A comparative study of the proteolytic enzymes of Trypanosoma brucei, T. equiperdum, T. evansi, T. vivax, Leishmania tarentolae and Crithidia fasciculata

Four types of proteolytic activity were detected in the bloodstream form of each of the four Trypanosoma species: (i) HPAase, active on hide powder azure and detected on polyacrylamide gels containing denatured haemoglobin; (ii) AZCase, active on azocasein; (iii) type 1, active on the chromogenic peptide N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine p-nitroanilide in the presence of dithiothreitol, and (iv) type 2, active against several nitroanilide derivatives in the absence of dithiothreitol. Studies of the pH optimum, dithiothreitol requirement and inhibitor sensitivities of the proteolytic activities suggested that: (a) HPAase and type 1 activities could be due to the same enzymes, probably a family of cysteine proteinases; (b) AZCase had some characteristics of a cysteine proteinase, but was not identical to HPAase, and (c) type 2 activity could be due to a serine proteinase. Procyclic T. brucei contained relatively low cysteine proteinase activities (HPAase, AZCase and type 1) but high type 2 activity. Their proteolytic enzymes thus were apparently more similar to those in Crithidia fasciculata and Leishmania tarentolae promastigotes than those in T. brucei bloodstream forms.     

Structural difference between the two forms of guinea-pig beta 2-microglobulin and their occurrence in inbred guinea-pig strains

The structural difference between two forms (basic and acidic) of guinea-pig beta 2-microglobulin (beta 2m) has been established. Both forms are present in urine from inbred guinea-pig strains. The beta 2m forms were each digested with carboxypeptidase Y and carboxypeptidase A contaminated with carboxypeptidase B. Released amino acids were separated from remaining protein, dansylated and analysed by 2-dimensional TLC on polyamide layer sheets. From the results it was concluded that the basic beta 2m form has lysine and the acidic beta 2m form has asparagine as their respective C-terminal amino acids. The acidic form is also 1 amino acid (lysine) shorter than the basic form, which is supported by electrophoretic studies on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of the 2 forms of beta 2m in urine from inbred guinea-pig strains 2 and 13, shown by gel filtration and ion exchange chromatography, makes it unlikely that the 2 forms are a result of genetic polymorphism.     

Guinea-pig peritoneal macrophage receptor for IgG--II. Purification of the receptor and its partial characterization

The Fc gamma receptor of guinea-pig peritoneal macrophages was purified by affinity chromatography by using rabbit IgG or guinea-pig IgG2 coupled to Sepharose. Lysates prepared by treatment of 125I-labeled macrophages with NP-40 were first applied to BSA-Sepharose and then to IgG-Sepharose and eluted with 0.5 M acetic acid containing 1% NP-40. The specific binding was determined by interaction of the 125I-labeled receptor with IgG-Sepharose in the presence and absence of soluble IgG. The specific binding of the purified receptor was 42-82%. Interactions of the purified receptor with IgG-Sepharose were equally well inhibited by soluble rabbit IgG or guinea-pig IgG2, but not by F(ab')2 fragments. Inclusion of NP-40 in the buffer used in the assay reduced nonspecific binding of the receptor to the affinity gels. The purified receptor can be stored for 20 days at 4 degrees C without a significant loss of the specific binding activity. Analysis of the receptor by SDS-polyacrylamide gel electrophoresis, under nonreducing and reducing conditions, revealed two major peaks of radioactivity corresponding to mol. wts of about 50,000 and 25,000, and one very minor peak corresponding to a mol. wt of about 30,000. The results obtained suggest that the protein of the second major peak is a product of the dissociation of the protein of the first major peak rather than a product of its reduction by 2-mercaptoethanol.     

Pronase and proteinase K digestion of human immunoglobulin M

Human 19S IgM was digested with pronase and proteinase K. Proteolysis was relatively fast, producing Fab2 mu-like fragments (approx. mol. wt 114,000) and Fab mu-like fragments (approx. mol. wt 46,500) as major products. Immunochemical analysis indicated that the fragments produced by either enzyme are very similar and that they are produced by cleavage at the C mu 2-C mu 3 and C mu 1-C mu 2 domain junctions respectively. An intermediate species of mol. wt 74,300, immunologically identical to F(ab)2 mu, was also identified. This is thought to represent an F(ab)2 mu fragment with one Fab mu fragment removed. Fc mu-related fragments were not identified in the digestion mixture with either enzyme. Covalently linked and non-covalently linked 7S human IgM (IgMs and IgMr respectively) were digested with pronase and proteinase K. IgMs was degraded very rapidly by either enzyme, producing relatively stable F(ab)2 mu- and Fab mu-like fragments. These fragments were similar in mol. wt and immunochemical properties to those produced from 19S IgM. IgMr was also degraded rapidly by either enzyme, in this case producing Fab mu-like fragments with no detectable F(ab)2 mu-like fragments. The kinetics of digestion and nature of the products suggest that cleavage of 19S IgM by pronase or proteinase K proceeds via an initial attack at the C mu 2-C mu 3 junction, followed by further degradation at the Cmu 1-C mu 2 junction. The results obtained using 7S IgM show that the intersubunit disulphide bonds, and the associated pentameric structure, are responsible for the relative resistance of 19S IgM to proteolysis. The inter-heavy-chain disulphide bonds, in particular the bond at cys 337, are responsible for the limited susceptibility of F(ab)2 mu-like fragments to proteolysis.     

