[Effects of salt stress on bacterial community composition and diversity in rhizosphere soil of Bletilla striata]. / ç›èƒè¿«å¯¹ç™½åŠæ ¹é™ ç»†èŒç¾¤è½ç»„æˆåŠå¤šæ ·æ€§çš„å½±å“.

Salinization environment affects the normal growth and development of plants, as well as the microbial community in the rhizosphere. To explore the succession dynamics of bacterial communities in the rhizosphere soil of Bletilla striata under salt stress condition, we performed 16S rRNA high-throughput sequencing to determine the bacterial community composition and diversity of B. striata in the rhizosphere under different salt stress concentrations, measured the effects of salt stress on the growth and development of B. striata and soil physicochemical pro-perties, and analyzed the correlation between community composition of rhizosphere bacteria and the soil environmental factors. The results showed that compared with the control, salt stress reduced growth rate and health degree of B. striata, and significantly decreased the content of soil organic matter, nitrogen and phosphorus. Under the salt stress treatment, species diversity and evenness of the bacterial communities in the rhizosphere of B. striata showed a trend of first decreasing and then increasing. There were significant differences in the relative abundance and variation trends of the dominant bacterial taxa in the rhizosphere soil of B. striata at the phylum and class levels between the control and the salt stress treatments. Salt stress intensity and duration were important factors affecting bacterial community composition in the rhizosphere soil of B. striata. Soil organic matter, available nitrogen, and total phosphorus content were key environmental factors affecting the structure of rhizosphere bacterial community composition. Functional genes related to cytoskeleton, cell motility, substance metabolism and signal transduction mechanisms may be involved in the adaptation and stress response of bacterial communities to salt stress. This study would provide theoretical basis and reference for the cultivation management of B. striatain saline area.

Comparison of Antioxidant Constituents of Agriophyllum squarrosum Seed with Conventional Crop Seeds.

Twelve chemical constituents were identified from the Agriophyllum squarrosum seed (ASS). ASS contained large amounts of flavonoids, which were more concentrated in the seed coat. ASS-coat (1 g) contained 335.7 µg flavonoids of rutin equivalent, which was similar to the flavonoid content in soybean (351.2 µg/g), and greater than that in millet, wheat, rice, peanut, and corn. By LC-MS analysis, the major constituents in ASS were 3-O-[α-L-rhamnopyranosyl-(1→6)-ß-D- glucopyranosyl]-7- O-(ß-D-glucopyranosyl)-quercetin (1), rutin (4), quercetin-3-O-ß-D- apiosyl(1→2)-[α-L-rhamnosyl(l→6)]-ß-D-glucoside (2), isorhamnetin-3-O-rutinoside (5), and allantoin (3), compared with isoflavonoids-genistin (16), daidzin (14), and glycitin (18) in soybean. Among constituents in ASS, compounds 1, 2, 4, protocatechuic acid (8), isoquercitrin (11), and luteolin-6-C-glucoside (12) potently scavenged DPPH radicals and intracellular ROS; strongly protected against peroxyl radical-induced DNA scission; and upregulated Nrf2, phosphorylated p38, phosphorylated JNK, and Bcl-2 in HepG2 cells. These results indicate that ASS is rich in antioxidant constituents that can enrich the varieties of food flavonoids, with significant beneficial implications for those who suffer from oxidative stress-related conditions. PRACTICAL APPLICATION: This study found that A. squarrosum seed contains large amounts of antioxidative flavonoids and compared its chemical constituents with those of conventional foods. These results should increase the interest in planting the sand-fixing A. squarrosum on a large scale, thus preventing desertification and providing valuable foods.

New cytotoxic triterpene glycoside from the East China Sea cucumber Holothuria nobilis.

A new cytotoxic triterpene glycoside named nobiliside E (1) has been obtained from the East China Sea cucumber Holothuria nobilis Selenka. Its structure was determined on the basis of NMR spectroscopic and MS analyses, together with chemical evidence. The new compound showed significant cytotoxicity to eight human tumor cell lines with IC50 values in the range of 0.53-4.06 µg/mL.

