ABSTRACT
Background/aim: Despite different regional anesthesia techniques used to provide intraoperative and postoperative analgesia in pediatric patients, the analgesic effectiveness of peripheral nerve blockades with minimal side effect profiles have not yet been fully determined. We aimed to compare the efficacy of ultrasound-guided transversus abdominis plane (TAP) block, quadratus lumborum (QL) block, and caudal epidural block on perioperative analgesia in pediatric patients aged between 6 months and 14 years who underwent elective unilateral lower abdominal wall surgery. Materials and methods: Ninety-four patients classified under the American Society of Anesthesiologists physical status classification system as ASA I or ASA II were randomly divided into 3 equal groups to perform TAP, QL or Caudal epidural block using 0.25% of bupivacaine solution (0.5 ml kg−1). Results: Postoperative analgesic consumption was highest in the TAP block group (P < 0.05). In the QL block group, Pediatric Objective Pain Scale (POAS) scores were statistically significantly lower after 2 and 4 h (P < 0.05). The length of hospital stay was significantly longer in the caudal block group than the QL block group (P < 0.05). Conclusion: We suggest that analgesia with ultrasound-guided QL block should be considered as an option for perioperative analgesia in pediatric patients undergoing lower abdominal surgery if the expertise and equipment are available.
Subject(s)
Abdominal Wall/surgery , Anesthesia, Caudal/methods , Nerve Block/methods , Ultrasonography, Interventional , Abdominal Muscles/innervation , Adolescent , Child , Child, Preschool , Female , Humans , Infant , Male , Ultrasonography, Interventional/methodsABSTRACT
Lumbar plexus block (LPB) is a suitable method for elder patients for lower extremity surgery. Many complications could be seen during LPB, but not as many as central block. In this case report, we aimed to report a total spinal block, an unusual complication. LPB with sciatic block was planned for a male patient, 76 years old, scheduled for total knee replacement due to gonarthrosis. The patient became unconscious after psoas compartment block with Chayen technique for LPB. The operation ended at 145th minute. The patient was admitted to intensive care unit until postoperative second day and discharged to home on fifth day of surgery. Main concern of patient monitorization should be an anesthesiologist. In this manner, we conclude that contacting to the patient should be ensured during these procedures.
Subject(s)
Arthroplasty, Replacement, Knee , Lumbosacral Plexus , Nerve Block , Aged , Humans , MaleABSTRACT
O bloqueio do plexo lombar (BPL) é um método adequado para uso em pacientes idosos e cirurgias na extremidade inferior. Muitas complicações podem ser observadas durante o BPL, mas não tanto quanto no bloqueio central. Neste relato de caso, nosso objetivo foi relatar uma raquianestesia total, uma complicação incomum. BPL com bloqueio ciático foi planejado para um paciente do sexo masculino, 76 anos, programado para artroplastia total do joelho por causa de gonartrose. O paciente ficou inconsciente após o bloqueio do compartimento do psoas com a técnica de Chayen para BPL. A operação terminou em 145 minutos. O paciente foi internado em unidade de terapia intensiva até o segundo dia pós-operatório e recebeu alta hospitalar no quinto dia pós-cirúrgico. A principal preocupação da monitoração do paciente deve ser a presença do anestesiologista. Dessa forma, conclui-se que o contato com o paciente deve ser garantido durante esses procedimentos.
Lumbar plexus block (LPB) is a suitable method for elder patients for lower extremity surgery. Many complications could be seen during LPB, but not as many as central block. In this case report, we aimed to report a total spinal block, an unusual complication. LPB with sciatic block was planned for a male patient, 76 years old, scheduled for total knee replacement due to gonarthrosis. The patient became unconscious after psoas compartment block with Chayen technique for LPB. The operation ended at 145th minute. The patient was admitted to intensive care unit until postoperative second day and discharged to home on fifth day of surgery. Main concern of patient monitorization should be an anesthesiologist. In this manner, we conclude that contacting to the patient should be ensured during these procedures.
