Arginine Deiminase: Current Understanding and Applications.

BACKGROUND: Arginine deiminase (ADI), an arginine catabolizing enzyme, is considered as an anti-tumor agent for the treatment of arginine auxotrophic cancers. However, some obstacles limit its clinical applications. OBJECTIVE: This review will summarize the clinical applications of ADI, from a brief history to its limitations, and will discuss the different ways to deal with the clinical limitations. METHOD: The structure analysis, cloning, expression, protein engineering and applications of arginine deiminase enzyme have been explained in this review. CONCLUSION: Recent patents on ADI are related to ADI engineering to increase its efficacy for clinical application. The intracellular delivery of ADI and combination therapy seem to be the future strategies in the treatment of arginine auxotrophic cancers. Applying ADIs with optimum features from different sources and or ADI engineering, are promising strategies to improve the clinical application of ADI.

The Variable Internal Structure of the Mycoplasma penetrans Attachment Organelle Revealed by Biochemical and Microscopic Analyses: Implications for Attachment Organelle Mechanism and Evolution.

Although mycoplasmas have small genomes, many of them, including the HIV-associated opportunist Mycoplasma penetrans, construct a polar attachment organelle (AO) that is used for both adherence to host cells and gliding motility. However, the irregular phylogenetic distribution of similar structures within the mycoplasmas, as well as compositional and ultrastructural differences among these AOs, suggests that AOs have arisen several times through convergent evolution. We investigated the ultrastructure and protein composition of the cytoskeleton-like material of the M. penetrans AO with several forms of microscopy and biochemical analysis, to determine whether the M. penetrans AO was constructed at the molecular level on principles similar to those of other mycoplasmas, such as Mycoplasma pneumoniae and Mycoplasma mobile We found that the M. penetrans AO interior was generally dissimilar from that of other mycoplasmas, in that it exhibited considerable heterogeneity in size and shape, suggesting a gel-like nature. In contrast, several of the 12 potential protein components identified by mass spectrometry of M. penetrans detergent-insoluble proteins shared certain distinctive biochemical characteristics with M. pneumoniae AO proteins, although not with M. mobile proteins. We conclude that convergence between M. penetrans and M. pneumoniae AOs extends to the molecular level, leading to the possibility that the less organized material in both M. pneumoniae and M. penetrans is the substance principally responsible for the organization and function of the AO.IMPORTANCEMycoplasma penetrans is a bacterium that infects HIV-positive patients and may contribute to the progression of AIDS. It attaches to host cells through a structure called an AO, but it is not clear how it builds this structure. Our research is significant not only because it identifies the novel protein components that make up the material within the AO that give it its structure but also because we find that the M. penetrans AO is organized unlike AOs from other mycoplasmas, suggesting that similar structures have evolved multiple times. From this work, we derive some basic principles by which mycoplasmas, and potentially all organisms, build structures at the subcellular level.

Binding of IgA by Mycoplasma penetrans.

The current study shows that Mycoplasma penetrans strain GTU binds human serum immunoglobulin A (IgA) and secretory IgA but not IgG. Binding of IgA was associated almost exclusively with the lipoprotein fraction obtained by Triton X-114 fractionation of isolated M. penetrans membranes. Western immunoblot analysis of isolated membranes of M. penetrans strain GTU and of the Triton X-114 fraction showed that the major IgA-binding component was a lipoprotein with a molecular mass of 38 kDa, one of the major lipoproteins of this organism. The authors suggest that the high IgA-binding capacity of M. penetrans strain GTU may serve as a defense mechanism, conferring on this microorganism the ability to evade clearance mediated by specific IgA antibodies.

Mycoplasma penetrans under nutritional stress: influence on lipid and lipoprotein profiles and on the binding to and invasion of HeLa cells.

By serial passages through media containing decreasing concentrations of horse serum, the sterol-requiring Mycoplasma penetrans strain HF-2 was adapted to grow in a serum-free medium supplemented with bovine serum albumin, cholesterol and free fatty acids. Chemical analysis of membrane preparations obtained from the native and adapted strains revealed two major differences. (1) The polar lipid fraction of the native strain contains, in addition to the de novo-synthesized phospholipids, exogenous lipids incorporated unchanged from the growth medium, whereas exogenous lipids were not detected in the adapted strain. (2) Protein analyses of the native and adapted strains showed that upon adaptation, the 42-kDa membrane lipoprotein, one of the two major lipoproteins of this organism, was missing. Studies on the adhesion to, and invasion of HeLa cells by the native and adapted strains revealed that whereas the adherence to HeLa cells of the adapted strain was almost the same as that of the native strain, the invasiveness of the adapted strain into HeLa cells was very low or nonexistent.

