters were measured. F205L shows impaired binding of the thiamin diphosphate cofactor, which may explain why patients carrying this mutation respond to high-dose vitamin B-1 therapy. T231A has very low activity and a greatly elevated K-m for pyruvate, and this combination of effects would be expected to result in severe lactic acidosis. The results lead to a better understanding of the consequences of these mutations on the functional and structural properties of the enzyme, which may lead to improved therapies for patients carrying these mutations.

Genetic characterization of pilin glycosylation and phase variation in Neisseria meningitidis

Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host. Pili are post-translationally modified by addition of either an O-linked trisaccharide, Gal (beta1-4) Gal (alpha1-3) 2,4-diacetamido-2,4,6-trideoxyhexose or an O-linked disaccharide Gal (alpha1,3) GlcNAc. The role of these structures in meningococcal pathogenesis has not been resolved. In previous studies we identified two separate genetic loci, pglA and pglBCD, involved in pilin glycosylation. Putative functions have been allocated to these genes; however, there are not enough genes to account for the complete biosynthesis of the described structures, suggesting additional genes remain to be identified. In addition, it is not known why some strains express the trisaccharide structure and some the disaccharide structure. In order to find additional genes involved in the biosynthesis. of these structures, we used the recently published group A strain Z2491 and group B strain MC58 Neisseria meningitidis genomes and the unfinished Neisseria meningitidis group C strain FAM18 and Neisseria gonorrhoeae strain FA1090 genomes to identify novel genes involved in pilin glycosylation, based on homology to known oligosaccharide biosynthetic genes. We identified a new gene involved in pilin glycosylation designated pglE and examined four additional genes pgIB/B2, pglF, pglG and pglH. A strain survey revealed that pglE and pglF were present in each strain examined. The pglG, pglH and pgIB2 polymorphisms were not found in strain C311#3 but were present in a large number of clinical isolates. Insertional mutations were constructed in pglE and pglF in N. meningitidis strain C311#3, a strain with well-defined lipopolysaccharide (LPS) and pilin-linked glycan structures. Increased gel migration of the pilin subunit molecules of pglE and pglF mutants was observed by Western analysis, indicating truncation of the trisaccharide structure. Antisera specific for the C311#3 trisaccharide failed to react with pilin from these pglE and pglF mutants. GC-MS analysis of the sugar composition of the pglE mutant showed a reduction in galactose compared with C311#3 wild type. Analysis of amino acid sequence homologies has suggested specific roles for pglE and pglF in the biosynthesis of the trisaccharide structure. Further, we present evidence that pglE, which contains heptanucleotide repeats, is responsible for the phase variation between trisaccharide and disaccharide structures in strain C311#3 and other strains. We also present evidence that pglG, pglH and pgIB2 are potentially phase variable.

Avaliação dos níveis séricos das vitaminas A, E, C e B2, de carotenóides e zinco, em idosos hospitalizados

Foram verificados os níveis séricos de zinco, carotenóides e vitaminas A, E, C, B2 em todos os idosos (n = 202) internados nas diversas enfermarias do hospital estudado, no período de fevereiro de 1986 a outubro de 1988. Foram estudados 130 homens e 72 mulheres que apresentaram média de idade de 67,8 anos, com variação entre 60 a 88. A percentagem de níveis séricos deficitários foi de 59,5 para o zinco, 56,5% para a vitamina C, 34,5% para a vitamina B2, 26% para a vitamina E, 13,2% para a vitamina A e 6,8% para os carotenóides. Os idosos portadores de leucoses, magaesôfogo, doença pulmonar obstrutiva crônica e insuficiência cardíaca congestiva constituíram-se no grupo de pacientes com grande prevalência de estado deficitário de zinco e das vitaminas estudadas, resultados que mostram a importância de se investigar as deficiências desses micronutrientes e dão subsídios para a abordagem terapêutica mais racional do paciente idoso internado.

