Nutritional composition of larvae of mealworm (Tenebrio molitor L) and crickets (Gryllus assimilis) with potential usage in feed / [Composição nutricional de larvas de Tenebrio molitor e de grilos (Gryllus assimilis) com potencial de uso na alimentação animal]

Atualmente, tem-se discutido a utilização de insetos na alimentação animal devido ao seu potencial para substituir as fontes tradicionais de proteína utilizadas. O objetivo deste trabalho foi determinar a composição nutricional de larvas de Tenebrio molitor e de grilos do gênero Gryllus assimilis. Os teores de energia bruta (kcal/kg), proteína bruta (g/kg), extrato etéreo (g/kg), cinza (g/kg), FDN (g/kg) e FDA (g/kg) encontrados nas larvas de Tenebrio molitor foram de 7.188,6, 490,2, 335,4, 36,8, 71,8, e 64,0 respectivamente; nos grilos (Gryllus assimilis), os valores foram de 5.942,6, 541,3, 75,2, 49,1, 277,8, e 193,0 respectivamente. Os macros e microminerais quantificados foram fósforo, potássio, sódio, cálcio, magnésio, ferro, manganês, zinco e cobre. Nas larvas de Tenebrio molitor, os valores encontrados foram de 8,56 g/kg, 8,39 g/kg, 1,39 g/kg, 0,44 g/kg, 2,3 g/kg, 48,4 mg/kg, 15 mg/kg, 189 mg/kg e 18 mg/kg respectivamente. Para os grilos (Gryllus assimilis), os teores encontrados foram respectivamente de 8,30 g/kg, 11,6 g/kg, 1,10 g/kg, 3,88 g/kg, 0,82 g/kg, 96,8 mg/kg, 23,7 mg/kg, 18,3 mg/kg e 21,7 mg/kg. Larvas de Tenebrio molitor e grilos do gênero Gryllus assimilis podem ser alternativas para reduzir o uso de fontes de proteína vegetal na alimentação animal.(AU)

Oral feeding with probiotic Lactobacillus rhamnosus attenuates cigarette smoke-induced COPD in C57Bl/6 mice: Relevance to inflammatory markers in human bronchial epithelial cells.

COPD is a prevalent lung disease with significant impacts on public health. Affected airways exhibit pulmonary neutrophilia and consequent secretion of pro-inflammatory cytokines and proteases, which result in lung emphysema. Probiotics act as nonspecific modulators of the innate immune system that improve several inflammatory responses. To investigate the effect of Lactobacillus rhamnosus (Lr) on cigarette smoke (CS)-induced COPD C57Bl/6 mice were treated with Lr during the week before COPD induction and three times/week until euthanasia. For in vitro assays, murine bronchial epithelial cells as well as human bronchial epithelial cells exposed to cigarette smoke extract during 24 hours were treated with Lr 1 hour before CSE addition. Lr treatment attenuated the inflammatory response both in the airways and lung parenchyma, reducing inflammatory cells infiltration and the production of pro-inflammatory cytokines and chemokines. Also, Lr-treated mice presented with lower metalloproteases in lung tissue and lung remodeling. In parallel to the reduction in the expression of TLR2, TLR4, TLR9, STAT3, and NF-κB in lung tissue, Lr increased the levels of IL-10 as well as SOCS3 and TIMP1/2, indicating the induction of an anti-inflammatory environment. Similarly, murine bronchial epithelial cells as well as human bronchial epithelial cells (BEAS) exposed to CSE produced pro-inflammatory cytokines and chemokines, which were inhibited by Lr treatment in association with the production of anti-inflammatory molecules. Moreover, the presence of Lr also modulated the expression of COPD-associated transcription found into BALF of COPD mice group, i.e., Lr downregulated expression of NF-κB and STAT3, and inversely upregulated increased expression of SOCS3. Thus, our findings indicate that Lr modulates the balance between pro- and anti-inflammatory cytokines in human bronchial epithelial cells upon CS exposure and it can be a useful tool to improve the lung inflammatory response associated with COPD.

Oocyte quality and heat shock proteins in oocytes from bovine breeds adapted to the tropics under different conditions of environmental thermal stress.

