Replacement of nonmuscle myosin II-B with II-A rescues brain but not cardiac defects in mice

The purpose of these studies was to learn whether one isoform of nonmuscle myosin II, specifically nonmuscle myosin II-A, could functionally replace a second one, nonmuscle myosin II-B, in mice. To accomplish this, we used homologous recombination to ablate nonmuscle myosin heavy chain (NMHC) II-B by inserting cDNA encoding green fluorescent protein (GFP)-NMHC II-A into the first coding exon of the Myh10 gene, thereby placing GFP-NMHC II-A under control of the endogenous II-B promoter. Similar to B(-)/B(-) mice, most B(a*)/B(a*) mice died late in embryonic development with structural cardiac defects and impaired cytokinesis of the cardiac myocytes. However, unlike B(-)/B(-) mice, 15 B(a*)/B(a*) mice of 172 F2 generation mice survived embryonic lethality but developed a dilated cardiomyopathy as adults. Surprisingly none of the B(a*)/B(a*) mice showed evidence for hydrocephalus that is always found in B(-)/B(-) mice. Rescue of this defect was due to proper localization and function of GFP-NMHC II-A in place of NMHC II-B in a cell-cell adhesion complex in the cells lining the spinal canal. Restoration of the integrity and adhesion of these cells prevents protrusion of the underlying cells into the spinal canal where they block circulation of the cerebral spinal fluid. However, abnormal migration of facial and pontine neurons found in NMHC II-B mutant and ablated mice persisted in B(a*)/B(a*) mice. Thus, although NMHC II-A can substitute for NMHC II-B to maintain integrity of the spinal canal, NMHC II-B plays an isoform-specific role during cytokinesis in cardiac myocytes and in migration of the facial and pontine neurons.     

Waist-to-height ratio is an accurate and easier index for evaluating obesity in children and adolescents

Objectives: The present study aims to evaluate the accuracy of the index of waist‐to‐height ratio (WHTR), and proposed the optimal thresholds of WHTR in the definition of childhood overweight and obesity in a bi‐ethnic Chinese school‐aged population. Research Methods and Procedures: Overweight and obese were identified by BMI for age and gender in a random sample including 2055 Han and 2132 Uygur ethnic school‐aged children (8 to 18 years old). WHTR was calculated by waist circumference divided by height on the basis of standard anthropometric measurements. Receiver operating characteristic (ROC) curve analyses were performed to assess the accuracy of WHTR as a diagnostic test for childhood overweight and obesity, compared with waist circumference. The optimal thresholds of WHTR for defining overweight and obesity were recommended respectively by gender. The correlation between WHTR and age was analyzed and compared with BMI. Results: A‐values (area under curve) of WHTR for diagnosing overweight and obesity were both over 0.90 in both genders and better than those of waist circumference. A threshold of 0.445 was identified for overweight in both genders, with the sensitivity and specificity >0.80. The thresholds for defining obesity was 0.485 in boys and 0.475 in girls, both having the sensitivity and specificity >0.90. WHTR showed less association with age than BMI. Conclusions: WHTR is a simple, easy, accurate, and non‐age‐dependent index with high applicability to screening overweight and obesity in children and adolescents. The use of WHTR in the general childhood population has been justified by this study.     

Inhibition of tumor growth in mice by endostatin derived from abdominal transplanted encapsulated cells

Endostatin,a C-terminal fragment of collagen 18a,inhibits the growth of established tumorsand metastases in vivo by inhibiting angiogenesis.However,the purification procedures required for large-scale production and the attendant cost of these processes,together with the low effectiveness in clinicaltests,suggest that alternative delivery methods might be required for efficient therapeutic use of endostatin.In the present study,we transfected Chinese hamster ovary(CHO)cells with a human endostatin geneexpression vector and encapsulated the CHO cells in alginate-poly-L-lysine microcapsules.The release ofbiologically active endostatin was confirmed using the chicken chorioallantoic membrane assay.The encap-sulated endostatin-expressing CHO cells can inhibit the growth of primary tumors in a subcutaneous B 16tumor model when injected into the abdominal cavity of mouse.These results widen the clinical applicationof the microencapsulated cell endostatin delivery system in cancer treatment.     

