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1.
Braz. j. biol ; 83: e246825, 2023. tab
Artigo em Inglês | LILACS-Express | MEDLINE, LILACSEXPRESS | ID: biblio-1285634

RESUMO

Abstract A study was conducted to evaluate the effect of Piper nigrum (black pepper) leaf extract on on the growth performance, proximate composition, hematological parameters, and immune response of Labeo rohita fingerlings with an average weight of 22.14 ± 0.98g. Aftrer acclimation for two weeks, fish (n=25) were randomly selected and placed in four glass aquaria (T0, T1, T2 and T3) at constant water temperature (30.0 ± 1.0 °C), pH (7.50 ± 0.5) and total hardness (200 ± 2.0 mgL,-1) for a period of 12 weeks, with three replicates each. Fish were fed with P. nigrum leaf extract supplemented feed @ 0.0%, 1.0%, 2.0% and 3.0% in T0, T1,T2 and T3, respectively. At the end of experiment, five fish were randomly selected from each aquaria for proximate composition, gut and skin microbial load, hematological parameters. Total proteins, albumins, and globulins were also recorded to evaluate immunological memory. The result revealed that fish in T2 showed better growth performance with an average weight gain of 56.11 ± 0.51 g. Thus, it had been concluded that Piper nigrum, a medicinal plant, can also be used to enhance the growth performance and immune response of Labeo rohita as attractive alternatives against antibiotics and vaccines and has shown no negative side effects on fish health as well as on its environment.


Resumo Um estudo foi conduzido para avaliar o efeito do extrato da folha de Piper nigrum (pimenta-do-reino) sobre o desempenho de crescimento, composição centesimal, parâmetros hematológicos e resposta imune de alevinos de Labeo rohita com peso médio de 22,14 ± 0,98g. Após aclimatação por duas semanas, os peixes (n = 25) foram selecionados aleatoriamente e colocados em quatro aquários de vidro (T0, T1, T2 e T3) em temperatura constante da água (30,0 ± 1,0 °C), pH (7,50 ± 0,5) e dureza total (200 ± 2,0 mgL-1) por um período de 12 semanas, com três repetições cada. Os peixes foram alimentados com ração suplementada com extrato de folha de P. nigrum @ 0,0%, 1,0%, 2,0% e 3,0% em T0, T1, T2 e T3, respectivamente. Ao final do experimento, cinco peixes foram selecionados aleatoriamente de cada aquário para composição centesimal, carga microbiana intestinal e cutânea e parâmetros hematológicos. Proteínas totais, albuminas e globulinas também foram registradas para avaliar a memória imunológica. O resultado revelou que os peixes em T2 apresentaram melhor desempenho de crescimento com ganho de peso médio de 56,11 ± 0,51 g. Assim, concluiu-se que Piper nigrum, uma planta medicinal, também pode ser usado para melhorar o desempenho de crescimento e resposta imunológica de Labeo rohita como alternativas atraentes contra antibióticos e vacinas e não mostrou efeitos colaterais negativos na saúde dos peixes, bem como sobre seu ambiente.

2.
Braz. j. biol ; 82: e243150, 2022. tab, graf
Artigo em Inglês | LILACS-Express | MEDLINE, LILACSEXPRESS | ID: biblio-1339334

RESUMO

Abstract Present study was conducted among school children to recognize the prevalence of IPIs in rural communities of district Dir Lower, Pakistan. A sum of 324 samples of stool were collected (210 boys and 114 girls). Used direct smear method and formol ether sedimentation concentration technique for processing the samples. The result shows that 82% (n=266) were found infected comprised 64.8% male and 35.1% females. Children of the age group 10 to 12 years were found extremely infected 94.2% while 4-6 year age group were having minimum ratio of infection 72%. Current study shows mono parasitism in 50.6% of the students while 22.2% were infected with 2 species and 7.40% were infected with three species of parasites. Seven species of intestinal parasites were reported include Ascaris lumbricoid in male (n=122) 58.0% and in female (n=65) 57.0% followed by Hook worm (n=88) 41.9% and (n=44) 38.5%; Tania saginata (n=44) 20.9% and (n=24) 21.0%; Entrobius vermicularis (n=32) 15.2% and (n=16) 14.0%; Trichuris trichura (n=25) 11.9% and (n=22) 19.2%; Hymenolepis nana (n=24) 11.4% and (n=18) 15.7% and Entameoba histolytica (n=16) 7.61% and (n=14) 12.2% in male and females respectively. The study indicates that most occurring intestinal parasite in the current study were Ascaris lumbricoides 58.0% (n=122) followed by hookworms 41.9% (n=88). Male students were more infected than females in the present study.