El bloqueo del plexo lumbar (BPL) es un método adecuado para usarlo en pacientes ancianos sometidos a cirugía de la extremidad inferior. Durante el BPL pueden observarse muchas complicaciones, pero no tantas como en el bloqueo central. En este relato de caso, nuestro objetivo fue exponer una raquianestesia total, una complicación no común. Se planificó un BPL con bloqueo ciático para un paciente del sexo masculino, de 76 años de edad, programado para artroplastia total de la rodilla debida a gonartrosis. El paciente quedó inconsciente después del bloqueo del compartimento del psoas con la técnica de Chayen para BPL. La operación terminó en 145 min. El paciente fue ingresado en la unidad de cuidados intensivos hasta el segundo día del postoperatorio y tuvo alta hospitalaria al quinto día poscirugía. La principal preocupación de la monitorización del paciente debe ser la presencia del anestesiólogo. Así se concluye que el contacto con el paciente debe estar garantizado durante esos procedimientos.
Subject(s)
Aged , Humans , Male , Arthroplasty, Replacement, Knee , Lumbosacral Plexus , Nerve BlockABSTRACT
The anesthesiologist must be aware of the causes, diagnosis and treatment of venous air embolism and adopt the practice patterns to prevent its occurrence. Although venous air embolism is a known complication of cesarean section, we describe an unusual inattention that causes iatrogenic near fatal venous air embolism during a cesarean section under spinal anesthesia. One of the reasons for using self-collapsible intravenous (IV) infusion bags instead of conventional glass or plastic bottles is to take precaution against air embolism. We also demonstrated the risk of air embolism for two kinds of plastic collapsible intravenous fluid bags: polyvinyl chloride (PVC) and polypropylene-based. Fluid bags without self-sealing outlets pose a risk for air embolism if the closed system is broken down, while the flexibility of the bag limits the amount of air entry. PVC-based bags, which have more flexibility, have significantly less risk of air entry when IV administration set is disconnected from the outlet. Using a pressure bag for rapid infusion can be dangerous without checking and emptying all air from the IV bag.
Subject(s)
Cesarean Section , Embolism, Air/etiology , Intraoperative Complications/etiology , Adult , Drug Packaging , Female , Fluid Therapy , Humans , Infusions, Intravenous , Polyvinyl Chloride , Risk FactorsABSTRACT
O anestesiologista deve estar ciente das causas, do diagnóstico e do tratamento de embolia venosa e adotar padrões de prática para prevenir sua ocorrência. Embora a embolia gasosa seja uma complicação conhecida da cesariana, descrevemos um caso raro de desatenção que causou embolia gasosa iatrogênica quase fatal durante uma cesariana sob raquianestesia. uma das razões para o uso de bolsas autorretráteis para infusão em vez dos frascos convencionais de vidro ou plástico é a precaução contra embolia gasosa. Também demonstramos o risco de embolia venosa com o uso de dois tipos de bolsas plásticas retráteis (à base de cloreto de polivinil [PVC] e de polipropileno) para líquidos intravenosos. As bolsas para líquidos sem saídas autovedantes apresentam risco de embolia gasosa se o sistema de fechamento estiver quebrado, enquanto a flexibilidade da bolsa limita a quantidade de entrada de ar. bolsas à base de pvc, que têm mais flexibilidade, apresentam risco significativamente menor de entrada de ar quando o equipo de administração intravenosa (IV) é desconectado da saída. usar uma bolsa pressurizada para infusão rápida sem verificar e esvaziar todo o ar da bolsa IV pode ser perigoso.
The anesthesiologist must be aware of the causes, diagnosis and treatment of venous air embolism and adopt the practice patterns to prevent its occurrence. Although venous air embolism is a known complication of cesarean section, we describe an unusual inattention that causes iatrogenic near fatal venous air embolism during a cesarean section under spinal anesthesia. One of the reasons for using self-collapsible intravenous (IV) infusion bags instead of conventional glass or plastic bottles is to take precaution against air embolism. We also demonstrated the risk of air embolism for two kinds of plastic collapsible intravenous fluid bags: polyvinyl chloride (PVC) and polypropylene-based. Fluid bags without self-sealing outlets pose a risk for air embolism if the closed system is broken down, while the flexibility of the bag limits the amount of air entry. PVC-based bags, which have more flexibility, have signifi cantly less risk of air entry when IV administration set is disconnected from the outlet. Using a pressure bag for rapid infusion can be dangerous without checking and emptying all air from the IV bag.