[Diagnosis and treatment of isolated methylmalonic acidemia].

OBJECTIVE: To explore the clinical feature, therapeutic effect and prognosis of isolated methylmalonic acidemia. METHODS: The clinical characteristics, laboratory findings, treatment and outcome of 40 patients were retrospectively analyzed. The main treatment was a low-protein diet supplemented with L-carnitine and special milk free of leucine, valine, threonine and methionine. Vitamin B12 was also given to cobalamin responders. The patients were followed up every 1-3 months. RESULTS: Mutations in the MUT gene were identified in 30 of 33 patients who had accepted DNA testing. Thirty cases were treated and followed up regularly for from 1 month to 8 years. Eight cases had died, 8 had developed normal intelligence, among whom 4 from newborn screening were asymptomatic. Psychomotor developmental delay and mental retardation were present in 14 cases. The propionylcarnitine level, ratio of propionylcarnitine/acetylcarnitine in blood, methylmalonic acid and methylcitric acid levels in urine have decreased significantly, with the median values reduced respectively from 24.15 (7.92-81.02) µmol/L, 1.08 (0.38-6.01), 705.34 (113.79-3078.60) and 7.71 (0.52-128.21) to 10.50 (3.00-30.92) µmol/L, 0.63 (0.25-2.89), 166.23 (22.40-3322.21) and 3.96 (0.94-119.13) (P < 0.05). CONCLUSION: The prognosis of isolated methylmalonic acidemia may be predicted with the enzymatic subgroup, age at onset and cobalamin responsiveness. Outcome is unfavorable in neonatal patients and those who were non-responsive to cobalamin.

[Outcomes of patients with combined methylmalonic acidemia and homocystinuria after treatment].

OBJECTIVE: Combined methylmalonic acidemia with homocystinuria is a common form of methylmalonic acidemia in China. Patients with this disease can progress to death without timely and effective treatment. This study aimed to analyze the treatment outcomes of patients with combined methylmalonic acidemia and homocystinuria. METHOD: From September 2004 to April 2012, 58 patients with combined methylmalonic acidemia and homocystinuria (34 males and 24 females) were diagnosed and treated in our hospital. Fifty cases were from clinical patients including 42 early-onset cases and 8 late-onset cases. Their age when they were diagnosed ranged from 18 days to 30.8 years. The other 8 cases were from newborn screening. All the patients were treated with vitamin B12, betaine, folic acid, vitamin B6, and L-carnitine. The physical and neuropsychological development, general laboratory tests, the levels of amino acids, acylcarnitines, and homocysteine in blood, and organic acids in urine were followed up. RESULT: The follow-up period ranged from 1 month to 7.1 years. Three cases died (all were early-onset cases). In the other patients after treatment, the symptoms such as recurrent vomiting, seizures, lethargy, and poor feeding disappeared, muscle strength and muscle tension were improved, and general biochemical abnormalities such as anemia and metabolic acidosis were corrected. Among the surviving 55 cases, 49 had neurological impairments such as developmental delay and mental retardation. The median levels of blood propionylcarnitine and its ratio with acetylcarnitine, serum homocysteine, and urine methylmalonic acid were significantly decreased (P < 0.01), from 7.73 µmol/L (ranged from 1.5 to 18.61 µmol/L), 0.74 (ranged from 0.29 to 2.06), 97.3 µmol/L (ranged from 25.1 to 250 µmol/L) and 168.55 (ranged from 3.66 to 1032.82) before treatment to 2.74 µmol/L (ranged from 0.47 to 12.09 µmol/L), 0.16 (ranged from 0.03 to 0.62), 43.8 µmol/L (ranged from 17 to 97.8 µmol/L) and 6.81 (ranged from 0 to 95.43) after treatment, respectively. CONCLUSION: Patients with combined methylmalonic acidemia and homocystinuria respond to a combined treatment consisting of supplementation of hydroxycobalamin, betaine, folic acid, vitamin B6 and L-carnitine with clinical and biochemical improvement. But the long-term outcomes are unsatisfactory, with neurological sequelae in most patients.