Development of fire-resistant (B1 & B2) winter 1 KPU foams applied by gun or straw adapter and optimization of a gun applied winter 1 KPU foam

A realização do presente trabalho teve como principais objectivos o desenvolvimento de espumas de poliuretano de um componente com propriedades de resistência à chama superiores (B1 & B2), aplicadas por pistola ou por adaptador/tubo e a optimização de uma espuma de poliuretano de um componente de inverno aplicada por pistola. Todo o trabalho desenvolvido está dividido em dois projectos distintos: i. O primeiro projecto consistiu em desenvolver espumas de um componente com propriedades de resistência à chama (classificadas como B1 e B2 de acordo com a norma alemã DIN 4102), aplicadas por pistola (GWB1 e GWB2) ou por adaptador/tubo (AWB), utilizando polióis poliésteres aromáticos modificados e aditivos retardantes de chama halogenados. Estas espumas deveriam apresentar também propriedades aceitáveis a baixas temperaturas. Após realizar várias formulações foi possível desenvolver uma espuma AWB2 com apenas 3,3% de poliol poliéster no pré-polímero e com propriedades equivalentes às da melhor espuma comercial mesmo a 5/-10 (temperatura da lata/cura da espuma em °C) e também com uma altura de chama de apenas 11 cm. A partir de duas formulações (AWB2) que passaram o Teste B2, foram obtidas também, uma espuma GWB2 e outra GWB1 com propriedades equivalentes às da melhor espuma da concorrência a -10/-10 e a 23/5, respectivamente, embora não tenham sido submetidas ao teste B2 e B1 após as modificações efectuadas. ii. O segundo projecto consistiu em optimizar uma espuma de poliuretano de um componente de inverno aplicada por pistola (GWB3). A espuma inicial tinha problemas de glass bubbles quando esta era dispensada a partir de uma lata cheia, sendo necessário ultrapassar este problema. Este problema foi resolvido diminuindo a razão de GPL/DME através do aumento da percentagem em volume de DME no pré-polímero para 14% no entanto, a estabilidade dimensional piorou um pouco. O reagente FCA 400 foi removido da formulação anterior (6925) numa tentativa de diminuir o custo da espuma, obtendo-se uma espuma aceitável a 23/23 e a 5/5, com uma redução de 4% no custo da produção e com uma redução de 5,5% no custo por litro de espuma dispensada, quando comparada com a sua antecessora. Por último, foi avaliada a influência da concentração de diferentes surfactantes na formulação 6925, verificando-se o melhoramento da estrutura celular da espuma para concentrções mais elevadas de surfactante, sendo este efeito mais notório a temperaturas mais baixas (5/5). Dos surfactantes estudados, o B 8871 mostrou o melhor desempenho a 5/5 com a concentração mais baixa, sendo portanto o melhor surfactante, enquanto o Struksilon 8003 demonstrou ser o menos adequado para esta formulação específica, apresentando piores resultados globais. Pode-se ainda acrescentar que os surfactantes L-5351, L-5352 e B 8526 também não são adequados para esta formulação uma vez que as espumas resultantes apresentam cell collapse, especialmente a 5/5. No caso dos surfactantes L-5351 e L-5352, esta propriedade piora com concentrações mais elevadas. Em cada projecto foram também efectuados testes de benchmark em determinadas espumas comerciais com o principal objectivo de comparar todos os resultados das espumas desenvolvidas, em ambos os projectos, com espumas da concorrência.

