Biodegradation of malachite green by Pseudomonas sp. strain DY1 under aerobic condition: characteristics, degradation products, enzyme analysis and phytotoxicity.

Malachite green (MG), a widely-used and recalcitrant dye, has been confirmed to be carcinogenic and mutagenic against many organisms. The main objective of this study is to investigate the capability of Pseudomonas sp. strain DY1 to decolorize MG, and to explore the possible mechanism. The results showed that this strain demonstrated high decolorizing capability (90.3-97.2%) at high concentrations of MG (100-1,000 mg/l) under shaking condition within 24 h. In static conditions, lower but still effective decolorization (78.9-84.3%) was achieved. The optimal pH and temperature for the decolorization was pH 6.6 and 28-30°C, respectively. Mg(2+) and Mn(2+) (1 mM) were observed to significantly enhance the decolorization. The intermediates of the MG degradation under aerobic condition identified by UV-visible, GC-MS and LC-MS analysis included malachite green carbinol, (dimethyl amino-phenyl)-phenyl-methanone, N,N-dimethylaniline, (methyl amino-phenyl)-phenyl-methanone, (amino phenyl)-phenyl methanone and di-benzyl methane. The enzyme analysis indicated that Mn-peroxidase, NADH-DCIP and MG reductase were involved in the biodegradation of MG. Moreover, phytotoxicity of MG and detoxification for MG by the strain were observed. Therefore, this strain could be potentially used for bioremediation of MG.

Isolation and characterization of Rhodococcus sp. NB5 capable of degrading a high concentration of nitrobenzene.

An efficient nitrobenzene degrading bacterium strain NB5, which was able to utilize nitrobenzene as a sole source of carbon, nitrogen and energy under aerobic condition, was isolated from activated sludge in an oil refinery at Hangzhou, China. Based on phenotypic features, 16S rDNA gene sequencing and G + C content analysis, strain NB5 was identified as Rhodococcus sp. NB5. Nitrobenzene degradation experiments using high performance liquid chromatograph (HPLC) showed that strain NB5 could tolerate a high nitrobenzene concentration and completely degrade nitrobenzene with initial concentration ranging from 100 mg · l(-1) to 1000 mg · l(-1) within 144 h. The optimal degradation and cell growth were observed at 30 °C, pH 7.0. The addition of second nitrogen source (0.1%) such as urea, peptone, yeast extract and beef extract generally enhanced degradation of nitrobenzene. Rhodococcus sp. strain NB5 could be an excellent candidate for biotreatment of industrial wastewater containing high concentration of nitrobenzene.

Ecofriendly decolorisation of Cr-complex dye Acid Black 172 by a newly isolated Pseudomonas sp. strain DY1.

Pseudomonas sp. strain DY1 was newly isolated from soil with rotten wood and identified as a member of the genus Pseudomonas based on 16S rDNA and biochemical tests. Acid Black 172, a water soluble Cr-complex dye, was then selected as a model dye to investigate the decolorisation ability of the strain. It was observed that the growth of the strain was not inhibited by high dose of metal ions (10 mM), and efficient decolorisation was still observed when high concentrations of Fe(2+), Fe(3+) and Ca(2+) existed. The optimal decolorising conditions obtained from Taguchi design were as follows: temperature 37˚C, pH 7.0, Fe(3+) and proline concentrations of 7 mM and 3.0 g/L, respectively. Under the optimal conditions, 94.5% of Acid Black 172 (100 mg/L) could be decolorised by the strain in 24 h. The kinetics of the decolorisation best fitted the first order kinetic model (R(2)=0.981). Besides, the phytotoxicity study demonstrated a good detoxification by the strain. This work has a certain practical value in microbial decolorisation of textile wastewater.

[The role of brain-derived neurotrophic factor in pain facilitation and spinal mechanism in rat model of bone cancer pain].