El anestesiólogo debe de estar consciente de las causas, del diagnóstico y del tratamiento de la embolia venosa, y adoptar los estándares de práctica para prevenir su aparecimiento. Aunque la embolia gaseosa sea una complicación conocida de la cesárea, describimos aquí un caso raro de falta de atención que causó embolia gaseosa iatrogénica casi fatal durante una cesárea bajo raquianestesia. Una de las razones para el uso de bolsas autoretráctiles para infusión en vez de los frascos convencionales de vidrio o plástico, es la precaución contra la embolia gaseosa. También demostramos riesgo de embolia venosa con el uso de dos tipos de bolsas plásticas retráctiles (a base de cloruro de polivinil [PVC] y de polipropileno) para líquidos intravenosos. Las bolsas para líquidos sin salidas de autosellado, tienen un riesgo de embolia gaseosa si el sistema de cierre está roto, mientras la flexibilidad de la bolsa limita la cantidad de entrada de aire. Bolsas hechas a base de PVC, y que tienen más flexibilidad, también tienen un riesgo signifi cativamente menor de entrada de aire cuando el equipo de administración intravenosa (IV) se apaga en la salida. Usar una bolsa de presión para la infusión rápida sin verifi car y vaciar todo el aire de la bolsa IV puede ser peligroso.
Subject(s)
Adult , Female , Humans , Cesarean Section , Embolism, Air/etiology , Intraoperative Complications/etiology , Drug Packaging , Fluid Therapy , Infusions, Intravenous , Polyvinyl Chloride , Risk FactorsABSTRACT
The anesthesiologist must be aware of the causes, diagnosis and treatment of venous air embolism and adopt the practice patterns to prevent its occurrence. Although venous air embolism is a known complication of cesarean section, we describe an unusual inattention that causes iatrogenic near fatal venous air embolism during a cesarean section under spinal anesthesia. One of the reasons for using self-collapsible intravenous (IV) infusion bags instead of conventional glass or plastic bottles is to take precaution against air embolism. We also demonstrated the risk of air embolism for two kinds of plastic collapsible intravenous fluid bags: polyvinyl chloride (PVC) and polypropylene-based. Fluid bags without self-sealing outlets pose a risk for air embolism if the closed system is broken down, while the flexibility of the bag limits the amount of air entry. PVC-based bags, which have more flexibility, have significantly less risk of air entry when IV administration set is disconnected from the outlet. Using a pressure bag for rapid infusion can be dangerous without checking and emptying all air from the IV bag.
Subject(s)
Cesarean Section , Embolism, Air/etiology , Intraoperative Complications/etiology , Adult , Drug Packaging , Female , Humans , Infusions, Intravenous , PregnancyABSTRACT
Myrosinases (ß-thioglucoside glucohydrolase, TGG; EC 3.2.1.147) catalyze the hydrolysis of glucosinolates, a structurally distinct group of nitrogen- and sulfur-containing secondary metabolites, to give a chemically unstable intermediate, glucose and sulfate. This catalysis initiates a chemical defense in crucifer plants as a response to the tissue-damaging activities of herbivores and pathogens. To characterize the individual and collective biochemical properties of the myrosinase enzymes found in the aerial tissues of Arabidopsis thaliana, we purified TGG1 and TGG2, which share 73% amino acid identity, individually from T-DNA insertion lines of Arabidopsis using lectin affinity and anion exchange chromatography. Electrophoresis under denaturing conditions and the mobility of nondenatured TGG1 and TGG2 protein on gel filtration chromatography indicated that the native proteins exist as dimers of 150 and 126 kDa, respectively. Despite their relatively similar kinetic parameters, both enzymes had distinct physicochemical properties such as extractability in low ionic strength buffer and electrophoretic mobility following deglycosylation treatment. Deglycosylation under nondenaturing conditions had limited effects on TGG1 and no effect on TGG2 activity. Both enzymes functioned across a broad range of temperatures (up to 60 °C) and pH values (5-10). These results demonstrate that myrosinases have the ability to function in environments like the digestive tract of insect herbivores that are significantly different from the environment in a damaged plant.