Resumo O presente estudo foi conduzido entre crianças em idade escolar para reconhecer a prevalência de IPIs em comunidades rurais do distrito de Dir Lower, Paquistão. Foram coletadas 324 amostras de fezes (210 meninos e 114 meninas). Método de esfregaço direto usado e técnica de concentração de sedimentação de formol éter para processar as amostras. O resultado mostra que 82% (n = 266) dos infectados eram 64,8% do sexo masculino e 35,1% do feminino. Crianças da faixa etária de 10 a 12 anos foram encontradas extremamente infectadas 94,2%, enquanto a faixa etária de 4 a 6 anos apresentava proporção mínima de infecção de 72%. O estudo atual mostra monoparasitismo em 50,6% dos alunos, enquanto 22,2% estavam infectados com 2 espécies e 7,40% estavam infectados com três espécies de parasitas. Sete espécies de parasitas intestinais foram relatadas incluindo Ascaris lumbricoid em machos (n = 122) 58,0% e em fêmeas (n = 65) 57,0% seguido por anzol (n = 88) 41,9% e (n = 44) 38,5%; Tania saginata (n = 44) 20,9% e (n = 24) 21,0%; Entrobius vermicularis (n = 32) 15,2% e (n = 16) 14,0%; Trichuris trichura (n = 25) 11,9% e (n = 22) 19,2%; Hymenolepis nana (n = 24) 11,4% e (n = 18) 15,7% e Entameoba histolytica (n = 16) 7,61% e (n = 14) 12,2% em homens e mulheres, respectivamente. O estudo indica que os parasitas intestinais que mais ocorreram no presente estudo foram Ascaris lumbricoides 58,0% (n = 122), seguidos por ancilóstomos 41,9% (n = 88). Estudantes do sexo masculino foram mais infectados do que do sexo feminino no presente estudo.

3.
Braz J Biol ; 83: e248122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34932614

RESUMO

Being vector of West Nile Virus and falariasis the control of Culex quinquefasciatus is likely to be essential. Synthetic insecticide treatment is looking most effective for vectors mosquito control. However, these products are toxic to the environment and non-target organisms. Consequently, ecofriendly control of vectors mosquito is needed. In this regard botanical insecticide is looking more fruitful. Therefore, the present research aimed to investigate the effectiveness of methanolic extract and various fractions, including, n-hexane, ethyl-acetate, chloroform, and aqueous fraction, obtained from methanolic extract of Ailanthus altissima, Artemisia scoparia, and Justicia adhatoda using separating funnel against larval, pupal, and adult stages of Culex quinquefasciatus. The larvae and pupae of Culex quinquefasciatus were exposed to various concentrations (31.25-1000 ppm) of methanolic extract and its fractions for 24 hours of exposure period. For knock-down bioassay (filter paper impregnation bioassay) different concentration of the methanolic extract and its various fractions (i.e. 0.0625, 0.125, 0.25, 0.5 and 1mg/mL) were applied for 1 hour exposure period. The results were statistically analysed using standard deviation, probit analysis, and linear regression. The R2 values of larvae, pupae, and adult range from 0.4 to 0.99. The values of LC50 (concentration causing 50% mortality) for late 3rd instar larvae after 24 hours exposure period range from 93-1856.7 ppm, while LC90 values range from 424 -7635.5ppm. The values of LC50for pupae range form 1326.7-6818.4ppm and and values of LC90 range from 3667.3-17427.9ppm, respectively. The KDT50 range from 0.30 to 2.8% and KDT90 values range from1.2 to 110.8%, respectively. In conclusion, Justicia adhatoda may be effective for controlling populations of vector mosquito.