OBJECTIVE: To investigate the role of brain-derived neurotrophic factor (BDNF) in pain facilitation and spinal mechanisms in the rat model of bone cancer pain. METHODS: The bone cancer pain model was developed by inoculated Walker 256 mammary gland carcinoma cells into the tibia medullary cavity. Sixty SD female rats were divided into 5 groups (n = 12 each) randomly; group I: control group (sham operation); group II: model group; group III: control group + anti-BDNF intrathecal (i.t.); group IV: model group + control IgG i.t.; group V: model group + anti-BDNF i.t.. Anti-BDNF or control IgG was injected i.t. during 7 to 9th day. Von-Frey threshold was measured one day before operation and every 2 days after operation. On the 9th day after threshold tested, rats were sacrificed after i.t. injection of either anti-BDNF or control IgG, the lumbar 4-6 spinal cord was removed. The expression of the spinal BDNF and the phosphorylation of extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) were detected by immunohistochemistry assay and Western-Blot. Co-expression pattern of BDNF and p-ERK1/2 were determined by double-labeling immunofluorescence. RESULTS: We demonstrated the coexistence of BDNF and p-ERK1/2 in the spinal cord of rats. From the 7 to 9th day after operation, von-Frey threshold in groups II and IV was significantly lower than that in group I and group V (P < 0.01), group V was remarkly higher than that in group IV (P < 0.01). The spinal BDNF and p-ERK1/2 expression in group II or IV were significantly increased compared with that in group I or V (P < 0.01), intrathecal anti-BDNF was significantly suppressed BDNF and p-ERK1/2 expression (P < 0.01). CONCLUSION: BDNF and p-ERK1/2 was coexistence in the spinal cord of rats, and it maybe involved in the bone cancer pain.

The Development and Validation of the Doctoral Student Identity Scale.

Objective: Based on how the identity of doctoral students is recognized and understood in the context of Chinese culture, we developed a doctoral identity scale using both qualitative and quantitative analyses. Methods: The initial project of the Scale was formed through qualitative analyses and expert consultation. Nine hundred and ninety-one doctoral students were officially tested, and 982 valid questionnaires were obtained. They were randomly divided into two parts, and 491 of which were assessed for item Response Theory (IRT) and exploratory factor analysis (EFA) and 491 of which were assessed for confirmatory factor analysis (CFA). The Subjective Well-Being Scale (SWB), the Rosenberg Self-Esteem Scale (RSE), and the Psychological Sense of School Membership Scale (PSSM) were used to test its the criterion-related validity. One hundred and forty-one students were selected for retesting after 8 weeks. Results: The doctoral student identity questionnaire consisted of two factors identity exploration and identity commitment, explaining 57% of the total variance. The results of CFA showed that the two-factor model fitted the data well. The two dimensions of the Doctoral Student Identity Scale were significantly and positively correlated with the two dimensions of the SWB scale (0.32-0.66), the latent factor of the RSE scale (0.42-0.55), and the latent factor of the PSSM scale (0.52-0.62). Composite reliability values for exploration and commitment were 0.79 and 0.83 respectively, and the values of McDonald's omega for exploration and commitment were 0.81 and 0.85 respectively. The test-retest reliability of the total questionnaire was 0.842. Conclusion: The Doctoral Student Identity Scale was developed with good reliability and validity, and can be used as a reliable tool for measuring the doctoral student identity. In addition, the questionnaire will provide corresponding ideas and methods for studying the identity issues of specific groups.

[Effects of Tianbingtiaodu capsule on improvement of learning and memory abilities and expression of NMDAR1 of epileptic rats in the hippocampus].