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Glycoside Hydrolases/metabolism , Plant Leaves/enzymology , Amino Acid Sequence , Arabidopsis/drug effects , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/isolation & purification , Ascorbic Acid/pharmacology , Biocatalysis/drug effects , Enzyme Activation/drug effects , Enzyme Stability/drug effects , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/isolation & purification , Glycosylation/drug effects , Hydrogen-Ion Concentration/drug effects , Hydrolysis/drug effects , Immunoblotting , Kinetics , Molecular Sequence Data , Mutation/genetics , Peptides/metabolism , Plant Leaves/drug effects , Protein Denaturation/drug effects , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Solubility/drug effects , Substrate Specificity/drug effects , TemperatureSubject(s)
Anesthetics, Intravenous , Piperidines , Anesthesia, General , Anesthesia, Intravenous , Child , Humans , Infusion Pumps , RemifentanilABSTRACT
Maize beta-glucosidase aggregating factor (BGAF) and its homolog Sorghum Lectin (SL) are modular proteins consisting of an N-terminal dirigent domain and a C-terminal jacalin-related lectin (JRL) domain. BGAF is a polyspecific lectin with a monosaccharide preference for galactose, whereas SL displays preference for GalNAc. Here, we report that deletion of the N-terminal dirigent domain in the above lectins dramatically changes their sugar-specificities. Deletions in the N-terminal region of the dirigent domain of BGAF abolished binding to galactose/lactose, but binding to mannose was unaffected. Glucose, which was a poor inhibitor of hemagglutinating activity of BGAF, displayed higher inhibitory effect on the hemagglutinating activity of deletion mutants. Deletion of the dirigent domain in SL abolished binding to GalNAc, but binding to mannose was not affected. Surprisingly, fructose, an extremely poor inhibitor (minimum inhibitory concentration (MIC) = 125 mM) of SL hemagglutinating activity, was found to be a very potent inhibitor (MIC = 1 mM) of hemagglutinating activity of its JRL domain. These results indicate that the dirigent domain in this class of modular lectins, at least in the case of maize BGAF and SL, influences sugar specificity.
Subject(s)
Carrier Proteins/genetics , Galactose/metabolism , Lactose/metabolism , Lectins/genetics , Plant Proteins/genetics , Acetylgalactosamine/metabolism , Binding Sites/genetics , Binding, Competitive , Carrier Proteins/metabolism , Fructose/metabolism , Glucose/metabolism , Lectins/metabolism , Mannose/metabolism , Mutation , Plant Proteins/metabolism , Protein Binding , Sequence Deletion , Sorghum/geneticsABSTRACT
ß-Glucosidases (3.2.1.21) are found in all domains of living organisms, where they play essential roles in the removal of nonreducing terminal glucosyl residues from saccharides and glycosides. ß-Glucosidases function in glycolipid and exogenous glycoside metabolism in animals, defense, cell wall lignification, cell wall ß-glucan turnover, phytohormone activation, and release of aromatic compounds in plants, and biomass conversion in microorganisms. These functions lead to many agricultural and industrial applications. ß-Glucosidases have been classified into glycoside hydrolase (GH) families GH1, GH3, GH5, GH9, and GH30, based on their amino acid sequences, while other ß-glucosidases remain to be classified. The GH1, GH5, and GH30 ß-glucosidases fall in GH Clan A, which consists of proteins with (ß/α)(8)-barrel structures. In contrast, the active site of GH3 enzymes comprises two domains, while GH9 enzymes have (α/α)(6) barrel structures. The mechanism by which GH1 enzymes recognize and hydrolyze substrates with different specificities remains an area of intense study.