Erwinia carotovora ssp. carotovora Infection Induced ＂Defense Lignin＂ Accumulation and Lignin Biosynthetic Gene Expression in Chinese Cabbage （Brassica rapa L. ssp. pekinensis）

Erwinia carotovora subsp, carotovora （Ecc） infects and causes soft rot disease in hundreds of crop species including vegetables, flowers and fruits. Lignin biosynthesis has been implicated in defensive reactions to injury and pathogen infection in plants. In this work, variations of lignin content and gene expression in the molecular interaction between Chinese cabbage and Ecc were investigated. H2O2 accumulation and peroxidase activity were detected by 3, 3＇- Dimethoxybenzidine staining at mocked and Ecc-inoculated sites of Chinese cabbage leafstalks. Klason lignin content in inoculated plants increased by about 7.84%, 40.37%, and 43.13% more than that of the mocked site at 12, 24 and 72 h after inoculation, respectively. Gas chromatography detected more p-coumaryl （H） and less coniferyl （G） and sinapyl （S） monolignins in leafstalks of Chinese cabbage. All three monomers increased in Ecc-infected leafstalks, and the Ecc-induced ＂defense lignin＂ were composed of more G and H monolignins, and less S monolignin. After searching the expressed sequence tags （EST） data of Chinese cabbage, 12 genes putatively encoding enzymes involved in lignin biosynthesis were selected to study their expression. All of these genes could be induced by mock inoculation and Ecc infection, while the gene expression lasted for several more hours in the infected samples than in mocked and untreated plants. Our results indicated that ＂defense lignin＂ was different from the developmental lignin in composition; G and S monolignins were significantly induced in plants in response to the soft rot Ecc; thus, lignin biosynthesis was differentially regulated and played a role in plant response to the soft rot Ecc.     

Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB

The biosynthesis of lovastatin in Aspergillus terreus requires two megasynthases. The lovastatin nonaketide synthase, LovB, synthesizes the intermediate dihydromonacolin L using nine malonyl-coenzyme A molecules, and is a reducing, iterative type I polyketide synthase. The iterative type I polyketide synthase is mechanistically different from bacterial type I polyketide synthases and animal fatty acid synthases. We have cloned the minimal polyketide synthase domains of LovB as standalone proteins and assayed their activities and substrate specificities. The didomain proteins ketosynthase-malonyl-coenzyme A:acyl carrier protein acyltransferase (KS-MAT) and acyl carrier protein-condensation (ACP-CON) domain were expressed solubly in Escherichia coli. The monodomains MAT, ACP and CON were also obtained as soluble proteins. The MAT domain can be readily labeled by [1,2-(14)C]malonyl-coenzyme A and can transfer the acyl group to both the cognate LovB ACP and heterologous ACPs from bacterial type I and type II polyketide synthases. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with k(cat)/K(m) values of 0.62 min(-1).mum(-1) and 0.032 min(-1).mum(-1), respectively. The LovB MAT domain was able to substitute the Streptomyces coelicolor FabD in supporting product turnover in a bacterial type II minimal polyketide synthase assay. The activity of the KS domain was assayed independently using a KS-MAT (S656A) mutant in which the MAT domain was inactivated. The KS domain displayed no activity towards acetyl groups, but was able to recognize malonyl groups in the absence of cerulenin. The relevance of these finding to the priming mechanism of fungal polyketide synthase is discussed.     

Development and validation of a LC-MS/MS method for the determination of viaminate in human plasma

A sensitive and specific method for determination of viaminate in human plasma by using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS) was developed in this study. The plasma samples were simply deproteinated, extracted, evaporated, and then reconstituted in 200 microl of methanol prior to analysis. Chromatographic separation was carried out on a Shimadzu VP-ODS column (250 mm x 2.0 mm, 5 microm) with a mobile phase of methanol-water (95:5, v/v) at a flow rate of 0.2 ml/min. Quantification was performed in the negative-ion electrospray ionization mode by selected ion monitoring of the product ions at m/z 164 for viaminate and m/z 109 for testosterone propionate which was used as the internal standard. The corresponding parent ions were m/z 446 and m/z 345. A linear calibration curve was observed within the concentration range of 0.10-200 ng/ml. The lowest limit of quantitation (LLOQ) was 0.1 ng/ml. The extraction-efficiency at three concentrations was 100.7, 93.6, and 99.7%. Practical utility of this new LC-MS/MS method was confirmed in pilot pharmacokinetic studies in humans following oral administration.     

Usefulness of multi-parameter opiates-amphetamines-cocainics analysis in hair of drug users for the evaluation of an abuse profile by means of LC-APCI-MS-MS

The report presents a segmental hair analysis for the retrospective multi-parameter evaluation of drugs of abuse including opioids, cocainics and amphetamines. The analysis was carried out with the use of liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS-MS). The authors have evaluated the differences in the contents of particular opiates in the hair as related to the origin of a sample taken from Polish drug users taking "Polish heroin", and also from heroin abusers from Western European countries taking "Western heroin". The results indicate distinct differences in the 6-MAM concentration values in the Polish and foreigners, suggesting that the foreigners take products containing high concentrations of heroin and the Polish take the poppy product "compote" characterized by its variable and low heroin content. An additional argument for a different abuse profile in the Polish and Western drug users is found in the presence of cocaine detected in hair samples originating from the latter, while cocaine is much less frequently detected in Polish drug users.