Caracterização do papel da adrenalina na maturação dos adrenorreceptores B2

As respostas pós-juncionais mediadas por adrenorreceptores β2 (ARβ2), responsáveis pelo relaxamento do músculo liso, na veia safena do cão, estão ausentes à nascença. Pelo contrário, no rato recém-nascido já se verifica a estimulação da adenilil ciclase pela activação dos ARβ2. Não existem ainda estudos no coelho recém-nascido. O principal objectivo deste trabalho é avaliar as respostas pós-juncionais mediadas pelos ARβ2 em coelhos recém-nascidos e jovens e relacionar essas respostas com a adrenalina produzida nas glândulas supra-renais. Traçaram-se curvas de dose-resposta à isoprenalina (agonista β) utilizando-se anéis de aorta montados em banho de órgãos isolados ligado a um transdutor de força isométrica. As catecolaminas das supra-renais foram quantificadas por RP-HPLC-ED. Em aortas pré-contraídas com fenilefrina (agonista α1), a isoprenalina causou relaxamento total apenas em coelhos recém-nascidos (n=10). O relaxamento máximo nos coelhos jovens foi de 21±4% (n=23). A potência da isoprenalina foi maior nos recém-nascidos (EC50=1.15×10-8±7.2×10-10 M, n=10) do que nos coelhos jovens (EC50=1.29×10-7 ±4.7×10-9 M, n=23). O relaxamento máximo com isoprenalina, em aortas pré-contraídas com prostaglandina F2α (PGF2α), no grupo de coelhos recém-nascidos foi de 95±3.6% (n=16). O relaxamento máximo nos coelhos jovens foi de 43.7±8.6% (n=9). Na pré-contracção com PGF2α a potência da isoprenalina registou-se maior nos recémnascidos (EC50=9.59×10-9±4.0×10-10 M, n=16) do que nos coelhos jovens (EC50=2.13×10- 8±3.8×10-9 M, n=9), estando concordante com os resultados da pré-contracção com fenilefrina. Nas supra-renais dos recém-nascidos, o conteúdo de noradrenalina foi de 586±128 nmol/mg e da adrenalina foi de 1915±356 nmol/mg (n=4) e nos coelhos jovens foi de 112±12 nmol/mg e de 3644±403 nmol/mg (n=6), respectivamente. As respostas mediadas por ARβ2 no coelho desenvolvem-se mais cedo do que no cão, pois já estão presentes no nascimento. Tal como no rato, no coelho a adrenalina é já a catecolamina em maior quantidade à nascença, enquanto no cão é vestigial. Há uma relação temporal entre a síntese da adrenalina, a única catecolamina biogénica com alta afinidade para os ARβ2 e a maturação das respostas pós-juncionais mediadas por esses receptores. Um protocolo para experiências futuras destinadas a testar esta hipótese, com base no knockdown da Feniletanolamina-N-metiltransferase por RNAi foi elaborado e incluído neste documento.

The Tetrahymena chaperonin subunit CCT eta gene is coexpressed with CCT gamma gene during cilia biogenesis and cell sexual reproduction

We report here the cloning and the characterization of the T. pyriformis CCT eta gene (TpCCT eta) and also a partial sequence of the corresponding T. thermophila gene (TtCCT eta). The TpCCt eta gene encodes a protein sharing a 60.3% identity with the mouse CCT eta. We have studied the expression of these genes in Tetrahymena exponentially growing cells, cells regenerating their cilia for different periods and during different stages of the cell sexual reproduction. These genes have similar patterns of expression to those of the previously identified TpCCt gamma gene. Indeed, the Tetrahymena CCT eta and CCT gamma genes are up-regulated at 60-120 min of cilia recovery, and in conjugation when vegetative growth was resumed and cell division took place. Our results seem to indicate that both CCT subunits play an important role in the biogenesis of the newly synthesized cilia of Tetrahymena and during its cell division.

An overview of the higher level classification of Pucciniomycotina based on combined analyses of nuclear large and small subunit rDNA sequences

Mycologia, Vol. 98, nº6

Novel FGFR1 Mutations in Kallmann Syndrome and Normosmic Idiopathic Hypogonadotropic Hypogonadism: Evidence for the Involvement of an Alternatively Spliced Isoform

OBJECTIVE: To determine the prevalence of fibroblast growth factor receptor 1 (FGFR1) mutations and their predicted functional consequences in patients with idiopathic hypogonadotropic hypogonadism (IHH). DESIGN: Cross-sectional study. SETTING: Multicentric. PATIENT(S): Fifty unrelated patients with IHH (21 with Kallmann syndrome and 29 with normosmic IHH). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Patients were screened for mutations in FGFR1. The functional consequences of mutations were predicted by in silico structural and conservation analysis. RESULT(S): Heterozygous FGFR1 mutations were identified in six (12%) kindreds. These consisted of frameshift mutations (p.Pro33-Alafs*17 and p.Tyr654*) and missense mutations in the signal peptide (p.Trp4Cys), in the D1 extracellular domain (p.Ser96Cys) and in the cytoplasmic tyrosine kinase domain (p.Met719Val). A missense mutation was identified in the alternatively spliced exon 8A (p.Ala353Thr) that exclusively affects the D3 extracellular domain of FGFR1 isoform IIIb. Structure-based and sequence-based prediction methods and the absence of these variants in 200 normal controls were all consistent with a critical role for the mutations in the activity of the receptor. Oligogenic inheritance (FGFR1/CHD7/PROKR2) was found in one patient. CONCLUSION(S): Two FGFR1 isoforms, IIIb and IIIc, result from alternative splicing of exons 8A and 8B, respectively. Loss-of-function of isoform IIIc is a cause of IHH, whereas isoform IIIb is thought to be redundant. Ours is the first report of normosmic IHH associated with a mutation in the alternatively spliced exon 8A and suggests that this disorder can be caused by defects in either of the two alternatively spliced FGFR1 isoforms.