[Primary carnitine deficiency in 17 patients: diagnosis, treatment and follow up].

OBJECTIVE: Many children were found to have low free carnitine level in blood by tandem mass spectrometry technology. In some of the cases the problems occurred secondary to malnutrition, organic acidemia and other fatty acid oxidation metabolic diseases, and some of cases had primary carnitine deficiency (PCD). In the present article, we discuss the diagnosis of PCD and evaluate the efficacy of carnitine in the treatment of PCD. METHOD: We measured the free carnitine (C0) and acylcarnitine levels in the blood of 270 000 neonates from newborns screening program and 12 000 children with suspected clinical inherited metabolic diseases by tandem mass spectrometry. The mutations of carnitine transporter protein were tested to the children with low C0 level and the diagnosis was made. The children with PCD were treated with 100 - 300 mg/kg of carnitine. RESULT: Seventeen children were diagnosed with PCD, 6 from newborn screening program and 11 from clinical patients. Mutations were found in all of them. The average C0 level [(2.9 ± 2.0) µmol/L] in patients was lower than the reference value (10 µmol/L), along with decreased level of different acylcarnitines. The clinical manifestations were diverse. For the 6 patients from newborn screening, 4 were asymptomatic, 1 showed hypoglycaemia and 1 showed movement intolerance from 2 years of age. For the 11 clinical patients, 8 showed hepatomegaly, 7 showed myasthenia, 6 showed cardiomyopathy, 1 showed chronic abdominal pain, and 1 showed restlessness and learning difficulty. Among these patients, 14 cases were treated with carnitine. Their clinical symptoms disappeared 1 to 3 months later. The C0 level in the blood rose to normal, with the average from (4.0 ± 2.7) µmol/L to (20.6 ± 8.3) µmol/L (P < 0.01). However, the level was still lower than the average level of healthy children [(27.1 ± 4.5) µmol/L, P < 0.01]. CONCLUSION: Seventeen patients were diagnosed with PCD by the test levels of free carnitine and acylcarnitines in blood with tandem mass spectrometry, and gene mutation test. Large dose of carnitine had a good effect in treatment of the PCD patients.

[Diagnosis, treatment and gene mutation analysis of the first case with dihydropteridine reductase deficiency in the mainland of China].

OBJECTIVE: The 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency is the most common type of tetrahydrobiopterin (BH4) deficiency. The reported patients with BH4 deficiency are all PTPS deficient found in the mainland of China previously. The activity of dihydropteridine reductase in BH4 metabolism has been determined for 902 patients with hyperphenylalaninemia in the authors' laboratory since 2003. The purposes of this study were to characterize the first case with DHPR deficiency who was diagnosed in June, 2007, to investigate the clinical manifestation, the differential diagnostic criteria, the effect of treatment as well as gene mutation of DHPR deficiency. METHODS: (1) A male patient presented with poor hand control, seizure, hypotonia and mental retardation since five-month after birth. His phenylalanine (Phe) level was 600 micromol/L and he was diagnosed as hyperphenylalaninemia at the age of one year and six-month. (2) This patient was subjected to combined Phe (100 mg/kg) and BH4 (20 mg/kg) loading test, to evaluate the degree of Phe level response to BH4. Urinary neopterin and biopterin analysis as well as the determination of DHPR activity in dried blood spot were also performed. (3) The blood DNA samples of the patient and his parents were collected to amplify the seven exons of QDPR gene using related primers, and the amplified products were directly sequenced for mutation analysis. (4) The patient was treated with BH4 or with a combined small amount of Phe-free special milk, neurotransmitter precursors and folic acid after the diagnosis and was followed up for clinical effects of treatment. RESULTS: (1) The basic Phe level was 476 micromol/L, then it increased to 1355 micromol/L at 3 h after taking Phe and slowly decreased to 610 micromol/L at 24h after taking BH4. (2) The basic urinary neopterin and biopterin were 2.92 mmol/mol Cr (normally < 2.61 mmol/mol Cr) and 7.44 mmol/molCr (normally < 2.67 mmol/mol Cr) respectively, and biopterin percentage was 71.79% (normally 42.7% - 75.9%). The patient had higher biopterin level. (3) The DHPR activity of this patient was (0.27 - 0.51) nmol/(min.5 mm disc) which were 6.11% - 10.6% of normal control, so he was diagnosed as DHPR deficiency. (4) The analysis of QDPR gene mutation showed that the patient carries missense mutation c.515C > T (P172L) from his father and nonsense mutation c.661C > T (R221X) from his mother. The c.515C > T is not reported before, we also did not find this mutation in 50 normal children. (5) The patient started to be treated with large dosage of BH4 (10 - 20) mg/(kg.d) or BH4 combined with small amounts of Phe-free milk, neurotransmitter precursors L-dopa (3 - 5) mg/(kg.d) plus carbidopa, 5-hydroxytryptophan (3 - 5) mg/(kg.d), and folic acid 15 mg/d as well at the age of one year and six-month after the diagnosis. The seizure has disappeared, the symptoms such as hypotonia have been obviously improved and the Phe level was 60 micromol/L at the six months after the treatment in this patient. CONCLUSION: (1) The patient with DHPR deficiency has common symptoms of BH4 deficiency (such as fair hair, hypotonia, mental retardation), and there is metabolic disturbance of folic acid in DHPR deficiency. (2) The higher Phe levels slowly decreased after BH4 loading test, the urinary biopterin level was very high and the DHPR activity was very low in the patient with DHPR deficiency. (3) The c.515C > T may be a new mutation of QDPR gene. (4) The DHPR deficient patient must be treated with higher dose of BH4 (8 - 20) mg/(kg.d), neurotransmitter precursors and folic acid as well.