Isolation and partial characterization of the tegumental outer membrane of schistosomula of Schistosoma mansoni

Separation of the external membranes from freshly converted mechanical schistosomula of Schistosoma mansoni was achieved by osmotic shock under hypertonic conditions, followed by mechanical shearing and ultracentrifugation. Prior to treatment, the schistosomula were surface labeled by introduction of N-DNP-epsilon-aminocaproylphosphatidylethanolamine molecules into their lipid bilayer followed by anti-DNP antibodies and stained with either 125I-protein-A or ferritin labeled secondary anti-DNP antibodies. This label provided a membrane marker by which the purity of the preparation could be assessed at each stage. Fluorescence staining with FITC-conjugated secondary antibodies prior to treatment revealed that the homogeneously stained membrane of the intact schistosomula became swollen and ruptured after the osmotic shock. The isolated membrane pellet was intensely fluorescent. Electron microscopical examination revealed mostly vesicles, some of them with organized multilayer assembly. The vesicles were ferritin labeled, indicating that they originated from the outer surface membrane of the schistosomula. A 100 fold enrichment in the alkaline phosphatase activity and about 300 fold enrichment in acetylcholinesterase activity in the membrane preparations, as compared to the intact schistosomula, was found. The isolated tegument was analyzed by SDS-polyacrylamide gel electrophoresis. The pattern obtained showed three major bands, of molecular weights 69 000, 45 000 and 12 000 alongside with a large number of minor bands. Immunoprecipitation of the isolated 125I-labeled membrane antigens with antisera from chronically infected mice revealed these three major bands together with three other bands of molecular weight 38 000, 23 000 and 16 000.     

Unique features of chicken Toll-like receptors

Toll-like receptors (TLRs) are a major class of innate immune pattern recognition receptors that have a key role in immune homeostasis and the defense against infections. The research explosion that followed the discovery of TLRs more than a decade ago has boosted fundamental knowledge on the function of the immune system and the resistance against disease, providing a rational for clinical modulation of the immune response. In addition, the conserved nature of the ancient TLR system throughout the animal kingdom has enabled a comparative biology approach to understand the evolution, structural architecture, and function of TLRs. In the present review we focus on TLR biology in the avian species, and, especially, on the unique functional properties of the chicken TLR repertoire.     

p53 protein, interferon-gamma, and NF-kappaB levels are elevated in the parkinsonian brain

We and other workers found markedly increased levels of proinflammatory cytokines and apoptosis-related proteins in parkinsonian brain. Although the pathogenesis of Parkinson's disease (PD) remains enigmatic, apoptosis might be involved in the degeneration of dopaminergic neurons in PD. To investigate the possible presence of other inflammatory cytokines and/or apoptosis-related protein, the levels of p53 protein, interferon-gamma, and NF-kappaB were measured for the first time in the brain (substantia nigra, caudate nucleus, putamen, cerebellum, and frontal cortex) from control and parkinsonian patients by a highly sensitive sandwich enzyme-linked immunosorbent assay. The p53 protein level in the caudate nucleus was significantly higher in parkinsonian patients than in controls (P<0.05), whereas this protein in the substantia nigra, putamen, and cerebral cortex showed no significant difference between parkinsonian and control subjects. The interferon-gamma level was significantly higher in the nigrostriatal dopaminergic regions (substantia nigra, caudate nucleus, and putamen) in parkinsonian patients than in the controls (P<0.05), but was not significantly different in the cerebellum or frontal cortex between the two groups. In accordance with previous immunohistochemical analysis, the NF-kappaB level in the nigrostriatal dopaminergic regions was significantly higher in parkinsonian patients than in the controls (P<0.05). These data suggest that the significant increase in the levels of p53 protein, interferon-gamma, and NF-kappaB reflect apoptosis and the inflammatory state in the parkinsonian brain and that their elevation is involved in the degeneration of the nigrostriatal dopaminergic neurons.     

Neuroprotective effect of gold nanoparticles composites in Parkinson's disease model

Parkinson’s disease (PD) is second most common neurodegenerative disorder worldwide. Although drugs and surgery can relieve the symptoms of PD, these therapies are incapable of fundamentally treating the disease. For PD patients, over-expression of α-synuclein (SNCA) leads to the death of dopaminergic neurons. This process can be prevented by suppressing SNCA over-expression through RNA interference. Here, we successfully synthesized gold nanoparticles (GNP) composites (CTS@GNP-pDNA-NGF) via the combination of electrostatic adsorption and photochemical immobilization, which could load plasmid DNA (pDNA) and target specific cell types. GNP was transfected into cells via endocytosis to inhibiting the apoptosis of PC12 cells and dopaminergic neurons. Simultaneously, GNP composites are also used in PD models in vivo, and it can successfully cross the blood-brain barrier by contents of GNP in the mice brain. In general, all the works demonstrated that GNP composites have good therapeutic effects for PD models in vitro and in vivo.