OBJECTIVE: To study effects of Tianbingtiaodu capsule on changement of learning and memory abilities and expression of NMDAR1 in epileptic rats. METHODS: Picrotoxin (1.5 mg/kg) was injected intraperitonrally in SD rats for thirty days. After the repeated attacks epilepsy model had been established successfully, model rats were randomly divided into model group, Tianbingtiaodu capsule low dosage (0.4 g/kg) group, Tianbingtiaodu capsules high dosage(0. 8 g/kg) group, Piracetam group and Piracetam and Valproate group. All groups were fed everyday. Sixty days later, the learning and memory abilities were tested with Morris water maze method. Expression of NMDAR1 in hippocampus was observed with western-blot and immunohistochemical method. The expression of NMDAR1 mRNA in hippocampus was determined by RT-PCR. RESULTS: Compared to the control group, the learning and memory abilities were significantly lower (P < 0.01), immune positive remarks of expression of NMDAR1 in hippocampus AC3 significantly increased in model group (P < 0.05); Compared with model group,the learning and memory abilities and expression of NMDAR1 in hippocampus was improved significantly in Tianbingtiaodu group (P < 0.05 or P < 0.01). CONCLUSION: Tianbingtiaodu capsule could improve the impairment of learning and memory in rats after repeated attacks of epilepsy through adjusting the NMDAR1 expression.

Characteristics of and strategies for patients with severe burn-blast combined injury.

BACKGROUND: Severe burn-blast combined injury is a great challenge to medical teams for its high mortality. The aim of this study was to elucidate the clinical characteristics of the injury and to present our clinical experiences on the treatment of such cases. METHODS: Five patients with severe burn-blast combined injuries were admitted to our hospital 77 hours post-injury on June 7, 2005. The burn extent ranged from 80% to 97% (89.6% +/- 7.2%) of TBSA (full-thickness burns 75% - 92% (83.4% +/- 7.3%)). All the patients were diagnosed as having blast injury and moderate or severe inhalation injury. Functions of the heart, liver, kidney, lung, pancreas and coagulation were observed. Autopsy samples of the heart, liver, and lungs were taken from the deceased. Comprehensive measures were taken during the treatment, including protection of organ dys function, use of antibiotics, early anticoagulant treatment, early closure of burn wounds, etc. All the data were analyzed statistically with t test. RESULTS: One patient died of septic shock 23 hours after admission (four days after injury), the others survived. Dysfunction of the heart, liver, lungs, pancreas, and coagulation were found in all the patients on admission, and the functions were ameliorated after appropriate treatments. CONCLUSIONS: Burn-blast combined injury may cause multiple organ dysfunctions, especially coagulopathy. Proper judgment of patients' condition, energetic anticoagulant treatment, early closure of burn wounds, rational use of antibiotics, nutritional support, intensive insulin treatment, timely and effective support and protection of organ function are the most important contributory factors in successful treatment of burn-blast combined injuries.

[Application of carbon fiber dressing on burn wounds].

OBJECTIVE: To observe the effects of carbon fiber dressing on burn wounds. METHODS: Two hundreds and seventy seven burn patients were randomly divided into treatment group (group T) and control group (group C). The burn wounds were covered with carbon fiber dressing in T group, and with povidone iodine gauze in C group, respectively. The absorption capability of the dressing, inflammatory reaction and bacteria quantitation of wound tissues and wound healing time were observed, and biopsy of wounds were performed. RESULTS: The absorption capability of the dressing was higher, the wound inflammatory reaction was milder, and bacteria quantitation of wound tissues was lower in the group T than that in group C. The wound healing time in the group T was shorter than that in group C. CONCLUSIONS: Carbon fiber dressing is a new model dressing, it can absorb wound exudation, lessen inflammatory reaction and improve wound healing.

Can HB vaccine yield a booster effect on individuals with positive serum anti-HBs and anti-HBc markers?

