Development of a multiplex polymerase chain reaction to detect five common Gram-negative bacteria of aquatic animals.

A multiplex polymerase chain reaction (m-PCR) technique was developed as a rapid and accurate diagnostic tool for identifying five major Gram-negative bacilli -Vibrio vulnificus, V. parahaemolyticus, Aeromonas hydrophila, Chryseobacterium meningosepticum and Edwardsiella tarda- that cause major diseases in cultured aquatic animals in Taiwan. The expected amplicons for V. vulnificus, V. parahaemolyticus, A. hydrophila, C. meningosepticum and E. tarda were 410, 368, 685, 180 and 230bp, respectively. The assay was shown to be specific for the target pathogens. The sensitivities of detection were estimated to be 20.5fg∼200pg of genomic DNA or 10(2) ∼10(4) colony-forming units (cfu) of bacterial isolates when adopted as PCR templates. The m-PCR was capable of simultaneously amplifying target fragments from bacterial genome DNA mixed with the DNA extracted from viscera and tissues taken from fish without affecting the performance of the method.

Progesterone inhibits human endothelial cell proliferation through a p53-dependent pathway.

Previous studies have shown that progesterone inhibits endothelial cell proliferation through a nuclear receptor-mediated mechanism. Here, we further demonstrate that progesterone at physiologic levels (5 - 500 nM) dose- and time-dependently inhibited DNA synthesis of cultured human umbilical vein endothelial cells (HUVEC). The mRNA and protein levels of p21, p27, and p53 in HUVEC were increased by progesterone. The formation of CDK2-p21 and CDK2-p27 were increased and the CDK2 activity was decreased in the progesterone-treated HUVEC. The progesterone-inhibited [3H]thymidine incorporation was completely blocked when the expressions of p21 and p27 were knocked-down together. Transfection of HUVEC with dominant negative p53 cDNA prevented the progesterone-induced increases in p21 and p27 promoter activity and protein level, decreases in thymidine incorporation, and capillary-like tube formation. Matrigel angiogenesis assay in mice demonstrated the antiangiogenic effect of progesterone in vivo. These findings demonstrate for the first time that progesterone inhibited endothelial cell proliferation through a p53-dependent pathway.

Investigating the role of the HLA-Cw*06 and HLA-DRB1 genes in susceptibility to psoriatic arthritis: comparison with psoriasis and undifferentiated inflammatory arthritis.

OBJECTIVE: Psoriasis of early onset (type I; age of onset 40 years). HLA-DRB1*07, in linkage disequilibrium with HLA-Cw*06, was also associated with patients with PsA having type I psoriasis (OR 2.7, 95% CI 2.1, 3.7, p<0.00001). HLA-DRB1*04 alleles and the SE were associated with undifferentiated inflammatory arthritis but not with PsA. CONCLUSIONS: The SE is not a PsA susceptibility locus. HLA-Cw*06 and HLA-DRB1*07 are associated with patients with PsA having type I psoriasis, suggesting that the primary association is with age of onset of psoriasis. Patients with PsA having type I psoriasis, therefore, have a genetic background different to those with type II psoriasis, and adjustment for this is necessary in future studies that investigate the genetic susceptibility of PsA.

Coracoclavicular ligament reconstruction using a gracilis tendon graft for acute type-III acromioclavicular dislocation.

PURPOSE: To review the functional and radiological results of patients after coracoclavicular ligament reconstruction. METHODS: Five patients aged 21 to 50 (mean, 37) years with acute Rockwood type-III acromioclavicular dislocation underwent coracoclavicular ligament reconstruction with autogenous gracilis tendon grafts. Patients were either active in sports or heavy manual workers. Assessments on shoulder function (using the Constant score), wound size, pain (using a visual analogue scale), and reduction (using radiographs of both acromioclavicular joints) were made. RESULTS: The mean follow-up period was 26 (range, 15-43) months; the mean time to return to work or sports was 14 (range, 12-20) weeks. The mean Constant score was 94 (range, 90-98). The mean donor-site scar size was 3 cm and the mean pain score was 0. No major complication or donor-site morbidity was noted. There was one subluxation. CONCLUSION: Coracoclavicular ligament reconstruction using an autogenous gracilis tendon graft was safe in physically active patients having acute type-III acromioclavicular dislocation.