Proteome of the bacterium Mycoplasma penetrans.

A proteome map of Mycoplasma penetrans has been constructed using two-dimensional gel electrophoresis in combination with mass spectrometry (MS). Mycoplasma penetrans infects the urogenital and respiratory tracts of humans. A total of 207 spots were characterized with MS and, in comparing the experimental data with the DNA sequence-derived predictions, it was possible to assign these 207 spots to 153 genes. The Pro-Q Diamond phosphoprotein dye technology was used for the fluorescent detection of 26 phosphoproteins in the 4-7 pH range.

Activation of nuclear factor kappaB and induction of inducible nitric oxide synthase by lipid-associated membrane proteins isolated from Mycoplasma penetrans.

BACKGROUND: This study was designed to investigate the potential pathogenicity of Mycoplasma penetrans (M. penetrans) and its molecular mechanisms responsible for the induction of iNOS gene expression in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. penetrans. METHODS: Mouse macrophages were stimulated with M. penetrans LAMPs to assay the production of nitric oxide (NO). The expression of inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western blotting. The activity of nuclear factor kappaB (NF-kappaB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, on the production of nitric oxide and the expression of iNOS were also assessed in mouse macrophages treated with M. penetrans LAMPs by indirect immunofluorescence and Western blotting. RESULTS: M. penetrans LAMPs stimulated mouse macrophages to produce nitric oxide in a dose- and time-dependent manner. The mRNA and protein levels of iNOS were also upregulated in response to LAMP stimulation and inhibited by PDTC treatment. M. penetrans LAMPs were found to trigger NF-kappaB activation, a possible mechanism for the induction of iNOS expression. CONCLUSION: This study demonstrated that M. penetrans may be an important etiological factor of certain diseases due to the ability of M. penetrans LAMPs to stimulate the expression of iNOS, which is probably mediated through the activation of NF-kappaB.

Identification of two glycosylated components of Mycoplasma penetrans: a surface-exposed capsular polysaccharide and a glycolipid fraction.

Among the wall-less mycoplasmas only a few species have been identified with a capsule at their cell surface. Mycoplasma penetrans is a recently identified mycoplasma with unique morphology, isolated from HIV-infected patients. Using transmission electron microscopy, it was found that M. penetrans is surrounded by capsular material 11 nm (strain GTU-54-6A1) to 30 nm (strain HF-2) thick, which can be stained with ruthenium red and labelled with cationized ferritin. The polysaccharide composition of this capsule was indicated by its staining with periodic acid-thiocarbohydrazide silver proteinate and the abolition of ruthenium red staining of the cell surface by neuraminidase treatment. In addition, proteinase K treatment of the M. penetrans cells resulted in removal of the capsule, suggesting that polypeptides may contribute in anchoring it to the membrane or in its stability. Two different types of glycosylated material were detected in mycoplasma extracts by SDS-PAGE and periodic acid-Schiff staining. The first component was a high-molecular-mass material, which was heat- and proteinase-K-labile and which probably constitutes the capsular polymer. The other component was a low-molecular-mass glycolipid fraction, which was proteinase-K-, heat- and EDTA-resistant. The identification of a capsule at the M. penetrans cell surface is of particular interest for a mycoplasma which has been shown to adhere to various host cells and to penetrate into their intracellular compartments. The capsule may have significance in the pathogenesis of disease associated with infection by this organism.

Characterization of a major Mycoplasma penetrans lipoprotein and of its gene.

A novel mycoplasmal species designated as Mycoplasma penetrans has been isolated recently from patients infected with human immunodeficiency virus. p35, a major antigen extracted from the membrane of this mycoplasma using Triton X-114 has been found to be a lipoprotein. After proteolytic treatment of p35, the sequence of one of the resulting peptides was determined and a corresponding oligonucleotide was deduced. Using this oligonucleotide as a probe the p35 gene was cloned and sequenced. Sequence analysis revealed an amino-terminal signal peptide with a potential acylation site which would result in a 35.3 kDa mature product. In addition, the p35 gene was followed by an open reading frame with a corresponding polypeptide partially homologous to p35, in particular to the N-terminus region.