AIM: To evaluate if HB vaccination can yield a booster effect on the anti-HBs level of those naturally acquired HBV positive markers. METHODS: Sera were collected from 1399 newly enrolled university students aged between 18-20 years at the entrance medical examination in 2001. Forty-four students (28 males and 16 females) with positive serum anti-HBs and anti-HBc markers served as an observation group and another 44 students (24 males and 20 females) without any HBV markers as the control. HB vaccination was given to all the students without positive serum HBsAg according to 0, 1, 6 month regimen and the peripheral venous blood was sampled from those of both observation and control groups for anti-HBs detection one month after the second and third doses. Anti-HBs levels were measured by ELISA. RESULTS: The seroconversion rate of anti-HBs in the control group was 100% after the second dose, but the geometric mean titers (GMTs) were low. The tendency of serum anti-HBs changes after the 3rd dose was completely different between the two groups. Although more than half of those with positive anti-HBs and anti-HBc showed a mild increase of anti-HBs levels after the 2nd boosting dose (mean anti-HBs level was 320:198 mIU), but the increase of serum anti-HBs titer was much smaller than that in the control group. The averages of their initial serum anti-HBs levels and the levels after the 2nd and 3rd doses were 198, 320 and 275 mIU respectively. All the subjects from the control group had an obvious increase in their serum anti-HBs levels which was nearly 4 times the baseline level (302:78 mIU). CONCLUSION: HB vaccination can not enhance anti-HBs levels in those with positive serum anti-HBs and anti-HBc markers.

Biodegradation of methyl red by Bacillus sp. strain UN2: decolorization capacity, metabolites characterization, and enzyme analysis.

Azo dyes are recalcitrant and refractory pollutants that constitute a significant menace to the environment. The present study is focused on exploring the capability of Bacillus sp. strain UN2 for application in methyl red (MR) degradation. Effects of physicochemical parameters (pH of medium, temperature, initial concentration of dye, and composition of the medium) were studied in detail. The suitable pH and temperature range for MR degradation by strain UN2 were respectively 7.0-9.0 and 30-40 °C, and the optimal pH value and temperature were respectively 8.0 and 35 °C. Mg(2+) and Mn(2+) (1 mM) were found to significantly accelerate the MR removal rate, while the enhancement by either Fe(3+) or Fe(2+) was slight. Under the optimal degradation conditions, strain UN2 exhibited greater than 98 % degradation of the toxic azo dye MR (100 ppm) within 30 min. Analysis of samples from decolorized culture flasks confirmed biodegradation of MR into two prime metabolites: N,N'dimethyl-p-phenyle-nediamine and 2-aminobenzoic acid. A study of the enzymes responsible for the biodegradation of MR, in the control and cells obtained during (10 min) and after (30 min) degradation, showed a significant increase in the activities of azoreductase, laccase, and NADH-DCIP reductase. Furthermore, a phytotoxicity analysis demonstrated that the germination inhibition was almost eliminated for both the plants Triticum aestivum and Sorghum bicolor by MR metabolites at 100 mg/L concentration, yet the germination inhibition of parent dye was significant. Consequently, the high efficiency of MR degradation enables this strain to be a potential candidate for bioremediation of wastewater containing MR.

Cloning, expression, and characterization of a wide-pH-range stable phosphite dehydrogenase from Pseudomonas sp. K in Escherichia coli.

A phosphite dehydrogenase gene (ptdhK) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdhK was expressed in Escherichia coli BL21 (DE3) and the corresponding recombinant enzyme was purified by metal affinity chromatography. The recombinant protein is a homodimer with a monomeric molecular mass of 37.2 kDa. The specific activity of PTDH-K was 3.49 U mg(-1) at 25 °C. The recombinant PTDH-K exhibited maximum activity at pH 3.0 and at 40 °C and displayed high stability within a wide range of pHs (5.0 to 10.5). PTDH-K had a high affinity to its natural substrates, with K (m) values for sodium phosphite and NAD of 0.475 ± 0.073 and 0.022 ± 0.007 mM, respectively. The activity of PTDH-K was enhanced by Na(+), NH (4) (+) , Mg(2+), Fe(2+), Fe(3+), Co(2+), and EDTA, and PTDH-K exhibited different tolerance to various organic solvents.

Treatment strategies for mass burn casualties.