A prospective case-control study of ankle fracture in postmenopausal women.

OBJECTIVES: To compare bone mineral density of women with postmenopausal ankle fractures with controls and review patient characteristics, injury mechanisms, and outcomes. DESIGN: Prospective case-control study. SETTING: University teaching hospital, Hong Kong. PARTICIPANTS: Women older than 60 years, admitted with ankle fractures between 2002 and 2003 and controls (age-matched women with femoral neck fractures). MAIN OUTCOME MEASURES: Demographic data, bone mineral density, mechanism of injury, fracture pattern, treatment, and the functional outcome. RESULTS: The mean age of the study group (18 ankle fracture patients) was 74 years. The fractures usually resulted from a low-energy trauma; isolated lateral malleolar fracture was the most common (8/18), whilst six had bimalleolar fractures. Their mean T-score bone mineral density values at the spine and hip were -1.67 and -1.70, respectively; corresponding Z-scores were +0.73 and +0.99. The bone mineral density of the study group was significantly higher than in patients with fractured neck of femur (controls) and the general population (P<0.05). Nine of the study group had diabetes and one had impaired glucose tolerance. Treatment comprised casting in 10 patients and operative fixation in seven. Good functional recovery was achieved; most patients were able to resume their premorbid level of independent daily activities with a good motor functional independence score (85.18/91) 1 year post-injury. CONCLUSION: In this case-control study, postmenopausal ankle fractures were not associated with osteoporosis. Diabetic neuropathy may have been a risk factor for such injury. The functional outcome of such patients was generally satisfactory, provided appropriate treatment was given.

Comparison of whole pelvis versus small-field radiation therapy for carcinoma of prostate.

One hundred thirty-six patients with adenocarcinoma of the prostate gland Stage A2 (12 patients), Stages B1 and B2 (26), Stage C (64), and Stages D1 and D2 (34 patients) were evaluated clinically and treated in a similar fashion at three hospitals. Megavoltage radiation therapy units were employed to deliver 4,600-5,000 cGy to the whole pelvis, and the prostatic area was treated for an additional 2,000 cGy (boost). Local recurrence was infrequent (8/136 = 6%), and the five-year actuarial survival and disease-free survival rates were 85 and 42 per cent, respectively. Adverse clinical parameters included poor histologic differentiation, age younger than sixty years, and diagnosis by transurethral resection of the prostate rather than needle biopsy in Stage C patients. Severe acute reactions occurred in only 2 patients, and only 2 patients were hospitalized for severe chronic (late) reactions. Whole pelvis radiation yielded a statistically significant improved five-year survival and three-year disease-free survival for similarly evaluated patients for Stage C but not for Stages A and B when compared with 116 patients treated with small-volume radiation (prostate area), previously reported from these three hospitals.

Production of L-DOPA by tyrosinase immobilized on modified polystyrene.

Mushroom tyrosinase was immobilized on modified polystyrene- polyamino styrene (PSNH) and polymethylchloride styrene (PSCL)-to produce L-DOPA from L-tyrosine. Glutaraldehyde was used as an activating agent for the PSNH to immobilize the tyrosinase, and 10% (w/v) glutaraldehyde was optimal in conferring the highest specific activity (11.96 U/g) to the PSNH. Methylchloride on the PSCL was directly linked with the tyrosinase, and 1.5 mmol of Cl/g was optimal in attaining the specific activity of 17.0 U/g. The temperature and optimal acidity were, respectively, 60 degrees C and pH 5.5 for the PSNH, and 70 degrees C and pH 3.0 for the PSCL. In a 50-mL batch reactor working over 36 h, the L-DOPA production rate at 30 degrees C was 1.44 mg/(L x h) for the PSNH and 2.33 mg/(L x h) for the PSCL. The production rate over 36 h was 3.86 mg/(L x h) for the PSNH at 60 degrees C and 5.54 mg/(L x h) for the PSCL at 70 degrees C. Both of the immobilized enzymes showed a remarkable stability with almost no change in activity after being stored wet. The operational stability study indicated a 22.4% reduction in L-DOPA production for the PSNH and an 8.63% reduction for the PSCL over seven runs (each run was for 144 h at 30 degrees C) when the immobilized enzymes were used under turnover conditions. The immobilized tyrosinase was more stable on the PSCL than on the PSNH.