Subject(s)
Cellulases/metabolism , Animals , Catalytic Domain , Cellulases/chemistry , Humans , Substrate SpecificityABSTRACT
Three beta-glucosidases (At1g66270-BGLU21, At1g66280-BGLU22, and At3g09260-BGLU23) were purified from the roots of Arabidopsis and their cDNAs were expressed in insect cells. In addition, two beta-glucosidase binding protein cDNAs (At3g16420; PBPI and At3g16430; PBPII) were expressed in Escherichia coli and their protein products purified. These binding proteins interact with beta-glucosidases and activate them. BGLU21, 22 and 23 hydrolyzed the natural substrate scopolin specifically and also hydrolyzed to some extent substrates whose aglycone moiety is similar to scopolin (e.g. esculin and 4-MU-glucoside). In contrast, they hydrolyzed poorly DIMBOA-glucoside and did not hydrolyze pNP- and oNP-glucosides. We determined the physicochemical properties of native and recombinant BGLUs, and found no differences between them. They were stable in a narrow pH range (5-7.5) and had temperature and pH optima for activity at 35 degrees C and pH 5.5, respectively. As for thermostability, >95% of their activity was retained at 40 degrees C but dramatically decreased at >50 degrees C. The apparent K(m) of native and recombinant enzymes for scopolin was 0.73 and 0.81 mM, respectively, and it was 5.8 and 9.7 mM, respectively, for esculin. Western blot analysis showed that all three enzymes were exclusively expressed in roots of seedlings but not in any other plant part or organ under normal conditions. Furthermore, spatial expression patterns of all eight genes belonging to subfamily 3 were investigated at the transcription level by RT-PCR.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Cellulases/metabolism , Coumarins/metabolism , Glucosides/metabolism , Plant Roots/enzymology , Amino Acid Sequence , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/isolation & purification , Carrier Proteins/genetics , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Cellulases/genetics , Cellulases/isolation & purification , Enzyme Activation/physiology , Esculin/metabolism , Gene Expression Regulation, Enzymologic/genetics , Gene Expression Regulation, Plant/genetics , Hydrogen-Ion Concentration , Hydrolysis , Hymecromone/analogs & derivatives , Hymecromone/metabolism , Molecular Sequence Data , Plant Roots/cytology , Plant Roots/genetics , Protein Binding/physiology , Protein Stability , TemperatureABSTRACT
The Arabidopsis genome contains 17 predicted beta-galactosidase genes, all of which belong to glycosyl hydrolase (GH) Family 35. These genes have been further grouped into seven subfamilies based on sequence similarity. The largest of these, subfamily a1, consists of six genes, Gal-1 (At3g13750), Gal-2 (At3g52840), Gal-3 (At4g36360), Gal-4 (At5g56870), Gal-5 (At1g45130), and Gal-12 (At4g26140), some of which were characterized in previous studies. We report here the purification and biochemical characterization of recombinant Gal-1, Gal-3, Gal-4 and Gal-12 from Pichiapastoris, completing the analysis of all six recombinant proteins, as well as the isolation and characterization of the native Gal-2 protein from Arabidopsis leaves. Comparison of the relative expression levels of the subfamily a1 beta-galactosidases at the mRNA and protein levels uncovered evidence of differential regulation, which may involve post-transcriptional and post-translational processes. In addition, this study provides further support for the proposed function of the subfamily a1 beta-galactosidases in cell wall modification based on analysis of the organ-specific expression and subcellular localization of Gal-1 and Gal-12. Our study suggests that, despite some differences in individual biochemical characteristics and expression patterns, each member of the family has the potential to contribute to the dynamics of the Arabidopsis plant cell wall.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Cell Wall/enzymology , Gene Expression Regulation, Plant , Genes, Plant , beta-Galactosidase/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/isolation & purification , Cell Wall/genetics , Genome, Plant , Multigene Family , Plant Structures , RNA, Messenger/metabolism , Recombinant Proteins , Saccharomycetales , beta-Galactosidase/genetics , beta-Galactosidase/isolation & purificationABSTRACT
Beta-glucosidases (Glu1 and Glu2) in maize specifically interact with a lectin called beta-glucosidase aggregating factor (BGAF). We have shown that the N-terminal (Glu(50)-Val(145)) and the C-terminal (Phe(466)-Ala(512)) regions of maize Glu1 are involved in binding to BGAF. Sequence comparison between sorghum beta-glucosidases (dhurrinases, which do not bind to BGAF) and maize beta-glucosidases, and the 3D-structure of Glu1 suggested that the BGAF-binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF-binding site, we constructed additional chimeric beta-glucosidases. The results showed that a region spanning 11 amino acids (Ile(72)-Thr(82)) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser(1)-Thr(29), together with C-terminal region Phe(466)-Ala(512), affects the size of Glu1-BGAF complexes. The dissociation constants (K(d)) of chimeric beta-glucosidase-BGAF interactions also demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile(72)-Thr(82) on Glu1 for BGAF binding, we constructed a chimeric sorghum beta-glucosidase, Dhr2 (C-11, Dhr2 whose Val(72)-Glu(82) region was replaced with the Ile(72)-Thr(82) region of Glu1). C-11 binds to BGAF, indicating that the Ile(72)-Thr(82) region is indeed a major interaction site on Glu1 involved in BGAF binding.