BACKGROUND: Mass burn casualties are always a great challenge to a medical team because a large number of seriously injured patients were sent in within a short time. Usually a high mortality is impending. Experiences gained from successful treatment of the victims may be useful in guiding the care of mass casualties in an armed conflict. METHODS: Thirty-five burn victims in a single batch, being transferred nonstop by air and highway from a distant province, were admitted 48 hours post-injury. All patients were male with a mean age of (22.4 +/- 8.7) years. The burn extent ranged from 4% to 75% ((13.6 +/- 12.9)%) total body surface area. Among them, thirty-two patients were complicated by moderate and severe inhalation injury, and tracheostomy had been performed in 15 patients. Decompression incisions of burn eschar on extremities were done in 17 cases before transportation. All the thirty-five patients arrived at the destination smoothly via 4-hour airlift and road transportation. Among them, twenty-five patients were in critical condition. RESULTS: These thirty-five patients were evacuated 6 hours from the scene of the injury, and they were transferred to a local hospital for primary emergency care. The patients were in very poor condition when admitted to our hospital because of the severe injury with delayed and inadequate treatment. Examination of these patients at admission showed that one patient was suffering from sepsis and multiple organ dysfunction syndrome. Dysfunction of the heart, lung, liver, kidney, and coagulation were all found in the patients. Forty-eight operations were performed in the 23 patients during one month together with comprehensive treatment, and the function of various organs was ameliorated after appropriate treatment. All the 35 patients survived. CONCLUSIONS: A well-organized team consisting of several cooperative groups with specified duties is very important. As a whole, the treatment protocol should be individualized, basing on the extent of the injury and the care that the patient had received at the spot. During airlift, the stretchers should be arranged perpendicular to the longitudinal axis of the cabin. The treatment protocol in our hospital consisted mainly of prompt effective relief of all life-threatening complications, followed by early closure of burn wounds, appropriate use of anti-infection therapy, emphasis on nutritional support, correction of metabolic disorders, alleviation of immunosuppression, correction of coagulopathy, and effective support and protection of organ function.

[Purification and characteristics of creatininase from Arthrobacter sp].

A creatininase produced from a Arthrobacter sp. was purified 145-fold by a series of steps including heat treatment, ammonium sulfate precipitation, DEAE-Cellulose ion-exchange and hydrophobic chromatography. The specific activity of the pure enzyme was 209u/mg. The subunit molecular mass of creatininase was estimated to be 33 700D by SDS-PAGE. The creatininase was stable in the pH range between 6.0 - 9.0 and below 60 degrees C . Its Km value for creatinine was estimated to be 21.14 mmol/L. The enzyme was markedly inactivated by incubation with 1 mmol/L of Hg2+, Ag2+, Li+, Cu2+ and 20 mmol/L of 1, 11-Phananthroline respectively. Activation was observed when the enzyme was incubated with 1 mmol/L of Co2+ and Mn2+.

[Study on the properties of a novel glycine amino peptidase from Actinomucor elegans].