In order to evaluate the influence of thermal stress on physiological parameters, and the oocyte quality of Girolando (n = 12) and adapted Pantaneira (n = 12) cattle, twelve sessions of ultrasound guided follicular aspiration (OPU) were performed, between January and November 2014 (during dry (May-September) and rainy season (October-April) in Brazil). The recovered cumulus-oocyte complexes (COCs) were selected and classified, according to quality, immediately after OPU. The oocytes were then stored in 3% paraformaldehyd before conducting immunofluorescence analysis under confocal microscopy to identify HSP70 and 90 proteins. Before each OPU session, the rectal temperature (RT) and respiratory frequency (RF) of each animal were measured. The black globe humidity index (BGHI) was calculated on the day of the OPUs and 90 days before each OPU session, and related to the thermal stress of the animals. The quality of oocytes from Girolando cattle, but not Pantaneira, showed a negative relationship with BGHI of 90 days before OPU. RT of both breeds did not exceed normal values for cattle below BGHI 95. BGHI variation on the day of OPU did not affect RF of the adapted Pantaneira breed (p = 0.3221). On the other hand, Girolando cattle showed a positive relationship between RF and BGHI (p = 0.0103). With increasing BGHI, the amount of HSP70 increased in Girolando oocytes, however, decreased in the Pantaneira breed. We have not observed a relationship between HSP 90 and BGHI, however Girolando cattle produced a greater amount of this protein in relation to the Pantaneira breed. In conclusion, higher BGHIs, 90 days before OPU session, negatively affect oocyte quality of Girolando cattle and positively affect oocyte quality of the Pantaneira breed. Higher BGHIs on the day of the OPU session negatively affected the respiratory frequency of the Girolando breed, and lead to a higher recruitment of HSP70 to protect oocyte maturation. The opposite pattern was observed for Pantaneira. In addition, Pantaneira cattle produced twice as much as HSP70 as Girolando cattle, suggesting that a natural higher production of this protein could be involved in the mechanisms of adaptation to heat conditions.

Efeito do estresse térmico calórico agudo e crônico sobre a qualidade oocitária de bovinos de raças adaptadas / Effect of acute and chronic caloric heat stress on oocyte quality of adapted breeds cattle

Nos trópicos, o uso de raças adaptadas tem sido uma estratégia para minimizar o efeito do estresse térmico calórico (ETC). No entanto, faltam informações que quantifiquem o estresse e o seu efeito sobre a reprodução dessas raças. O objetivo deste estudo foi avaliar a qualidade do oócito recuperado e alguns parâmetros fisiológicos indicadores de ETC em bovinos de raças adaptadas. Animais Bos taurus x Bos indicus (n=6) e Bos taurus (raça Pantaneira; n=12), localizados na região de transição entre o Cerrado e o Pantanal brasileiro, foram submetidos à aspiração folicular guiada por ultrassonografia (OPU) em diferentes condições climáticas. Foram realizadas oito sessões de OPU, com intervalo mínimo de sete dias e máximo de 54 dias entre as coletas. Para caracterização climática, foi realizado o cálculo do índice de temperatura e umidade (ITU). Foram quantificados os ITUs do dia da OPU, sete dias antes e 60 dias antes de cada sessão. Os parâmetros fisiológicos e a viabilidade oocitária de fêmeas das raças Girolando e Pantaneira não foram afetados negativamente por ITUs entre 72 e 78. O ETC crônico (60 dias) parece afetar a viabilidade oocitária de doadoras na raça Pantaneira quando ITU é superior a 75.(AU)

In tropical regions, the use of adapted breeds has been a strategy to minimize the effect of heat stress (HS) in cattle. However, information quantifying stress and its effect on reproduction of these breeds is lacking. The aim of this study was to evaluate the quality of the recovered oocyte and some physiological parameters that indicate HS in adapted breed. Bos taurus x Bos indicus (n=6) and Pantaneira (n=12) cows, located in the transition region between Cerrado and Brazilian Pantanal, underwent follicular aspiration guided by ultrasound (OPU) in different weather conditions. Eight sessions of OPU were carried out, with a minimum interval of 7 days and maximum 54 days between sessions. For weather characterization, the temperature and humidity index (THI) was calculated. THI of the day of OPU, 7 days before and 60 days before each session were calculated. The physiological parameters and oocyte viability of Girolando and Pantaneira cows were not negatively influenced under ITU between 72 and 78. The chronic HS (60 days)may affect the oocyte viability of Pantaneira donors when ITU is over 75.(AU)

Anchoring-driven spontaneous rotations in active gel droplets.

