[Discussion of the Roles of Medical Social Workers in the Response to the Explosion Incident at Formosa Fun Coast].

Medical social workers apply the theories of "person in the environment" (PIE) and "ecological perspective" as practical foundations. Furthermore, they emphasize the people, the environment, and the interactions between these two. When burn patients from the explosion at Formosa Fun Coast were sent to hospitals, social workers not only provided care and assessed the impact on burn patients but also assisted in supporting the family members of these patients. This article discusses the various roles of social workers within different systems. In the individual system, we use Eric Erickson's theory of psychosocial development to evaluate the patient's crisis and the tasks of social workers. Secondly, in the systems of family, school, and work, we assess the relationships between a patient, his/her significant others, and caregivers as well as the interactions among sub-systems in the family. In the community and cultural systems, we focus on the social resources that may be utilized by the burn patients after discharge. Moreover, we add a time frame to examine our major tasks, including the initial stage, the middle stage, and the preparation-for-discharge stage. We explore the roles of social workers, the applicable theories, and the goals for each stage.

Differential expression of genes encoding acid invertases in multiple shoots of bamboo in response to various phytohormones and environmental factors.

The promoter regions of two cell wall invertase genes, Boßfruct1 and Boßfruct2, and a vacuolar invertase gene, Boßfruct3, in Bambusa oldhamii were cloned, and putative regulatory cis-elements were identified. The expression of these three genes in multiple shoots of bamboo that were cultured in vitro under different conditions was analyzed by real-time PCR. The two cell wall invertase genes were upregulated by indole-3-acetic acid and cytokinins but responded differently to other phytohormones and different temperatures. Boßfruct1 was also upregulated by sucrose and glucose. In contrast, the Boßfruct2 expression was induced by the depletion of sucrose, and this induction could be suppressed by glucose and sucrose. The expression of Boßfruct3 was light-dependent; however, abscisic acid (ABA) could induce its expression in the dark. ABA and light exhibited an additive effect on the expression of Boßfruct3. Our results suggest that these three Boßfruct genes have individual roles in the adaption of the plant to environmental changes. Boßfruct2 might also have an essential role in the immediate response of cells to sucrose availability and in the maintenance of sink activity. Moreover, Boßfruct3 might be one of the interacting nodes of the light and ABA signaling pathways.

Purification and characterization of two chitosanase isoforms from the sheaths of bamboo shoots.

Two thermally stable chitosanase isoforms were purified from the sheaths of chitosan-treated bamboo shoots. Isoforms A and B had molecular masses of 24.5 and 16.4 kDa and isoelectric points of 4.30 and 9.22, respectively. Using chitosan as the substrate, both isoforms functioned optimally between pH 3 and 4, and the optimum temperatures for the activities of isoforms A and B were 70 and 60 °C, respectively. The kinetic parameters K(m) and V(max) for isoform A were 0.539 mg/mL and 0.262 µmol/min/mg, respectively, and for isoform B were 0.183 mg/mL and 0.092 µmol/min/mg, respectively. Chitosans were susceptible to degradation by both enzymes and could be converted to low molecular weight chitosans between 28.2 and 11.7 kDa. Furthermore, the most susceptible chitosan substrates were 50-70 and 40-80% deacetylated for isoforms A and B, respectively. Both enzymes could also degrade chitin substrates with lower efficacy. N-Bromosuccinimide and Woodward's reagent K strongly inhibited both enzymes.

Insights into the catalytic properties of bamboo vacuolar invertase through mutational analysis of active site residues.

Plant acid invertases, which are either associated with the cell wall or present in vacuoles, belong to family 32 of glycoside hydrolases (GH32). Homology modeling of bamboo vacuolar invertase Bobetafruct3 using Arabidopsis cell-wall invertase AtcwINV1 as a template showed that its overall structure is similar to GH32 enzymes, and that the three highly conserved motifs, NDPNG, RDP and EC, are located in the active site. This study also used site-directed mutagenesis to examine the roles of the conserved amino acid residues in these three motifs, which include Asp135, Arg259, Asp260, Glu316 and Cys317, and a conserved Trp residue (Trp159) that resides between the NDPNG and RDP motifs. The mutants W159F, W159L, E316Q and C317A retained acid invertase activity, but no invertase activity was observed for the mutant E316A or mutants with changes at Asp135, Arg259, or Asp260. The apparent K(m) values of the four mutants with invertase activity were all higher than that of the wild-type enzyme. The mutants W159L and E316Q exhibited lower k(cat) values than the wild-type enzyme, but an increase in the k(cat) value was observed for the mutants W159F and C317A. The results of this study demonstrate that these residues have individual functions in catalyzing sucrose hydrolysis.