Assuntos
Anopheles , Culex , Inseticidas , Animais , Inseticidas/farmacologia , Larva , Mosquitos Vetores , Extratos Vegetais/farmacologia , Folhas de Planta
4.
Braz J Biol ; 83: e247583, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34932616

RESUMO

The study was undertaken from September 2018 to April 2019 to determine the prevalence of cutaneous leishmaniasis in human beings living in six districts of Karachi. Suspected persons were screened for the disease and positive cases were identified on the basis of skin lesions and blood samples. Samples were observed by mounting their smear. A total of 207 subjects of different ages and sex groups were investigated, however, only 192 (92%) of the suspected cases were found to have the disease 64% of cases were male which were significantly high (p 0.05), than female 36%. The lesion was more frequently detected among the youth ages of 21-30 years (31%) as compared to other groups. In both sexes, legs were found to be more infected (25% male + 20% female) followed by arms (20% male + 0% female) and face (15% male +11% female). The mixed body parts had shown the lowest infections such as (4% in males + 5%) in females. In conclusion, the highest and lowest leishmaniasis infections were observed in District West (23% male + 9% female) followed by District East (15% male + 7% female), District Malir (11% male+ 4% female), District Central (7% male + 5% female), District Korangi (4% male + 7% female) and District South (4% male + 4% female) respectively.


Assuntos
Leishmaniose Cutânea , Adolescente , Adulto , Feminino , Humanos , Leishmaniose Cutânea/epidemiologia , Masculino , Paquistão/epidemiologia , Prevalência , Adulto Jovem
5.
Braz J Biol ; 82: e242897, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34816975

RESUMO

Cyprinus carpio is the member of family cyprinidae commonly called common carp. This study was aimed to find out the comparison of brain of wild (river system) and captive (hatchery reared) population of common carp. A total of thirty samples (15 from hatchery and 15 from river Swat) were collected. All the specimens were examined in Laboratory of Parasitoloy, Zoology Department, University of Malakand. Findings indicated that wild population were greater in brain size and weight as compared to hatchery reared population. The fish samples collected from captive environment (hatchery) were showing more weight and length as compared to wild population of common carps. The mean value of total weight of hatchery fishes 345±48.68 and the mean value of brain weight of hatchery reared fishes 0.28±0.047. The mean value of wild fish's total body weight 195.16±52.58 and the mean value of brain weight of wild fishes are 0.45±0.14. Present research calls for the fact that fish in dependent environmental conditions possess brain larger in size as compared to its captive population, it is due to use and disuse of brain in their environmental requirements.


Assuntos
Carpas , Cyprinidae , Animais , Encéfalo , Rios
6.
Braz J Biol ; 83: e249211, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34730605

RESUMO

This study was conducted to estimate the diversity and the occurrence of commercially important finfish species collected by twenty fish sampling site of Sindh and Baluchistan coasts of the Arabian Sea in Pakistan from January to December 2019. Additionally, physicochemical characteristics of seawater were analyzed from these selected sites and found to be within suitable ranges required for fish growth and survive. A total of 81287 fish individuals were collected and identified as 49 species belonging to 26 families in our study. The most diversified family was Sparidae (13 species) followed by Carangidae and Lutjanidae (4 species), Mullidae, Serranidae, Ariidae (3 species), and Sciaenidae (2 species). The remaining 20 families were represented by only one species. The values of Shannon diversity index calculated for the four selected habitats revealed that high fish diversity was reported at Sonmiani Coast (H'=1.81), while less at Ormara Coast (H'=0.23). Likewise, Evenness index (E) was high at Sonmiani Coast (E=0.50) and less fish diversity was reported at Ormara Coast (E=0.06). Reducing risks to threatened marine species in coastal habitats also requires conservation actions at multiple scales. Thus, it was concluded that our study could be valuable in providing the more information's regarding to the diversity of finfish species and their occurrence along the Pakistan Coast. Further, to better understand the effects, regular monitoring and conservation measures should be taken to mitigate the influence of anthropogenic activities and protect finfish diversity from further decline.


Assuntos
Biodiversidade , Água , Animais , Ecossistema , Peixes , Humanos , Água do Mar
7.
Braz J Biol ; 82: e243150, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34644727