We study the dynamics of an active gel droplet with imposed orientational anchoring (normal or planar) at its surface. We find that if the activity is large enough droplets subject to strong anchoring spontaneously start to rotate, with the sense of rotation randomly selected by fluctuations. Contractile droplets rotate only for planar anchoring and extensile ones only for normal anchoring. This is because such a combination leads to a pair of stable elastic deformations which creates an active torque to power the rotation. Interestingly, under these conditions there is a conflict between the anchoring promoted thermodynamically and that favoured by activity. By tuning activity and anchoring strength, we find a wealth of qualitatively different droplet morphologies and spatiotemporal patterns, encompassing steady rotations, oscillations, and more irregular trajectories. The spontaneous rotations we observe are fundamentally different from previously reported instances of rotating defects in active fluids as they require the presence of strong enough anchoring and entail significant droplet shape deformations.

Higher visceral to subcutaneous fat ratio is associated with small intestinal bacterial overgrowth.

BACKGROUND AND AIMS: There is a lack of studies evaluating the association between small intestinal bacterial overgrowth (SIBO) and abdominal fat. The aim of this study was to evaluate whether visceral fat area (VFA), subcutaneous fat area (SFA) or visceral to subcutaneous fat ratio (VFA/SFA ratio) were associated with SIBO. METHODS AND RESULTS: In this case-control study, 152 eligible patients submitted to glucose hydrogen/methane breath test who also had computed tomography (CT) of the abdomen performed were included. Clinical and demographic information was obtained. VFA and SFA were measured using Image J software at lumbar 3 level on CT cross-sectional image of the 152 patients included in this study, 68 patients (44.7%) tested positive for SIBO. In the univariate analysis, the presence of SIBO was associated with older age (65.2 ± 1.5 vs. 59.3 ± 1.5, p = 0.007); type 2 diabetes mellitus (33.8% vs. 17.9%; p = 0.019); hypertension (63.2% vs. 39.3%; p = 0.003); metabolic syndrome (85.3% vs. 64.3%; p = 0.003); and higher VFA/SFA ratio (1.0 ± 0.1 vs. 0.7 ± 0.1; p < 0.001). In multivariate analysis, metabolic syndrome (odds ratio [OR]: 2.5; 95% confidence interval [CI]: 1.1-5.7; p = 0.035) and higher VFA/SFA ratio (OR: 3.3; 95% CI: 1.6-7.2; p = 0.002) remained independently associated with SIBO. CONCLUSION: The presence of SIBO was found to be associated with high VFA/SFA ratio measured from cross-sectional CT image.

Fish oils against Burkholderia and Pseudomonas aeruginosa: in vitro efficacy and their therapeutic and prophylactic effects on infected Galleria mellonella larvae.

AIM: This study investigates the antimicrobial effects of fish oil-based formulas rich in omega-3 fatty acids (free fatty acids, ethyl esters or triacylglycerols), against cystic fibrosis (CF) pathogens (Burkholderia cenocepacia K56-2 and Pseudomonas aeruginosa PAO1), often resistant to multiple antibiotics. METHODS AND RESULTS: The fish oils have shown antibacterial efficacy, although activity was highest for the one containing the fatty acid EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) in their free form (MIC value is 1·87% v/v for both pathogens). To test whether the fish oils could have a therapeutic and prophylactic potential in vivo, we assessed its efficacy using a Galleria mellonella caterpillar model of infection. The treatment of infected larvae with a single dose (7 h post infection) enhances the survival of larvae, being more pronounced with the free fatty acid form (EPAX 6000 FA). Moreover, we observed that the prophylactic food provision of the fish oil EPAX 6000 FA during 12 days prior to bacterial infection extended the life of the infected larvae. CONCLUSION: The fish oils, particularly in the free fatty acid form, are active in killing Burkholderia and Ps. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: The possibility of using fish oils for the treatment of bacterial infections in CF patients.