Subject(s)
Cellulases/chemistry , Lectins/chemistry , Zea mays/enzymology , Amino Acid Sequence , Binding Sites , Isoenzymes , Lectins/metabolism , Molecular Sequence Data , Sequence Homology, Amino Acid , Sorghum/enzymology , Sorghum/genetics , Zea mays/chemistryABSTRACT
The RNAs for the storage proteins of rice (Oryza sativa), prolamines and glutelins, which are stored as inclusions in the lumen of the endoplasmic reticulum (ER) and storage vacuoles, respectively, are targeted by specific cis-localization elements to distinct subdomains of the cortical ER. Glutelin RNA has one or more cis-localization elements (zip codes) at the 3' end of the RNA, whereas prolamine has two cis-elements; one located in the 5' end of the coding sequence and a second residing in the 3'-untranslated region (UTR). We had earlier demonstrated that the RNAs for the maize zeins ('prolamine' class) are localized to the spherical protein body ER (PB-ER) in developing maize endosperm. As the PB-ER localization of the 10-kDa delta-zein RNA is maintained in developing rice seeds, we determined the number and proximate location of their cis-localization elements by expressing GFP fusions containing various zein RNA sequences in transgenic rice and analyzing their spatial distribution on the cortical ER by in situ RT-PCR and confocal microscopy. Four putative cis-localization elements were identified; three in the coding sequences and one in the 3'-UTR. Two of these zip codes are required for restricted localization to the PB-ER. Using RNA targeting determinants we show, by mis-targeting the storage protein RNAs from their normal destination on the cortical ER, that the coded proteins are redirected from their normal site of deposition. Targeting of RNA to distinct cortical ER subdomains may be the underlying basis for the variable use of the ER lumen or storage vacuole as the final storage deposition site of storage proteins among flowering plant species.
Subject(s)
Endoplasmic Reticulum/metabolism , RNA Transport , RNA, Plant/metabolism , Zea mays/genetics , Zein/genetics , 3' Untranslated Regions , Base Sequence , Gene Expression Regulation, Plant , Microscopy, Confocal , Molecular Sequence Data , Oryza/genetics , Oryza/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, RNA , Zea mays/metabolism , Zein/metabolismABSTRACT
In certain maize genotypes (nulls), beta-glucosidase specifically interacts with a chimeric lectin called beta-glucosidase aggregating factor (BGAF), resulting in high molecular weight complexes. Previously, we showed that three regions (S1-T29, E50-N127, and F466-A512) on the maize beta-glucosidase isozyme Glu1 are involved in interaction and aggregation with BGAF. Recently, we found that the peptide span I72-T82 within E50-N127 is essential and sufficient for BGAF binding, whereas the S1-T29 and F466-A512 regions are required for formation of large complexes. To define the contribution of individual amino acids in the above three regions to BGAF binding, we constructed mutant beta-glucosidases based on sequence differences between maize beta-glucosidase and sorghum beta-glucosidase (dhurrinase 2, Dhr2), which does not bind BGAF. Binding was evaluated by gel-shift assay and affinity by frontal affinity chromatography (FAC). In the gel-shift assay, Glu1 mutants K81E and T82Y failed to bind BGAF, and their FAC profiles were essentially similar to that of Dhr2, indicating that these two amino acids within the I72-T82 region are important for BGAF binding. Substitution of N481 with E (as in Dhr2) lowered affinity for BGAF, whereas none of the mutations in the S1-T29 region showed any effect on BGAF binding. To further confirm the importance of K81 and T82 for BGAF binding, we produced a number of Dhr2 mutants, and the results showed that all four amino acids (I72, N75, K81, and T82) that differ between Glu1 and Dhr2 in the peptide span I72-T82 are required to impart BGAF-binding ability to Dhr2.