RESUMO

Present study was conducted among school children to recognize the prevalence of IPIs in rural communities of district Dir Lower, Pakistan. A sum of 324 samples of stool were collected (210 boys and 114 girls). Used direct smear method and formol ether sedimentation concentration technique for processing the samples. The result shows that 82% (n=266) were found infected comprised 64.8% male and 35.1% females. Children of the age group 10 to 12 years were found extremely infected 94.2% while 4-6 year age group were having minimum ratio of infection 72%. Current study shows mono parasitism in 50.6% of the students while 22.2% were infected with 2 species and 7.40% were infected with three species of parasites. Seven species of intestinal parasites were reported include Ascaris lumbricoid in male (n=122) 58.0% and in female (n=65) 57.0% followed by Hook worm (n=88) 41.9% and (n=44) 38.5%; Tania saginata (n=44) 20.9% and (n=24) 21.0%; Entrobius vermicularis (n=32) 15.2% and (n=16) 14.0%; Trichuris trichura (n=25) 11.9% and (n=22) 19.2%; Hymenolepis nana (n=24) 11.4% and (n=18) 15.7% and Entameoba histolytica (n=16) 7.61% and (n=14) 12.2% in male and females respectively. The study indicates that most occurring intestinal parasite in the current study were Ascaris lumbricoides 58.0% (n=122) followed by hookworms 41.9% (n=88). Male students were more infected than females in the present study.


Assuntos
Enteropatias Parasitárias , Parasitos , Animais , Criança , Fezes , Feminino , Humanos , Enteropatias Parasitárias/epidemiologia , Masculino , Paquistão/epidemiologia , Prevalência , Instituições Acadêmicas
8.
Braz J Biol ; 83: e246825, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34431909

RESUMO

A study was conducted to evaluate the effect of Piper nigrum (black pepper) leaf extract on on the growth performance, proximate composition, hematological parameters, and immune response of Labeo rohita fingerlings with an average weight of 22.14 ± 0.98g. Aftrer acclimation for two weeks, fish (n=25) were randomly selected and placed in four glass aquaria (T0, T1, T2 and T3) at constant water temperature (30.0 ± 1.0 °C), pH (7.50 ± 0.5) and total hardness (200 ± 2.0 mgL,-1) for a period of 12 weeks, with three replicates each. Fish were fed with P. nigrum leaf extract supplemented feed @ 0.0%, 1.0%, 2.0% and 3.0% in T0, T1,T2 and T3, respectively. At the end of experiment, five fish were randomly selected from each aquaria for proximate composition, gut and skin microbial load, hematological parameters. Total proteins, albumins, and globulins were also recorded to evaluate immunological memory. The result revealed that fish in T2 showed better growth performance with an average weight gain of 56.11 ± 0.51 g. Thus, it had been concluded that Piper nigrum, a medicinal plant, can also be used to enhance the growth performance and immune response of Labeo rohita as attractive alternatives against antibiotics and vaccines and has shown no negative side effects on fish health as well as on its environment.


Assuntos
Cyprinidae , Piper nigrum , Animais , Dieta , Suplementos Nutricionais , Extratos Vegetais/farmacologia
9.
Anal Methods ; 9(28): 4112-4134, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29151901

RESUMO

Surface acoustic waves (SAWs), are electro-mechanical waves that form on the surface of piezoelectric crystals. Because they are easy to construct and operate, SAW devices have proven to be versatile and powerful platforms for either direct chemical sensing or for upstream microfluidic processing and sample preparation. This review summarizes recent advances in the development of SAW devices for chemical sensing and analysis. The use of SAW techniques for chemical detection in both gaseous and liquid media is discussed, as well as recent fabrication advances that are pointing the way for the next generation of SAW sensors. Similarly, applications and progress in using SAW devices as microfluidic platforms are covered, ranging from atomization and mixing to new approaches to lysing and cell adhesion studies. Finally, potential new directions and perspectives on the field as it moves forward are offered, with a specific focus on potential strategies for making SAW technologies for bioanalytical applications.

10.
Front Microbiol ; 5: 474, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25250022

RESUMO

Myxococcus xanthus is a bacterial micro-predator known for hunting other microbes in a wolf pack-like manner. Outer membrane vesicles (OMVs) are produced in large quantities by M. xanthus and have a highly organized structure in the extracellular milieu, sometimes occurring in chains that link neighboring cells within a biofilm. OMVs may be a vehicle for mediating wolf pack activity by delivering hydrolytic enzymes and antibiotics aimed at killing prey microbes. Here, both the protein and small molecule cargo of the OMV and membrane fractions of M. xanthus were characterized and compared. Our analysis indicates a number of proteins that are OMV-specific or OMV-enriched, including several with putative hydrolytic function. Secondary metabolite profiling of OMVs identifies 16 molecules, many associated with antibiotic activities. Several hydrolytic enzyme homologs were identified, including the protein encoded by MXAN_3564 (mepA), an M36 protease homolog. Genetic disruption of mepA leads to a significant reduction in extracellular protease activity suggesting MepA is part of the long-predicted (yet to date undetermined) extracellular protease suite of M. xanthus.