Disease-based modeling to predict fluid response in intensive care units.

OBJECTIVE: To compare general and disease-based modeling for fluid resuscitation and vasopressor use in intensive care units. METHODS: Retrospective cohort study involving 2944 adult medical and surgical intensive care unit (ICU) patients receiving fluid resuscitation. Within this cohort there were two disease-based groups, 802 patients with a diagnosis of pneumonia, and 143 patients with a diagnosis of pancreatitis. Fluid resuscitation either progressing to subsequent vasopressor administration or not was used as the primary outcome variable to compare general and disease-based modeling. RESULTS: Patients with pancreatitis, pneumonia and the general group all shared three common predictive features as core variables, arterial base excess, lactic acid and platelets. Patients with pneumonia also had non-invasive systolic blood pressure and white blood cells added to the core model, and pancreatitis patients additionally had temperature. Disease-based models had significantly higher values of AUC (p < 0.05) than the general group (0.82 ± 0.02 for pneumonia and 0.83 ± 0.03 for pancreatitis vs. 0.79 ± 0.02 for general patients). CONCLUSIONS: Disease-based predictive modeling reveals a different set of predictive variables compared to general modeling and improved performance. Our findings add support to the growing body of evidence advantaging disease specific predictive modeling.

Reducing unnecessary lab testing in the ICU with artificial intelligence.

OBJECTIVES: To reduce unnecessary lab testing by predicting when a proposed future lab test is likely to contribute information gain and thereby influence clinical management in patients with gastrointestinal bleeding. Recent studies have demonstrated that frequent laboratory testing does not necessarily relate to better outcomes. DESIGN: Data preprocessing, feature selection, and classification were performed and an artificial intelligence tool, fuzzy modeling, was used to identify lab tests that do not contribute an information gain. There were 11 input variables in total. Ten of these were derived from bedside monitor trends heart rate, oxygen saturation, respiratory rate, temperature, blood pressure, and urine collections, as well as infusion products and transfusions. The final input variable was a previous value from one of the eight lab tests being predicted: calcium, PTT, hematocrit, fibrinogen, lactate, platelets, INR and hemoglobin. The outcome for each test was a binary framework defining whether a test result contributed information gain or not. PATIENTS: Predictive modeling was applied to recognize unnecessary lab tests in a real world ICU database extract comprising 746 patients with gastrointestinal bleeding. MAIN RESULTS: Classification accuracy of necessary and unnecessary lab tests of greater than 80% was achieved for all eight lab tests. Sensitivity and specificity were satisfactory for all the outcomes. An average reduction of 50% of the lab tests was obtained. This is an improvement from previously reported similar studies with average performance 37% by [1-3]. CONCLUSIONS: Reducing frequent lab testing and the potential clinical and financial implications are an important issue in intensive care. In this work we present an artificial intelligence method to predict the benefit of proposed future laboratory tests. Using ICU data from 746 patients with gastrointestinal bleeding, and eleven measurements, we demonstrate high accuracy in predicting the likely information to be gained from proposed future lab testing for eight common GI related lab tests. Future work will explore applications of this approach to a range of underlying medical conditions and laboratory tests.

Outbreak of acute gastroenteritis in young children with death due to rotavirus genotype G9 in Rio Branco, Brazilian Amazon region, 2005.

BACKGROUND: An epidemic of acute gastroenteritis occurred in Rio Branco City, Acre State, in Brazil's Amazon region in 2005. An investigation was conducted to confirm the etiology and identify possible risk factors for death. METHODS: Rio Branco municipality surveillance data for the period May to October 2005 were reviewed. In a case-control study, children who died following acute gastroenteritis were compared to age-matched controls with acute gastroenteritis who survived. Rotavirus A (RV-A) was investigated in 799 stool samples and genotyped by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The cumulative incidence of diarrhea in children aged <5 years was 21%. A fatal outcome was significantly associated with uncovered household water storage containers. RV-A was identified in 88% of samples and G9 was the prevalent genotype (71%). CONCLUSIONS: Oral rehydration solution and boiling or chlorinating drinking water likely limited mortality. This epidemic was caused by RV-A genotype G9. After the outbreak, a rotavirus vaccine was introduced into the official childhood immunization schedule in Brazil.

