Leukodystrophy and CSF Purine Abnormalities Associated with Isolated 3-Methylcrotonyl-CoA Carboxylase Deficiency

We report the first case of isolated biotin resistant 3-methylcrotonyl-CoA carboxylase (MCC) deficiency in Argentina. The diagnosis was established at 14 months of age by urinary organic-acid analysis and confirmed by enzyme assay in fibroblasts. The patient suffered from severe psychomotor retardation, hypotonia, areflexia, and failure to thrive, and died unexpectedly at 3 years 4 months of life. Brain MRI at 14 months showed signals of the white matter on cerebral T₂-weighted, which were indicative of confluent and multiple foci of leukodystrophy, a pattern not previously described in this entity. In addition, high levels of oxypurines were detected in cerebrospinal fluid. This might be related to energetic consequences of the enzyme deficiency in the brain. This case extends the phenotype of isolated MCC deficiency in infancy and suggests this entity should be considered to be one of the possible causes of metabolic leukodystrophies.     

Admission screening by thyroid function tests in an acute general care teaching hospital.

To determine the incidence of unrecognized thyroid disease among admissions to a large acute care university teaching hospital, 364 samples taken on consecutive admissions were assayed for thyroid-stimulating hormone (TSH) and free thyroxine index (FTI). Patients with abnormal test results were further evaluated by determination of antimicrosomal and antithyroglobulin antibodies, and charts were reviewed for evidence of prior diagnosis of thyroid disease, especially severe illness, drug treatment that might affect thyroid function tests, and prior diagnosis of thyroid disease. Results of subsequent thyroid function tests performed during the patient's hospitalization were correlated with the admission serum assays, and data on subsequent testing during the following 6 months were also obtained. A total of 3.9% of patients had significantly depressed TSH, and 11.1% of values were significantly elevated. A total of 11.3% of patients had significantly low FTI values, and 1% had significantly elevated values. A total of 7.4% appeared to have the euthyroid sick syndrome, 5.8% appeared to have unrecognized or undertreated primary thyroid failure, 6% had apparent subclinical hypothyroidism, 2% were thyrotoxic, and 2.8% (all women) had suppressed TSH levels for inapparent reasons. Limiting testing to patients over 49 years of age, or to women, would have missed many individuals with abnormal test results. Considering widespread availability of tests, relative costs, and value of the information obtained, it is suggested that the FTI determination would provide an appropriate screening test for patients in a population such as this entering a large, acute care general hospital.     

The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.     

Inhibition of the replication of parvovirus X14 by 5-iodo-2'-deoxyuridine pre-treatment of cell cultures.

Pre-treatment of rat embryo cell cultures with 5-iodo-2'-deoxyuridine (IdUrd) inhibits the replication of parvovirus X14. Reduced yields of haemagglutinating and infectious particles were observed. Adsorption of virus to cells was not affected, but both virus protein and DNA synthesis were inhibited. Fewer cells were capable of supporting protein or antigen synthesis as determined by immunofluorescence. Virus-specific DNA was detected in IdUrd pre-treated cells, but the amount synthesized was considerably less than that from control cultures. Cellular DNA synthesis was also inhibited in IdUrd pre-treated cells. Therefore, the replication of parvoviruses appears dependent upon host cell factors involved in cellular DNA synthesis.     

力竭运动大鼠心室肌蛋白质组表达特征

目的:采用蛋白质组学技术,建立安静和递增运动负荷训练后力竭大鼠心室肌蛋白质组的差异性表达谱,初步筛选出心室肌对力竭运动产生反应的目标蛋白质。方法:实验于2007-03在湖南师范大学生命科学学院蛋白质化学与蛋白质组学国家教育部重点实验室和省级运动人体科学实验室完成。①实验分组:10只SD雄性大鼠随机分为对照组和运动组,每组5只。②实验方法:运动组经过7周的大强度递增运动负荷训练后(最后一次力竭),对两组心室肌组织的全蛋白进行双向凝胶电泳分离。结果:经图像分析,在运动组的电泳图谱上共展现蛋白质点(338±17)个,对照组展现蛋白质点(352±17)个。运动后差异表达的蛋白质点共有99个。对其中差异表达的9个蛋白质点进行质谱鉴定,共鉴定出7个蛋白质,Stress-70protein,NADH-ubiquinone oxidoreductase Mr75000subnunit,Long-chain specific acyl-CoA dehydrogenase,Tropomyosin-1alphachain在运动后"缺失",Nitrilase family,member2在运动后表达上调在5倍以上,一个相对分子质量为21000的未知蛋白在运动后表达下调在5倍以上,另外有两个点经鉴定均为Myosin-6,在运动后表达量相反。这些蛋白质属于收缩蛋白、能量代谢酶、分子伴侣等。结论:递增运动负荷训练后力竭时,大鼠心室肌蛋白质组明显地发生了反应。运动后"缺失"和下调的蛋白质点与心肌收缩的调控和能量代谢的方式转变以及细胞的应激反应有关,其中,成功筛选出6种在运动医学领域尚未涉足的、具有运动应激特点的目标蛋白质。     

Reduced GRK2 level in T cells potentiates chemotaxis and signaling in response to CCL4

Chemokine receptors belong to the family of G-protein-coupled receptors (GPCR). Phosphorylation of GPCR by GPCR kinases (GRKs) is considered to play an important role in desensitization of these receptors. We have recently shown in patients with rheumatoid arthritis that the level of GRK2 in lymphocytes is reduced by approximately 50%. However, the physiological relevance of reduced GRK2 levels in lymphocytes is not known. Here, we investigated whether reduced GRK2 expression changes the chemotactic response of T cells to the chemokines CCL3, CCL4, and CCL5. Activated T cells from GRK2+/- mice, which have a 50% reduction in GRK2 protein levels, showed a significant 40% increase in chemotaxis toward the CCR5 ligand CCL4. In addition, chemotaxis toward the CCR1 and CCR5 ligands CCL3 and CCL5 was also increased. Binding of CCL4 to activated T cells from GRK2+/- and wild-type (WT) mice was similar, but agonist-induced CCR5 phosphorylation was attenuated in GRK2+/- cells. Moreover, the calcium response and phosphorylation of protein kinase B and extracellular-regulated kinase in response to CCL4 were significantly increased in GRK2+/- T cells, showing that signaling is increased when the level of GRK2 is reduced. GRK2+/- and WT cells do become refractory to restimulation with CCL4. In conclusion, a 50% decrease in T cell GRK2 expression results in increased responsiveness to CCL3, CCL4, and CCL5, suggesting that the 50% reduction in lymphocyte GRK2 level as observed during inflammation can have functional consequences for the response of these cells to chemokines.     

Molecular characterization of methicillin-resistant Staphylococcus aureus isolates collected in Asunción, Paraguay

We characterized 34 methicillin-resistant Staphylococcus aureus strains isolated in Paraguay in 2005. The strains belonged to two clones. The major clone (sequence type 5 [ST5] or ST221, spa type t149, staphylococcal cassette chromosome mec [SCCmec] type I) was similar to the Cordobes/Chilean clone spreading through South America, and the minor clone (ST239 or ST889, spa type t037, SCCmec type IIIA) was related to the Brazilian clone.