11.
Mol Biol Int ; 2014: 287430, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25197572

RESUMO

As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.

12.
Plant J ; 79(3): 517-29, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24905498

RESUMO

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate-Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell-wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full-length coding sequences without termination codons and are Gateway® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/.


Assuntos
Genômica , Glicosiltransferases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/metabolismo , Parede Celular/metabolismo
13.
PLoS One ; 9(6): e100836, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24971883

RESUMO

Three lignocellulosic pretreatment techniques (ammonia fiber expansion, dilute acid and ionic liquid) are compared with respect to saccharification efficiency, particle size and biomass composition. In particular, the effects of switchgrass particle size (32-200) on each pretreatment regime are examined. Physical properties of untreated and pretreated samples are characterized using crystallinity, surface accessibility measurements and scanning electron microscopy (SEM) imaging. At every particle size tested, ionic liquid (IL) pretreatment results in greater cell wall disruption, reduced crystallinity, increased accessible surface area, and higher saccharification efficiencies compared with dilute acid and AFEX pretreatments. The advantages of using IL pretreatment are greatest at larger particle sizes (>75 µm).


Assuntos
Biomassa , Carboidratos/biossíntese , Parede Celular/efeitos dos fármacos , Líquidos Iônicos/farmacologia , Ácidos/farmacologia , Parede Celular/ultraestrutura , Cristalização , Microscopia Eletrônica de Varredura , Nitrogênio/química , Nitrogênio/metabolismo , Tamanho da Partícula , Poaceae/química , Poaceae/metabolismo
14.
PLoS One ; 8(11): e79725, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244549

RESUMO

Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25-42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.


Assuntos
Celulase/metabolismo , Líquidos Iônicos , Poaceae , Thermotoga maritima/enzimologia , Biomassa , Celulase/química , Celulase/genética , Ativação Enzimática , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Mutagênese , Conformação Proteica , Thermotoga maritima/genética
15.
Plant J ; 76(6): 1016-29, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24128328

RESUMO

We have characterized a ß-glucuronosyltransferase (AtGlcAT14A) from Arabidopsis thaliana that is involved in the biosynthesis of type II arabinogalactan (AG). This enzyme belongs to the Carbohydrate Active Enzyme database glycosyltransferase family 14 (GT14). The protein was localized to the Golgi apparatus when transiently expressed in Nicotiana benthamiana. The soluble catalytic domain expressed in Pichia pastoris transferred glucuronic acid (GlcA) to ß-1,6-galactooligosaccharides with degrees of polymerization (DP) ranging from 3-11, and to ß-1,3-galactooligosaccharides of DP5 and 7, indicating that the enzyme is a glucuronosyltransferase that modifies both the ß-1,6- and ß-1,3-galactan present in type II AG. Two allelic T-DNA insertion mutant lines showed 20-35% enhanced cell elongation during seedling growth compared to wild-type. Analyses of AG isolated from the mutants revealed a reduction of GlcA substitution on Gal-ß-1,6-Gal and ß-1,3-Gal, indicating an in vivo role of AtGlcAT14A in synthesis of those structures in type II AG. Moreover, a relative increase in the levels of 3-, 6- and 3,6-linked galactose (Gal) and reduced levels of 3-, 2- and 2,5-linked arabinose (Ara) were seen, suggesting that the mutation in AtGlcAT14A results in a relative increase of the longer and branched ß-1,3- and ß-1,6-galactans. This increase of galactosylation in the mutants is most likely caused by increased availability of the O6 position of Gal, which is a shared acceptor site for AtGlcAT14A and galactosyltransferases in synthesis of type II AG, and thus addition of GlcA may terminate Gal chain extension. We discuss a role for the glucuronosyltransferase in the biosynthesis of type II AG, with a biological role during seedling growth.


Assuntos
Arabidopsis/enzimologia , Galactanos/biossíntese , Glucuronosiltransferase/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabinose/genética , Arabinose/metabolismo , Transporte Biológico , Domínio Catalítico , Parede Celular/metabolismo , Expressão Gênica , Glucuronosiltransferase/genética , Complexo de Golgi/metabolismo , Modelos Estruturais , Mutagênese Insercional , Fenótipo , Filogenia , Pichia/enzimologia , Pichia/genética , Proteínas Recombinantes , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Especificidade por Substrato , Tabaco/enzimologia , Tabaco/genética
16.
Front Microbiol ; 4: 282, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24069019

RESUMO

Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26%) cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase (GH) families were detected. Using tetranucleotide frequency (TNF) binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts.