Evaluating a bioremediation tool for atrazine contaminated soils in open soil microcosms: the effectiveness of bioaugmentation and biostimulation approaches.

A previously developed potential cleanup tool for atrazine contaminated soils was evaluated in larger open soil microcosms for optimization under more realistic conditions, using a natural crop soil spiked with an atrazine commercial formulation (Atrazerba FL). The doses used were 20x or 200x higher than the recommended dose (RD) for an agricultural application, mimicking over-use or spill situations. Pseudomonas sp. strain ADP was used for bioaugmentation (around 10(7) or 10(8) viable cells g(-1) of soil) and citrate for biostimulation (up to 4.8 mg g(-1) of soil). Bioremediation treatments providing fastest and higher atrazine biodegradation proved to differ according to the initial level of soil contamination. For 20x RD of Atrazerba FL, a unique inoculation with Pseudomonas sp. ADP (9 +/- 1 x 10(7) CFU g(-1)) resulted in rapid atrazine removal (99% of the initial 7.2 +/- 1.6 microg g(-1) after 8d), independent of citrate. For 200x RD, an inoculation with the atrazine-degrading bacteria (8.5 +/- 0.5 x 10(7) CFU g(-1)) supplemented with citrate amendment (2.4 mg g(-1)) resulted in improved biodegradation (87%) compared with bioaugmentation alone (79%), even though 7.8 +/- 2.1 microg of atrazine g(-1) still remained in the soil after 1 wk. However, the same amount of inoculum, distributed over three successive inoculations and combined with citrate, increased Pseudomonas sp. ADP survival and atrazine biodegradation (to 98%, in 1 wk). We suggest that this bioremediation tool may be valuable for efficient removal of atrazine from contaminated field soils thus minimizing atrazine and its chlorinated derivatives from reaching water compartments.

Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate.

The cloning, expression, purification, crystallization and preliminary crystallographic analysis of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate are reported. Diffraction data sets were obtained from seven crystal forms in five different space groups, with highest resolutions ranging from 4.20 to 2.65 A. The phase problem was solved for a P2(1) crystal form using multiple isomorphous replacement with anomalous scattering from an osmium derivative and a SeMet derivative. The best native crystal in space group P2(1) has unit-cell parameters a = 105.5, b = 85.7, c = 151.8 A, beta = 105.2 degrees . Model building and refinement are currently under way.

Reverse transcription-polymerase chain reaction assay for rabies virus detection

Este estudo teve por objetivo implantar um protocolo de amplificação genômica, precedida de transcrição reversa (RT-PCR) para o gene da nucleoproteína do vírus da raiva, para a utilização dessa metodologia em laboratórios onde são realizadas investigações para a detecção do vírus rábico. Foram utilizadas 50 amostras de tecido encefálico de animais (44 bovinos, 5 eqüinos e 1 quiróptero) oriundos do Estado do Rio de Janeiro, positivos por imunofluorescência direta e/ou prova biológica para o vírus rábico. A extração do RNA foi feita a partir da suspensão a 10 por cento em PBS pH7,2 do tecido encefálico utilizando-se a metodologia de TRIzolTM (Life Technologies) e o protocolo de RT-PCR descrito por Heaton et al. (1997), incluindo algumas modificações. Dentre as 50 amostras analisadas, 50 foram positivas pela prova biológica e pela RT-PCR e destas, 49 foram positivas pela imunofluorescência direta. Estes resultados demonstram ser este protocolo de RT-PCR uma metodologia sensível, específica, rápida e extremamente valiosa, podendo ser utilizada como rotina em laboratórios que trabalham no diagnóstico de vírus rábico.

Cloning and sequence analysis of the ces10 gene encoding a Sphingomonas paucimobilis esterase.