The glycine amino peptidase of Actinomucor elegans was studied in this work. For the enzyme production Actinomucor elegans was cultured with an enzyme producing medium. Then the cells were collected and subjected to enzyme purification. The glycine aminopeptidase was purified 592 times by a DEAE-Toyopearl column, a Toyopearl HW 65-C column and a Superdex 200 column subsequently and the purified enzyme had a specific activity of 14.2 u/mg. The enzyme was estimated to have molecular mass of 320kD by gel filtration and a subunit size of 56.5kD by SDS-PAGE. It hydrolyzes glycine residue containing substrates such as glycine-betanaphthylamine more efficiently than those containing other amino acid residue. Addition to Gly-betaNA, the enzyme could also hydrolyze Ala-betaNA, Met-betaNA, Leu-betaNA, Arg-betaNA and Ser-betaNA but it had no activity on the substrates such as Trp-betaNA, Pyr-betaNA, Pro-betaNA, Asp-betaNA, Lys-betaNA, Val-betaNA. It was also observed when the glycine-betanaphthylamine concentration was higher than 2mmol/L the enzyme showed a substrate inhibition, and at the 20 mmol/L the enzyme only showed about 55% activity as it showed at the 2mmol/L. Whereas no such phenomenon was observed on the other substrate such as alanine-betanaphthylamine. The optimal temperature and pH for the reaction of this enzyme is 30 degrees C and pH 8.0, respectively. The Km and Kcat of the enzyme for glycine-betanaphthylamine is 0.24 mmol/L and 100.8 s(-1), respectively. Zn2+, Cu2+ and Cd2+ suppress almost all activities of the enzyme at the concentration of 1.0 mmol/L. Based on the study of chelating reagents, GAP belongs to the metalloenzyme. When a gelatin solution was hydrolyzed with 0.5% of alkaline proteinase together with glycine aminopeptidase at 50 degrees C for 18 hours, the glycine aminopeptidase could improve the hydrolysis degree of the protease. The total free amino acid was improved about 13% and although the enzyme mainly had the activity to hydrolyze the glycine residue, individual amino acids analysis with an amino acid analyzer showed that the contents of glycine, proline, alanine, arginine and glutamate were considerably increased. The results of this study showed that the glycine aminopeptidase would be useful in the food industry.

Phylogenetic study on Shiraia bambusicola by rDNA sequence analyses.

In this study, 18S rDNA and ITS-5.8S rDNA regions of four Shiraia bambusicola isolates collected from different species of bamboos were amplified by PCR with universal primer pairs NS1/NS8 and ITS5/ITS4, respectively, and sequenced. Phylogenetic analyses were conducted on three selected datasets of rDNA sequences. Maximum parsimony, distance and maximum likelihood criteria were used to infer trees. Morphological characteristics were also observed. The positioning of Shiraia in the order Pleosporales was well supported by bootstrap, which agreed with the placement by Amano (1980) according to their morphology. We did not find significant inter-hostal differences among these four isolates from different species of bamboos. From the results of analyses and comparison of their rDNA sequences, we conclude that Shiraia should be classified into Pleosporales as Amano (1980) proposed and suggest that it might be positioned in the family Phaeosphaeriaceae.

[Application of digital subtraction angiography and type B ultrasonography in the evaluation of vascular injury in patients with high voltage electrical injury].

OBJECTIVE: To compare the difference between digital subtraction angiography (DSA) and type B ultrasonography in the evaluation of vascular injury in patients inflicted with high voltage electrical injury. METHODS: Nineteen patients with high voltage electrical injury of upper limbs were enrolled in the study as burn group, and another 12 healthy volunteers as controls. The endovascular membrane, vascular wall thickness, intra-vascular blood flow and endovascular thrombosis formation of ulnar and radial arteries at wound site and in regions 5, 10 and 15 cm proximal to the wounds were examined by DSA and type B ultrasonography and compared with imagings of healthy volunteers as control. The injury degree of the ulnar and radial arteries was examined during operation for evaluation to corroborate with DSA and ultrasonography findings. Necrotic and/or thrombotic vessels were excised and sent for pathomorphological examination. RESULTS: By DSA images abnormal signs as thrombosis, vascular lumen stenosis and blood flow deceleration were found in 14 ulnar and 11 radial arteries, and the signs were more pronounced in ulnar arteries. By type B ultrasonography, abnormal signs as roughing of tunica intima, swelling or exfoliation, thickening of vascular wall, lumen stenosis, decreased blood flow, even necrosis of vascular wall and thrombosis were identified in 19 ulnar and 16 radial arteries in burn group (P < 0.05 approximately 0.01). The blood flow in ulnar artery 5 cm to the approximal part of the wound edge was obvious lower than that of the control (31.60 +/- 13.90 ml/min vs 47.70 +/- 9.60 ml/min, P < 0.05). CONCLUSION: Type B ultrasonography and DSA could be helpful in the evaluation of vascular injury in patients inflicted with high voltage electrical injury.