17.
Biotechnol Biofuels ; 6(1): 93, 2013 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-23819686

RESUMO

INTRODUCTION: Cellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive. To further improve these enzymes for application at industrial conditions, it is critical to gain a better understanding of not only the details of the degradation process, but also the function of accessory modules. METHOD: We fused a carbohydrate-binding module (CBM) from family 2a to two thermophilic endoglucanases. We then applied neutron reflectometry to determine the mechanism of the resulting enhancements. RESULTS: Catalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type. Importantly, we demonstrate that the wild type enzymes affect primarily the surface properties of an amorphous cellulose film, while the chimeras containing a CBM alter the bulk properties of the amorphous film. CONCLUSION: Our findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film.

18.
Iran J Public Health ; 41(9): 25-30, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23193502

RESUMO

BACKGROUND: Pre-operative informed consent is an important aspect of surgery, yet there has been no formal training regarding it in Pakistan. This study was done to assess the preoperative informed consent practice. METHODS: After taking informed permission, a questionnaire was filled in during an interview with 350 patients, who have undergone elective surgical procedures under routine practice conditions from July to October 2010. All the patients were asked a set of standard questions which related to the information they were provided before the operation as a part of standard informed consent practice. RESULTS: Most i.e. 307 (87.7%) patients were informed about their condition but very few 12 (3.4%) were briefed regarding complications. Only 17 (4.9%) patients said they knew about the risks and complications of proposed anesthesia. One hundred thirty-eight (39.4%) patients said that they were allowed to ask questions while giving consent. Most of the time 196 (56%) consent was taken one day before surgery but in few 2 (0.6%) instances it was taken on the morning of surgery and on operation table in some cases 3 (0.9%) as reported by patients. The consent form was signed by the patients themselves in only 204 (58.3%) cases and by their relatives in the rest. About half the number of patients 171 (48.9%) interviewed were satisfied from the information they received as informed consent process. CONCLUSION: This study highlights the poor quality of patient knowledge about surgical procedures and the inadequate information provided.

19.
BMC Biotechnol ; 12: 38, 2012 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-22759983

RESUMO

BACKGROUND: Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals. RESULTS: Twenty-two putative ORFs (open reading frames) were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in E. coli and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed in vivo, purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to ~60-70°C. CONCLUSIONS: Seven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Metagenoma , Microbiologia do Solo , Bactérias/química , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Fases de Leitura Aberta , Solo
20.
Plant Physiol ; 159(4): 1408-17, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22706449

RESUMO

Xylan is a major component of the plant cell wall and the most abundant noncellulosic component in the secondary cell walls that constitute the largest part of plant biomass. Dicot glucuronoxylan consists of a linear backbone of ß(1,4)-linked xylose residues substituted with α(1,2)-linked glucuronic acid (GlcA). Although several genes have been implicated in xylan synthesis through mutant analyses, the biochemical mechanisms responsible for synthesizing xylan are largely unknown. Here, we show evidence for biochemical activity of GUX1 (for GlcA substitution of xylan 1), a member of Glycosyltransferase Family 8 in Arabidopsis (Arabidopsis thaliana) that is responsible for adding the glucuronosyl substitutions onto the xylan backbone. GUX1 has characteristics typical of Golgi-localized glycosyltransferases and a K(m) for UDP-GlcA of 165 µm. GUX1 strongly favors xylohexaose as an acceptor over shorter xylooligosaccharides, and with xylohexaose as an acceptor, GlcA is almost exclusively added to the fifth xylose residue from the nonreducing end. We also show that several related proteins, GUX2 to GUX5 and Plant Glycogenin-like Starch Initiation Protein6, are Golgi localized and that only two of these proteins, GUX2 and GUX4, have activity as xylan α-glucuronosyltransferases.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Glucuronosiltransferase/metabolismo , Glicosiltransferases/metabolismo , Xilanos/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Microssomos/enzimologia , Modelos Moleculares , Filogenia , Transporte Proteico , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Frações Subcelulares/enzimologia , Tabaco/metabolismo
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