The ces10 gene of the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461 was cloned and sequenced. Multi-sequence alignment of the deduced protein indicated that Ces10 belongs to the serine hydrolase family with a potential catalytic triad comprising Ser(153) (within the G-X-S-X-G consensus sequence), His(75) and Asp(125). The mixed block results obtained following pattern search and the low identities detected in a BLAST analysis indicate that Ces10 is significantly different from other characterised bacterial esterases/lipases. Nevertheless, the Ces10 amino acid sequence showed 45% similarity with Rhodococcus sp. heroin esterase and 48% with Bacillus subtilis p-nitrobenzyl esterase. Ces10, with a predicted molecular mass of 30,641 Da, was overproduced in Escherichia coli and purified to homogeneity in a histidine-tagged form. Enzyme assays using p-nitrophenyl-esters (p-NP-esters) with different acyl chain-lengths as the substrate confirmed the anticipated esterase activity. Ces10 exhibited a marked preference for short-chain fatty acids, yielding the highest activity with p-NP-propionate (optimal pH 7.4, optimal temperature 37 degrees C).

Characterization of the ugpG gene encoding a UDP-glucose pyrophosphorylase from the gellan gum producer Sphingomonas paucimobilis ATCC 31461.

The ugpGgene, which codes for a UDP-glucose pyrophosphorylase (UGP) (or glucose-1-phosphate uridylyltransferase; EC 2.7.7.9) in Sphingomonas paucimobilis ATCC 31461, was cloned and sequenced. This industrial strain produces the exopolysaccharide gellan, a new commercial gelling agent, and the ugpG gene may convert glucose-1-phosphate into UDP-glucose in the gellan biosynthetic pathway. The ugpG gene is capable of restoring the capacity of an Escherichia coli galU mutant to grow on galactose by functional complementation of its deficiency for UDP-glucose pyrophosphorylase activity. As expected, the predicted gene product shows strong homology to UDP-glucose pyrophosphorylases from several bacterial species. The N-terminal region of UgpG exhibits the motif GXGTRXLPXTK, which is highly conserved among bacterial XDP-sugar pyrophosphorylases, and a lysine residue (K(192)) is located within a VEKP motif predicted to be essential for substrate binding or catalysis. UgpG was purified to homogeneity as a heterologous fusion protein from crude cell extracts prepared from IPTG-induced cells of E. coli, using affinity chromatography. Under denaturing conditions, the fusion protein S-UgpG-His(6) migrated with an estimated molecular mass of 36 kDa [corresponding to the predicted molecular mass of native UgpG (31.2 kDa) plus 5 kDa for the S and histidine tags). Kinetic analysis of UgpG in the reverse reaction (pyrophosphorolysis) showed a typical Michaelis-Menten substrate saturation pattern. The apparent K(m) and V(max) values estimated for UDP-glucose were 7.5 microM and 1275 micromol/min/g.

Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: genes, enzymes and exopolysaccharide production engineering.

The commercial gelling agent, gellan, is an extracellular polysaccharide (EPS) produced by Sphingomonas paucimobilis ATCC 31461. In recent years, significant progress in understanding the relationship between gellan structure and properties and elucidation of the biosynthesis and engineering of this recent product of biotechnology has been made. This review focuses on recent advances in this field. Emphasis is given to identification and characterization of genes and enzymes involved, or predicted to be involved, in the gellan biosynthetic pathway, at the level of synthesis of sugar-activated precursors, of the repeat unit assembly and of gellan polymerization and export. Identification of several genes, biochemical characterization of the encoded enzymes and elucidation of crucial steps of the gellan pathway indicate that possibilities now exist for exerting control over gellan production at any of the three levels of its biosynthesis. However, a better knowledge of the poorly understood steps and of the bottlenecks and regulation of the pathway, the characterization of the composition, structure and functional properties of gellan-like polymers produced either by the industrial strain under different culture conditions or by mutants are still required for eventual success of the metabolic engineering of gellan production.

Biochemical characterization of the beta-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461.

