Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean

The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.     

Identification of an integral plasma membrane pool of protein kinase C in mammalian tissues and cells

Protein kinase C (PKC) is a family of serine/threonine protein kinases that are pivotal in cellular regulation. Since its discovery in 1977, PKCs have been known as cytosolic and peripheral membrane proteins. However, there are reports that PKC can insert into phospholipids vesicles in vitro. Given the intimate relationship between the plasma membrane and the activation of PKC, it is important to determine whether such "membrane-inserted" form of PKC exists in mammalian cells or tissues. Here, we report the identification of an integral plasma membrane pool for all the 10 PKC isozymes in vivo by their ability to partition into the detergent-rich phase in Triton X-114 phase partitioning, and by their resistance to extractions with 0.2 M sodium carbonate (pH 11.5), 2 M urea and 2 M sodium chloride. The endogenous integral membrane pool of PKC in mouse fibroblasts is found to be acutely regulated by phorbol ester or diacylglycerol, suggesting that this pool of PKC may participate in cellular processes known to be regulated by PKC. At least for PKC(alpha), the C2-V3 region at the regulatory domain of the kinase is responsible for membrane integration. Further exploration of the function of this novel integral plasma membrane pool of PKC will not only shed new light on molecular mechanisms underlying its cellular functions but also provide new strategies for pharmaceutical modulation of this important group of kinases.     

Determination and occurrence of polybrominated diphenyl ethers in maternal adipose tissue from inhabitants of Singapore

Polybrominated diphenyl ethers (PBDEs), as a specific group of brominated flame retardants (BFR), are used in a variety of consumer products including electronics and household furnishings. In recent years, a marked increase in the levels of PBDEs in human biological tissues and fluids, especially breast milk, has been reported in several countries. However, few data are available from countries in the Asia-pacific region, including Singapore. This study presents a validated method procedure and the first available data of the concentrations of PBDE congeners: PBDE-47 (2,2,4,4-Tetrabromodiphenyl ether), PBDE-99 (2,2',4,4',5-Pentabromodiphenyl ether), PBDE-100 (2,2',4,4',6-Pentabromodiphenyl ether), PBDE-153 (2,2',4,4',5,5'-Hexabromodiphenyl ether), PBDE-154 (2,2',4,4',5,6'-Hexabromodiphenyl ether) in maternal adipose tissue collected from inhabitants of Singapore. Microwave-assisted extraction (MAE) of PBDEs spiked adipose tissues coupled with GC-MS analysis achieved comparable recoveries to a conventional Soxhlet Extraction (SE) procedure of between 70 and 130%. MAE also yielded comparable precision data (variance less than 13%) relative to the SE procedure. Spiked Carbon-13 PBDE congeners were also used as surrogates for MAE quality assurance and confirmed the efficiency of the procedure. PBDE congeners were detected in all of 16 maternal adipose tissues collected in Singapore, where levels were comparable to available data from Belgium.     

Structural and dynamic properties of cytochrome P450 BM-3 in pure water and in a dimethylsulfoxide/water mixture

Solvent molecules play an important role for the structural and dynamical properties of proteins. A major focus of current protein engineering is the development of enzymes that are catalytically active in the presence of organic solvents. The monooxygenase P450 BM-3 is one of the best-studied enzymes and promising for industrial applications but with limited activity in the presence of organic solvents or cosolvents. To gain insights into the structural and dynamical properties of the heme domain of this enzyme in solution, molecular dynamics simulations in pure water and in a 14% DMSO/water mixture were performed. The results of the simulations show overall similar structural fluctuations in both solvent systems, with no indication of partial or global unfolding. In 14% DMSO, the regions comprising the helices E, F, and the EF loop (implicated in controlling the entry to the active site channel) undergo a large shift. Significant changes were also observed near the active site access channel at the residue R47. During the simulation, no DMSO molecule penetrated the active site. However, a significant accumulation of DMSO molecules close to the substrate-binding site and to the Flavin Mononucleotide (FMN) reductase domain interface was observed.     

Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents

Cytochrome p450 BM-3 (EC 1.14.14.1) catalyzes the hydroxylation and/or epoxidation of a broad range of substrates, including alkanes, alkenes, alcohols, fatty acids, amides, polyaromatic hydrocarbons, and heterocycles. For many of these notoriously water-insoluble compounds, p450 BM-3's K(m) values are in the millimolar range. Polar organic cosolvents are therefore added to increase substrate solubility and achieve high catalytic efficiency. Using p450 BM-3 as a catalyst for these important transformations requires that we improve its ability to tolerate the cosolvents. By directed evolution, we improved the activity of p450 BM-3 in the presence of dimethylsulfoxide (DMSO) and tetrahydrofuran (THF), achieving increases in specific activity up to 10-fold in 2% (v/v) THF and 6-fold in 25% (v/v) DMSO. The engineered p450 BM-3's are also significantly more resistant to acetone, acetonitrile, dimethylformamide, and ethanol as cosolvents in the reaction.     