Cloning and characterization of an antifungal class III chitinase from suspension-cultured bamboo ( Bambusa oldhamii ) cells.

A class III chitinase cDNA (BoChi3-1) was cloned using a cDNA library from suspension-cultured bamboo ( Bambusa oldhamii ) cells and then transformed into yeast ( Pichia pastoris X-33) for expression. Two recombinant chitinases with molecular masses of 28.3 and 35.7 kDa, respectively, were purified from the yeast's culture broth to electrophoretic homogeneity using sequential ammonium sulfate fractionation, Phenyl-Sepharose hydrophobic interaction chromatography, and Con A-Sepharose chromatography steps. N-Terminal sequencing and immunoblotting revealed that both recombinant chitinases were encoded by BoChi3-1, whereas SDS-PAGE and glycoprotein staining showed that the 35.7 kDa isoform (35.7 kDa BoCHI3-1) was glycosylated and the 28.3 kDa isoform (28.3 kDa BoCHI3-1) was not. For hydrolysis of ethylene glycol chitin (EGC), the optimal pH values were 3 and 4 for 35.7 and 28.3 kDa BoCHI3-1, respectively; the optimal temperatures were 80 and 70 degrees C, and the K(m) values were 1.35 and 0.65 mg/mL. The purified 35.7 kDa BoCHI3-1 hydrolyzed EGC more efficiently than the 28.3 kDa isoform, as compared with their specific activity and activation energy. Both recombinant BoCHI3-1 isoforms showed antifungal activity against Scolecobasidium longiphorum and displayed remarkable thermal (up to 70 degrees C) and storage (up to a year at 4 degrees C) stabilities.

Molecular cloning and functional identification of invertase isozymes from green bamboo Bambusa oldhamii.

Three Bo beta fruct cDNAs encoding acid invertases were cloned from shoots of the green bamboo Bambusa oldhamii. On the basis of the amino acid sequences of their products and phylogenetic analyses, Bo beta fruct1 and Bo beta fruct2 were determined to encode cell wall invertases, whereas Bo beta fruct3encodes a vacuolar invertase. The recombinant proteins encoded by Bo beta fruct2 and Bo beta fruct3 were produced in Pichia pastoris and purified to near homogeneity using ammonium sulfate fractionation and immobilized metal affinity chromatography. The pH optima, pI values, and substrate specificities of the isolated enzymes were consistent with those of plant cell wall or vacuolar invertases. The growth-dependent expression of Bo beta fruct1 and Bo beta fruct2 in the base regions of shoots underscores their roles in sucrose unloading and providing substrates for shoot growth. Its high sucrose affinity suggests that the Bo beta fruct2-encoded enzyme is important for maintaining the sucrose gradient between source and sink organs, while the predominant expression of Bo beta fruct3 in regions of active cell differentiation and expansion suggests functions in osmoregulation and cell enlargement.

Characterization of a chitosanase isolated from a commercial ficin preparation.

A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).

Vacuolar invertases in sweet potato: molecular cloning, characterization, and analysis of gene expression.

Two cDNAs (Ib beta fruct2 and Ib beta fruct3) encoding vacuolar invertases were cloned from sweet potato leaves, expressed in Pichia pastoris, and the recombinant proteins were purified by ammonium sulfate fractionation and chromatography on Ni-NTA agarose. The deduced amino acid sequences encoded by the cDNAs contained characteristic conserved elements of vacuolar invertases, including the sequence R[G/A/P]xxxGVS[E/D/M]K[S/T/A/R], located in the prepeptide region, Wxxx[M/I/V]LxWQ, located around the starting site of the mature protein, and an intact beta-fructosidase motif. The pH optimum, the substrate specificity, and the apparent K(m) values for sucrose exhibited by the recombinant proteins were similar to those of vacuolar invertases purified from sweet potato leaves and cell suspensions, thus confirming that the proteins encoded by Ib beta fruct2 and Ib beta fruct3 are vacuolar invertases. Moreover, northern analysis revealed that the expression of the two genes was differentially regulated. With the exception of mature leaves and sprouting storage roots, Ib beta fruct2 mRNA is widely expressed among the tissues of the sweet potato and is more abundant in young sink tissues. By contrast, Ib beta fruct3 mRNA was only detected in shoots and in young and mature leaves. It appears, therefore, that these two vacuolar invertases play different physiological roles during the development of the sweet potato plant.