Subject(s)
Carrier Proteins/metabolism , Lysine/metabolism , Plant Proteins/metabolism , Threonine/metabolism , Zea mays/enzymology , beta-Glucosidase/chemistry , beta-Glucosidase/metabolism , Amino Acid Sequence , Chromatography, Affinity , Cloning, Molecular , Electrophoretic Mobility Shift Assay , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/isolation & purification , Mutant Proteins/metabolism , Protein Binding , Protein Structure, Secondary , Sequence Alignment , Sequence Analysis, Protein , Structure-Activity Relationship , beta-Glucosidase/isolation & purificationABSTRACT
Recently, we identified the maize beta-glucosidase aggregating factor (BGAF) as a jacalin-related lectin (JRL) and showed that its lectin domain is responsible for beta-glucosidase aggregation. By searching for BGAF homologs in sorghum, we identified and obtained an EST clone and determined its complete sequence. The predicted protein had the same modular structure as maize BGAF, shared 67% sequence identity with it, and revealed the presence of two potential carbohydrate-binding sites (GG...ATYLQ, site I and GG...GVVLD, site II). Maize BGAF1 is the only lectin from a class of modular JRLs containing an N-terminal dirigent and a C-terminal JRL domain, whose sugar specificity and beta-glucosidase aggregating activity have been studied in detail. We purified to homogeneity a BGAF homolog designated as SL (Sorghum lectin) from sorghum and expressed its recombinant version in Escherichia coli. The native protein had a molecular mass of 32 kD and was monomeric. Both native and recombinant SL-agglutinated rabbit erythrocytes, and inhibition assays indicated that SL is a GalNAc-specific lectin. Exchanging the GG...GVVLD motif in SL with that of maize BGAF1 (GG...GIAVT) had no effect on GalNAc-binding, whereas binding to Man was abolished. Substitution of Thr(293) and Gln(296) in site I to corresponding residues (Val(294) and Asp(297)) of maize BGAF1 resulted in the loss of GalNAc-binding, indicating that site I is responsible for generating GalNAc specificity in SL. Gel-shift and pull-down assays after incubating SL with maize and sorghum beta-glucosidases showed no evidence of interaction nor were any SL-protein complexes detected in sorghum tissue extracts, suggesting that the sorghum homolog does not participate in protein-protein interactions.
Subject(s)
Carrier Proteins/chemistry , Plant Lectins/chemistry , Plant Proteins/chemistry , Sorghum/metabolism , beta-Glucosidase/chemistry , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Clone Cells , Databases, Factual , Escherichia coli/genetics , Expressed Sequence Tags , Hemagglutination , Molecular Sequence Data , Molecular Structure , Molecular Weight , Open Reading Frames , Phylogeny , Plant Lectins/isolation & purification , Protein Structure, Tertiary , Rabbits , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Homology, Amino Acid , Sorghum/geneticsABSTRACT
The proteins present in the extracellular polymeric substances (EPS) of activated sludge flocs were investigated using three cation-associated extraction methods. The subproteomes generated from four full-scale activated sludges were subsequently fractionated by ammonium sulfate precipitation and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that each extraction method led to unique SDS-PAGE protein profiles, which provided strong evidence that the extracted proteins are uniquely associated with specific cations in activated sludge flocs. The comparison of protein profiles across sludges from different treatment plants revealed that extracts obtained using a cation-exchange resin exhibited similar protein banding patterns while sulfide- and base-extracted EPS led to more variable protein profiles. Analysis of several SDS-PAGE bands by liquid chromatography-tandem mass spectrometry of tryptic digests led to the identification of several bacterial proteins as well as sewage-derived polypeptides (human elastase IIIA and keratins). Their putative roles in activated sludges and their association with targeted cations are proposed.
Subject(s)
Proteins/chemistry , Sewage/chemistry , Ammonium Sulfate/chemistry , Chemical Precipitation , Electrophoresis, Polyacrylamide GelABSTRACT
The Arabidopsis genes At1g45130 and At3g52840 encode the beta-galactosidase isozymes Gal-5 and Gal-2 that belong to Glycosyl Hydrolase Family 35 (GH 35). The two enzymes share 60% sequence identity with each other and 38-81% with other plant beta-galactosidases that are reported to be involved in cell wall modification. We studied organ-specific expression of the two isozymes. According to our western blot analysis using peptide-specific antibodies, Gal-5 and Gal-2 are most highly expressed in stem and rosette leaves. We show by dot-immunoblotting that Gal-5 and Gal-2 are associated with the cell wall in Arabidopsis. We also report expression of the recombinant enzymes in P. pastoris and describe their substrate specificities. Both enzymes hydrolyze the synthetic substrate para-nitrophenyl-beta-d-galactopyranoside and display optimal enzyme activity between pH 4.0 and 4.5, similar to the pH optimum reported for other well-characterized plant beta-galactosidases. Both Gal-5 and Gal-2 show a broad specificity for the aglycone moiety and a strict specificity for the glycone moiety in that they prefer galactose and its 6-deoxy analogue, fucose. Both enzymes cleave beta-(1,4) and beta-(1,3) linkages in galacto-oligosaccharides and hydrolyze the pectic fraction of Arabidopsis cell wall. These findings suggest that Gal-5 and Gal-2 could be involved in the modification of cell wall polysaccharides.