A novel non-catalytic mechanism employed by the C-terminal Src-homologous kinase to inhibit Src-family kinase activity

Although C-terminal Src kinase (CSK)-homologous kinase (CHK) is generally believed to inactivate Src-family tyrosine kinases (SFKs) by phosphorylating their consensus C-terminal regulatory tyrosine (Tyr(T)), exactly how CHK inactivates SFKs is not fully understood. Herein, we report that in addition to phosphorylating Tyr(T), CHK can inhibit SFKs by a novel non-catalytic mechanism. First, CHK directly binds to the SFK members Hck, Lyn, and Src to form stable protein complexes. The complex formation is mediated by a non-catalytic Tyr(T)-independent mechanism because it occurs even in the absence of ATP or when Tyr(T) of Hck is replaced by phenylalanine. Second, the non-catalytic CHK-SFK interaction alone is sufficient to inactivate SFKs by inhibiting the catalytic activity of autophosphorylated SFKs. Third, CHK and Src co-localize to specific plasma membrane microdomains of rat brain cells, suggesting that CHK is in close proximity to Src such that it can effectively inactivate Src in vivo. Fourth, native CHK.Src complex exists in rat brain, and recombinant CHK.Hck complex exists in transfected HEK293T cells, implying that CHK forms stable complexes with SFKs in vivo. Taken together, our findings suggest that CHK inactivates SFKs (i) by phosphorylating their Tyr(T) and (ii) by this novel Tyr(T)-independent mechanism involving direct binding of CHK to SFKs. It has been documented that autophosphorylated SFKs can still be active, in some cases even when their Tyr(T) is phosphorylated. Thus, the ability of the Tyr(T)-independent mechanism to suppress the activity of both non-phosphorylated and autophosphorylated SFKs represents a fail-safe measure employed by CHK to down-regulate SFK signaling under all circumstances.     

Overexpression of 7a, a protein specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a caspase-dependent pathway

Besides genes that are homologous to proteins found in other coronaviruses, the severe acute respiratory syndrome coronavirus genome also contains nine other potential open reading frames. Previously, we have characterized the expression and cellular localization of two of these "accessory" viral proteins, 3a (previously termed U274) and 7a (previously termed U122). In this study, we further examined whether they can induce apoptosis, which has been observed clinically. We showed that the overexpression of 7a, but not of 3a or the viral structural proteins, nucleocapsid, membrane, and envelope, induces apoptosis. 7a induces apoptosis via a caspase-dependent pathway and in cell lines derived from different organs, including lung, kidney, and liver.     

AAV8-mediated expression of glucocerebrosidase ameliorates the storage pathology in the visceral organs of a mouse model of Gaucher disease

BACKGROUND: Gaucher disease is the most common of the lysosomal storage disorders. The primary manifestation is the accumulation of glucosylceramide (GL-1) in the macrophages of liver and spleen (Gaucher cells), due to a deficiency in the lysosomal hydrolase glucocerebrosidase (GC). A Gaucher mouse model (D409V/null) exhibiting reduced GC activity and accumulation of GL-1 was used to evaluate adeno-associated viral (AAV)-mediated gene therapy. METHODS: A recombinant AAV8 serotype vector bearing human GC (hGC) was administered intravenously to the mice. The levels of hGC in blood and tissues were determined, as were the effects of gene transfer on the levels of GL-1. Histopathological evaluation was performed on liver, spleen and lungs. RESULTS: Vector administration to pre-symptomatic Gaucher mice resulted in sustained hepatic secretion of hGC at levels that prevented GL-1 accumulation and the appearance of Gaucher cells in the liver, spleen and lungs. AAV administration to older mice with established disease resulted in normalization of GL-1 levels in the spleen and liver and partially reduced that in the lung. Analysis of the bronchoalveolar lavage fluid (BALF) from treated mice showed significant correction of the abnormal cellularity and cell differentials. No antibodies to the expressed hGC were detected following a challenge with recombinant enzyme suggesting the animals were tolerized to human enzyme. CONCLUSIONS: These data demonstrate the effectiveness of AAV-mediated gene therapy at preventing and correcting the biochemical and pathological abnormalities in a Gaucher mouse model, and thus support the continued consideration of this vector as an alternative approach to treating Gaucher disease.