Cloning and characterization of a plant defensin VaD1 from azuki bean.

A recombinant mungbean defensin VrD1 was previously shown to exhibit antifungal and bruchid-resistant activity. To study the function and regulation of VrD1, genomic DNAs of plant defensins were isolated from Vigna radiata VC6089A and azuki bean Vigna angularis Kao Hsiung No. 6. The azuki bean defensin genomic DNA VaD1 was sequenced and converted to VaD1 cDNA. VaD1 defensin was purified from Vigna angularis Kao Hsiung No. 6 to apparent homogeneity. The complete amino acid sequence of the purified VaD1 was determined and was found to be exactly the same as the sequence deduced from VaD1 cDNA. VaD1 is a basic protein containing 46 amino acids with four conserved disulfide bonds and shares high sequence homology (78.3%) with VrD1. VaD1 inhibited the growth of Fusarium oxysporum, Fusarium oxysporum f. sp. pisi, Staphylococcus epidermidis, and Salmonella typhimurium. VaD1 also inhibited in vitro protein synthesis and bruchid larval development, but was less active than the recombinant VrD1.

A cDNA encoding vacuolar type beta-D-fructofuranosidase (Os beta fruct3) of rice and its expression in Pichia pastoris.

A vacuolar type beta-D-fructofuranosidase (Os beta fruct3) was cloned from etiolated rice seedlings cDNA library. It encodes an open reading frame of 688 residues. The deduced amino acid sequence had 58% identity to the vacuolar type beta-D-fructofuranosidase of maize (Ivr1). Os beta fruct3 exists as a single copy per genome. Northern analyses showed that Os beta fruct3 undergoes organ-specific expression and is involved in the adjustment of plant responses to environmental signals and metabolizable sugars. Os beta fruct3 was also heterologously expressed in Pichia pastoris. The recombinant proteins were confirmed to be a vacuolar type beta-D-fructofuranosidase.

Cloning, characterization and functional expression of a new beta-D-fructofuranosidase (Os beta fruct2) cDNA from Oryza sativa.

A new cDNA (Os beta fruct2) encoding an acid beta-D-fructofuranosidase from rice has been cloned, sequenced and expressed in Pichia pastoris. The full-length cDNA is 2453 base pairs long and encodes a pre-pro-protein of 682 amino acids. The cDNA fragment coding for mature enzyme was sub-cloned into vector pPICZalphaA for extracellular expression in the methylotrophic yeast. The recombinant product was purified by Ni2+-nitrilotriacetic acid agarose column and biochemically characterized. The enzyme could hydrolyze sucrose and raffinose. Molecular mass is 66 kDa. The activity optimum was at pH 4.8 and 40 degrees C.

Expression and characterization of sweet potato invertase in Pichia pastoris.

An invertase cDNA (Ibbetafruct1) was cloned from sweet potato leaves and characterized. The deduced amino acid sequence of the Ibbetafruct1-encoded protein was closely related to vacuolar invertases and included the WECVD catalytic domain characteristic of them. An expression plasmid containing the coding region of Ibbetafruct1 under the control of the alcohol oxidase promoter was used to transform the methylotrophic yeast Pichia pastoris. The biochemical properties for the expressed recombinant enzyme, which was determined to be the acid beta-fructofuranosidase with an acidic pI value (5.1), were similar to those of vacuolar invertases purified from sweet potato. Periodic acid/Schiff staining and Con A-Sepharose gel-binding experiments revealed the recombinant invertase to be a glycoprotein containing glucose and/or mannose residues. Furthermore, the carbohydrate moiety appears to be a key determinant of the enzyme's sucrose hydrolysis activity, substrate affinity, and thermal stability.

Characterization of three chitosanase isozymes isolated from a commercial crude porcine pepsin preparation.