Subject(s)
Arabidopsis/enzymology , Arabidopsis/genetics , Cell Wall/metabolism , Polysaccharides/metabolism , beta-Galactosidase/genetics , beta-Galactosidase/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Blotting, Western , Cell Wall/chemistry , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Phylogeny , Pichia/genetics , Polysaccharides/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Seeds/enzymology , Seeds/genetics , Substrate Specificity , beta-Galactosidase/chemistryABSTRACT
The structures of rice BGlu1 beta-glucosidase, a plant beta-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 A and 1.55 A resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GH1) beta-glucosidases, but showed several differences in the loops around the active site, which lead to an open active site with a narrow slot at the bottom, compatible with the hydrolysis of long beta-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polymyxa beta-glucosidase B, which hydrolyzes similar oligosaccharides, molecular docking studies indicate that the residues interacting with the substrate beyond the conserved -1 site are completely different, reflecting the independent evolution of plant and microbial GH1 exo-beta-glucanase/beta-glucosidases. The complex with the 2-fluoroglucoside included a glycerol molecule, which appears to be in a position to make a nucleophilic attack on the anomeric carbon in a transglycosylation reaction. The coordination of the hydroxyl groups suggests that sugars are positioned as acceptors for transglycosylation by their interactions with E176, the catalytic acid/base, and Y131, which is conserved in barley BGQ60/beta-II beta-glucosidase, that has oligosaccharide hydrolysis and transglycosylation activity similar to rice BGlu1. As the rice and barley enzymes have different preferences for cellobiose and cellotriose, residues that appeared to interact with docked oligosaccharides were mutated to those of the barley enzyme to see if the relative activities of rice BGlu1 toward these substrates could be changed to those of BGQ60. Although no single residue appeared to be responsible for these differences, I179, N190 and N245 did appear to interact with the substrates.
Subject(s)
Oligosaccharides/metabolism , Oryza/enzymology , beta-Glucosidase/chemistry , beta-Glucosidase/metabolism , Amino Acid Sequence , Binding Sites , Crystallization , Glycosylation , Hordeum/enzymology , Hydrolysis , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Binding , Sequence Homology, Amino Acid , Substrate Specificity , Zea mays/enzymology , Zinc/pharmacology , beta-Glucosidase/geneticsABSTRACT
Catalysing the hydrolysis of terminal beta-galactosyl residues from carbohydrates, galactolipids, and glycoproteins, glycoside hydrolase family 35 (beta-galactosidases; BGALs) are widely distributed in plants and believed to play many key roles, including modification of cell wall components. Completion of the Arabidopsis thaliana genome sequencing project has, for the first time, allowed an examination of the total number, gene structure, and evolutionary patterns of all Family 35 members in a representative (model) angiosperm. Reiterative database searches established a multigene family of 17 members (designated BGAL1-BGAL17). Using these genes as query sequences, BLAST and Hidden Markov Model searches identified BGAL genes among 22 other eukaryotes, whose genomic sequences are known. The Arabidopsis (n=17) and rice (n=15) BGAL families were much larger than those of Chlamydomonas, fungi, and animals (n=0-4), and a lineage-specific expansion of BGAL genes apparently occurred after divergence of the Arabidopsis and rice lineages. All plant BGAL genes, with the exception of Arabidopsis BGAL17 and rice Os 9633.m04334, form a monophyletic group. Arabidopsis BGAL expression levels are much higher in mature leaves, roots, flowers, and siliques but are lower in young seedlings. BGAL8, BGAL11, BGAL13, BGAL14, and BGAL16 are expressed only in flowers. Catalytically active BGAL4 was produced in the E. coli and baculoviral expression systems, purified to electrophoretic homogeneity, and partially characterized. The purified enzyme hydrolyzed p- and o-nitrophenyl-beta-d-galactosides. It also cleaved beta-(1,3)-, beta-(1,4)-, and beta-(1,6)-linked galactobiosides and galactotriosides, showing a marked preference for beta-(1,3)- and beta-(1,4)-linkages.