Biosynthesis of bacterial polysaccharide-repeat units proceeds by sequential transfer of sugars, from the appropriate sugar donor to an activated lipid carrier, by committed glycosyltransferases (GTs). Few studies on the mechanism of action for this type of GT are available. Sphingomonas paucimobilis A.T.C.C. 31461 produces the industrially important polysaccharide gellan gum. We have cloned the gelK gene from S. paucimobilis A.T.C.C. 31461. GelK belongs to family 1 of the GT classification [Campbell, Davies, Bulone, Henrissat (1997) Biochem. J. 326, 929-939]. Sequence similarity studies suggest that GelK consists of two protein modules corresponding to the -NH(2) and -CO(2)H halves, the latter possibly harbouring the GT activity. The gelK gene and the open reading frames coding for the -NH(2) (GelK(NH2)) and -CO(2)H (GelK(COOH)) halves were overexpressed in Escherichia coli. GelK and GelK(NH2) were present in both the soluble and membrane fractions of E. coli, whereas GelK(COOH) was only present in the soluble fraction. GelK catalysed the transfer of [(14)C]glucuronic acid from UDP-[(14)C]glucuronic acid into a glycolipid extracted from S. paucimobilis or E. coli, even in the presence of EDTA, and the radioactive sugar was released from the glycolipid by beta-1,4-glucuronidase. GelK was not able to use synthetic glucosyl derivatives as acceptors, indicating that the PP(i)-lipid moiety is needed for enzymic activity. Recombinant GelK(NH2) and GelK(COOH) did not show detectable activity. Based on the biochemical characteristics of GelK and on sequence similarities with N-acetylglucosaminyltransferase, we propose that GT families 1 and 28 form a superfamily.

Rotavirus genotypes P[4]G9, P[6]G9, and P[8]G9 in hospitalized children with acute gastroenteritis in Rio de Janeiro, Brazil.

Fifty-three rotavirus-positive fecal specimens from children with diarrhea admitted to a Rio de Janeiro, Brazil, children's hospital between January 1997 and December 1998 were characterized for P and G types by using reverse transcription-PCR. Genotype P[4]G2 accounted for 21% of isolates, while uncommon genotypes P[8]G9, P[6]G9, and P[4]G9 accounted for 13% of the isolates.

Identification of the Pseudomonas aeruginosa glmM gene, encoding phosphoglucosamine mutase.

A search for a potential algC homologue within the Pseudomonas aeruginosa PAO1 genome database has revealed an open reading frame (ORF) of unknown function, ORF540 in contig 54 (July 1999 Pseudomonas genome release), that theoretically coded for a 445-amino-acid-residue polypeptide (I. M. Tavares, J. H. Leitão, A. M. Fialho, and I. Sá-Correia, Res. Microbiol. 150:105-116, 1999). The product of this gene is here identified as the phosphoglucosamine mutase (GlmM) which catalyzes the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the formation of the cell wall precursor UDP-N-acetylglucosamine. The P. aeruginosa gene has been cloned into expression vectors and shown to restore normal peptidoglycan biosynthesis and cell growth of a glmM Escherichia coli mutant strain. The GlmM enzyme from P. aeruginosa has been overproduced to high levels and purified to homogeneity in a six-histidine-tagged form. Beside its phosphoglucosamine mutase activity, the P. aeruginosa enzyme is shown to exhibit phosphomannomutase and phosphoglucomutase activities, which represent about 20 and 2% of its GlmM activity, respectively.

Identification of the pgmG gene, encoding a bifunctional protein with phosphoglucomutase and phosphomannomutase activities, in the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461.

The pgmG gene of Sphingomonas paucimobilis ATCC 31461, the industrial gellan gum-producing strain, was cloned and sequenced. It encodes a 50,059-Da polypeptide that has phosphoglucomutase (PGM) and phosphomannomutase (PMM) activities and is 37 to 59% identical to other bifunctional proteins with PGM and PMM activities from gram-negative species, including Pseudomonas aeruginosa AlgC. Purified PgmG protein showed a marked preference for glucose-1-phosphate (G1P); the catalytic efficiency was about 50-fold higher for G1P than it was for mannose-1-phosphate (M1P). The estimated apparent K(m) values for G1P and M1P were high, 0.33 and 1.27 mM, respectively. The pgmG gene allowed the recovery of alginate biosynthetic ability in a P. aeruginosa mutant with a defective algC gene. This result indicates that PgmG protein can convert mannose-6-phosphate into M1P in the initial steps of alginate biosynthesis and, together with other results, suggests that PgmG may convert glucose-6-phosphate into G1P in the gellan pathway.