Three chitosanases designated PSC-I, PSC-II, and PSC-III were purified from commercial pepsin preparation by sequentially applying pepstatin A-agarose affinity chromotography, DEAE-Sephacel ion-exchange chromatography, Mono Q column chromatography, and Mono P chromatofocusing. With respect to chitosan hydrolysis, the optimal pHs were 5.0, 5.0, and 4.0 for PSC-I, PSC-II, and PSC-III, respectively; optimal temperatures were 40, 40, and 30 degrees C; and the Km's were 5.2, 4.0, and 5.6 mg/mL. The molecular masses of the three isozymes were approximately 40 kDa, as estimated by both gel filtration and SDS-PAGE, and the isoelectric points were 4.9, 4.6, and 4.4, respectively, as estimated by isoelectrofocusing electrophoresis. All three chitosanase isozymes showed activity toward chitosan polymer and N,N",N' "-triacetylchitotriose oligomer. Most effectively hydrolyzed were chitosan polymers that were 68-88% deacetylated.

Invertase inhibitors from sweet potato (Ipomoea batatas): purification and biochemical characterization.

Two proteinaceous invertase inhibitors, designated ITI-L and ITI-R, were purified to electrophoretic homogeneity. ITI-L was purified from acetone powder of sweet potato leaves through sequential steps entailing buffer extraction, acid treatment, DEAE-Sephacel ion-exchange chromatography, and Sephacryl S-100 gel filtration. ITI-R was purified from sweet potato tuberous roots by sequentially applying buffer extraction, Con A-Sepharose affinity chromatography, DEAE-Sephacel ion-exchange chromatography, Sephacryl S-200, and Superose 12 gel filtration. The optimal pHs for interaction between ITI-L and ITI-R and acid invertase from sweet potato leaves were 5.5 and 5.0, respectively. The molecular masses of ITI-L and ITI-R were 10 and 22 kDa, respectively, as estimated by both gel filtration and SDS-PAGE. Both inhibitors were thermostable (90% of the activity remained after incubation at 100 degrees C for 20 min), and Western blotting showed them to be immunologically related.

A novel defensin encoded by a mungbean cDNA exhibits insecticidal activity against bruchid.

A cDNA encoding a small cysteine-rich protein designated VrCRP was isolated from a bruchid-resistant mungbean. VrCRP encodes a protein of 73 amino acids containing a 27 amino acid signal peptide and 8 cysteines. On the basis of the amino acid sequence similarity and conserved residues, it is suggested that VrCRP is a member of the plant defensin family. VrCRP protein was obtained by overexpression of VrCRP with a truncated signal peptide in an IMPACT system. Artificial seeds containing 0.2% (w/w) of the purified VrCRP-TSP were lethal to larvae of the bruchid Callosobruchus chinensis. VrCRP is apparently the first reported plant defensin exhibiting in vitro insecticidal activity against C. chinensis.

Purification and characterization of hydrolase with chitinase and chitosanase activity from commercial stem bromelain.

A hydrolase with chitinase and chitosanase activity was purified from commercial stem bromelain through sequential steps of SP-Sepharose ion-exchange adsorption, HiLoad Superdex 75 gel filtration, HiLoad Q Sepharose ion-exchange chromatography, and Superdex 75 HR gel filtration. The purified hydrolase was homogeneous, as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited chitinase activity for hydrolysis of glycol chitin and 4-methylumbelliferyl beta-D-N,N',N' '-triacetylchitotrioside [4-MU-beta-(GlcNAc)(3)] and chitosanase activity for chitosan hydrolysis. For glycol chitin hydrolysis, the enzyme had an optimal pH of 4, an optimal temperature of 60 degrees C, and a K(m) of 0.2 mg/mL. For the 4-MU-beta-(GlcNAc)(3) hydrolysis, the enzyme had an optimal pH of 4 and an optimal temperature of 50 degrees C. For the chitosan hydrolysis, the enzyme had an optimal pH of 3, an optimal temperature of 50 degrees C, and a K(m) of 0.88 mg/mL. For hydrolysis of chitosans with various N-acetyl contents, the enzyme degraded 30-80% deacetylated chitosan most effectively. The enzyme split chitin or chitosan in an endo-manner. The molecular mass of the enzyme estimated by gel filtration was 31.4 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 5.9. Heavy metal ions of Hg(2+) and Ag(+), p-hydroxymercuribenzoic acid, and N-bromosuccinimide significantly inhibited the enzyme activity.