Efeitos do carvedilol (bloqueador b1,b2,a1) na insuficiência cardíaca refratária

OBJETIVO: Os efeitos dos beta-bloqueadores na insuficiência cardíaca (IC) refratária não têm sido adequadamente estudados. Investigamos os efeitos do carvedilol (bloqueador b1,b2,a1) nos sintomas e na função ventricular de portadores de IC refratária. MÉTODOS: Foram estudados 21 pacientes, idade média de 56±10 anos, 9 em classe funcional (CF) IV, e 12 em CF III intermitente com IV. A dose inicial de carvedilol foi de 6,25mg e, se tolerada, aumentada progressivamente. A dose média final foi 42±11mg. Os pacientes foram submetidos a avaliações clínicas e eletrocardiográficas seriadas. Realizaram-se, antes e com 196±60 dias de evolução, ecocardiograma e ventriculografia radioisotópica. RESULTADOS: O medicamento foi tolerado em 16 (76%) pacientes. Um paciente está em fase de titulação em CF II. Com 196±60 dias de evolução observaram-se 8 pacientes em CF I e 7 em II; redução da freqüência cardíaca de 96±15 para 67±10bpm (p<0,0001); redução do diâmetro diastólico final de ventrículo esquerdo (VE) de 73±13 para 66±12mm (ecocardiograma) (p<0,009); e aumento da fração de ejeção de VE de 0,21±0,06 para 0,34±0,12 (p<0,0003). CONCLUSÃO: O carvedilol devido aos seus efeitos benéficos na função ventricular, remodelamento e CF é, se tolerado, uma potencial alternativa terapêutica no tratamento medicamentoso da IC refratária. Entretanto, estudos adicionais são necessários para definição do efeito a longo prazo neste específico subgrupo de pacientes.

Protease-activated receptor (PAR)-1 and PAR-2 protect rat astrocytes from apoptotic cell death via differentially regulating JNK isoform-specific release of the chemokine GRO/CINC-1 : two novel protective pathways in brain

Protease-activated receptor; c-Jun N-terminal kinase (JNK); thrombin; neuroprotection; siRNA

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop.

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.

The fourth transmembrane segment of the Na,K-ATPase alpha subunit: a systematic mutagenesis study.

The Na,K-ATPase is a major ion-motive ATPase of the P-type family responsible for many aspects of cellular homeostasis. To determine the structure of the pathway for cations across the transmembrane portion of the Na,K-ATPase, we mutated 24 residues of the fourth transmembrane segment into cysteine and studied their function and accessibility by exposure to the sulfhydryl reagent 2-aminoethyl-methanethiosulfonate. Accessibility was also examined after treatment with palytoxin, which transforms the Na,K-pump into a cation channel. Of the 24 tested cysteine mutants, seven had no or a much reduced transport function. In particular cysteine mutants of the highly conserved "PEG" motif had a strongly reduced activity. However, most of the non-functional mutants could still be transformed by palytoxin as well as all of the functional mutants. Accessibility, determined as a 2-aminoethyl-methanethiosulfonate-induced reduction of the transport activity or as inhibition of the membrane conductance after palytoxin treatment, was observed for the following positions: Phe(323), Ile(322), Gly(326), Ala(330), Pro(333), Glu(334), and Gly(335). In accordance with a structural model of the Na,K-ATPase obtained by homology modeling with the two published structures of sarcoplasmic and endoplasmic reticulum calcium ATPase (Protein Data Bank codes 1EUL and 1IWO), the results suggest the presence of a cation pathway along the side of the fourth transmembrane segment that faces the space between transmembrane segments 5 and 6. The phenylalanine residue in position 323 has a critical position at the outer mouth of the cation pathway. The residues thought to form the cation binding site II ((333)PEGL) are also part of the accessible wall of the cation pathway opened by palytoxin through the Na,K-pump.