[The analysis of the content of bile acids in ox-bile powder and goat gall powder of Mongolian medicine].

The present study was to detect and compare the content of bile acids in ox bile powder and goat gall powder of Mongolia medicine by UV. Cholic acid with sulphuric acid were heated and dehydrated, and they produced conjugated double bond. The conjugated bond showed the same absorption peak in the ultraviolet range. The method of ultraviolet spectrophotometry can be used to detect and compare the content of bile acids in ox bile powder and goat gall powder. The result showed that the linear range was 0.003 3-0.016 7 mg x mL(-1) (r = 0.999 7). The average recovery (n = 5) of standard addition method of ox bile powder and goat gall powder was 98.48% (RSD = 1.79%) and 96.46% (RSD = 2.50%) respectively. The result of determination of five different samples showed that the content of bile acids in ox bile powder and goat gall powder was 40.85%-43.03% and 30.88%-32.64% respectively. The RSD of the analysis of ox bile powder and goat ball powder was 2.40% and 2.92% respectively, the RSD of stationary test of ox bile powder and goat ball powder in eight hours was 0.55% and 0.59% respectively, and the RSD of reproducibility of the analysis of ox bile powder and goat ball powder was 2.11% and 2.68% respectively. The method was simple, accurate, fast and easy to generalize and apply in many fields. It can be used to control the quality of ox bile powder and goat gall powder.

[Effects of long-term petroleum and heavy metals pollution on the diversity and community structure of Pesudomonas populations in agricultural soils].

By using PCR-DGGE method, this paper studied the diversity and community structure of Pseudomonas populations in long-term petroleum- and heavy metals-contaminated agricultural soils in Northeast China. The results showed that the Shannon diversity index of Pseudomonas was significantly higher in petroleum- than in heavy metals-contaminated soils. The diversity of Pseudomonas in petroleum-contaminated soil was approached to that in clean soil but lower than that in polluted lowland rice soil, suggesting that contaminant type and cultivation mode were the main factors affecting the diversity of Pseudomonas in agricultural soils. The sequences of V6/V7 regions in 16S rRNA gene indicated that P. mendocina, P. stutzeri and P. aeruginosa were the dominant species in both petroleum- and heavy metals-contaminated soils, demonstrating that these three species were enriched under the stress of long-term pollution, which might correlate with the natural degradation of petroleum and the resistance of Pseudomonas to heavy metals.