Mercury and methylmercury levels in the main traded fish species in Hong Kong.

Levels of total mercury (tHg) and mono-methylmercury (MeHg) were measured in 280 different fish, including fresh/frozen raw whole fish of 89 different species and canned tuna fish of three different species, that are traded mainly in Hong Kong, China. These samples were purchased from different commercial outlets between April and August 2007. All samples of raw whole fish were identified at species level by the Agriculture, Fisheries and Conservation Department. The range for tHg and MeHg of all samples were 3-1370 and 3-1010 µg kg(-1), respectively, with medians of 63 and 48 µg kg(-1), respectively. The results show that, according to Hong Kong legislation, the products on the market are generally 'safe'. A total of 277 samples (99?) contained tHg and MeHg below the legal limit of 500 µg kg(-1). The remaining three samples of alfonsino (species: Beryx splendens) were found to contain tHg and MeHg at levels higher than 500 µg kg(-1) (tHg: 609-1370 µg kg(-1); MeHg: 509-1010 µg kg(-1)). The ratios of MeHg to tHg in the different fish species ranged from 0.46 to 0.99.

High-performance liquid chromatographic assay for catecholamines and metanephrines using fluorimetric detection with pre-column 9-fluorenylmethyloxycarbonyl chloride derivatization.

A convenient HPLC-fluorescent assay of norepinephrine (NE), epinephrine (E), dopamine (DA) and their 3-O-methylated metabolites, normetanephrine (NM) and metanephrine (MN) was developed. These analytes were coupled to 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) before assays. Results showed that using a linear gradient elution, peaks of FMOC-NE, FMOC-E, FMOC-DA, FMOC-NM, FMOC-MN and FMOC-DHBA (3,4-dihydroxybenzylamine, internal standard) were simultaneously resolved within 40 min. Optimization of the chromatographic and derivatization conditions, and validation of the assay were further discussed in the paper. The structures of these derivatives were confirmed by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The molecular ions [M+H]- of FMOC-NE, FMOC-E, FMOC-DA, FMOC-NM and FMOC-MN were m/z 836, 850, 820, 628 and 642, respectively. Based on these findings, the FMOC-derivatives of metanephrines and catecholamines were confirmed to be bi-substituted and tri-substituted respectively at the amino and catechol functional groups. Finally, the assay was successfully applied to the measurement of urinary E, DA, NM and MN after direct derivatization and simple cleaning.

Site of vitellogenin synthesis determined from a cDNA encoding a vitellogenin fragment in the freshwater giant prawn, Macrobrachium rosenbergii.

Vitellogenesis is an important part of reproductive process in crustaceans, and the process is characterized by the synthesis and accumulation of yolk protein in the developing oocytes. The yolk proteins in crustaceans mainly consist of vitellogenin (Vg) and vitellin (Vn), which are respectively present in extra-oocyte tissues and intra-oocytes. The site and the process of yolk protein synthesis in crustaceans are still controversial. The synthesis site of Vg in a crustacean species, Macrobrachium rosenbergii, is determined by immunological and immunohistochemical techniques, and molecular cloning of a cDNA encoding the primary structure of Vn in this study. The hepatopancrease is clearly shown to be the synthesis site of Vg in this species. The length of Vg mRNA was estimated as about 6 kb from Northern blotting analysis. The partial primary structure of Vg gene is presented, and the post-translational processing are further discussed. For the first time, the partial primary structure of Vg gene and the synthesis site of Vg approached by molecular cloning in crustaceans are presented.

Left ventricular long axis function in diastolic heart failure is reduced in both diastole and systole: time for a redefinition?

OBJECTIVE: To test the hypothesis that, when measured in the long axis, left ventricular systolic function is abnormal in patients with diastolic heart failure. DESIGN: A case-control study. SETTING: University teaching hospital (tertiary referral centre). PATIENTS: 68 patients with heart failure, 29 with a left ventricular ejection fraction (LVEF) of > 0.45 and diastolic dysfunction (diastolic heart failure), 39 with an LVEF of