Characterization of isolates and clones of leishmania by analysis of kinetoplast DNA

The genetic characterization of pathogenic isolates of Leishmania was attempted by analysis of the molecular properties of kinetoplast DNA (kDNA) minicircles. Unit minicircle size is not conserved during speciation of Leishmania since the minicircles of strains and clones of L t major are smaller (700 bp) than those found in certain strains of L mexicana ssp (820 bp), L donovani (850 bp) or L t tropica (900 bp). Schizodeme analysis of minicircles reveals a high degree of sequence divergence in kDNA of Leishmania with the degree of microheterogeneity varying between species. This sequence divergence allows the discrimination of species, strains, and clones of Leishmania into schizodemes. Southern blot hybridization experiments reveal that at high stringency overall minicircle sequence homology is conserved among clones and strains of one species (L t major) but not between different species. This property of minicircle DNA permits the use of kDNA probes as a species-specific diagnostic test for the identification of unknown Leishmania isolates. The properties of kDNA from an L t tropica strain LRC-L32 (a “recidiva” organism) are so diverged from those of L t major strains as to support the classification [22,23] of L t tropica and L t major as separate species of Leishmania rather than subspecies of L tropica.     

Leishmania major: expression and gene structure of the glycoprotein 63 molecule in virulent and avirulent clones and strains

Two Leishmania membrane glycoconjugates, gp63 and lipophosphoglycan, have been implicated in parasite attachment and uptake into the host macrophage. Moreover, recent data suggest that parasite virulence is associated with high expression of gp63. In this study we have surveyed gp63 gene copy number, in addition to the level of expression of gp63 mRNA and protein in several Leishmania major isolates, as well as virulent and avirulent strains and clones. The highest level of gp63 expression was found in the avirulent cloned line LRC-L119.3G7, which expresses about a 15-fold higher level of gp63 RNA and protein than the virulent cloned line LRC-L137/7/V121, suggesting that large amounts of gp63 are not sufficient for infectivity and do not correlate with virulence. L119.3G7 has eight copies of the gp63 gene compared to five copies in the virulent cloned line V121 and its parental virulent isolate LRC-L137. A series of avirulent clones derived from LRC-L137 also had five copies of the gene, suggesting that gp63 copy number is maintained among closely related parasites. Different virulent isolates of L. major from different geographic regions exhibited six copies of the gp63 gene. The variation in total gene copy number is due to different numbers of the tandemly repeated gp63 isogene in different strains. Our data show that there is wide variability between strains of L. major in the copy number of gp63 genes as well as in the amount of RNA and protein expressed.     

A putative kinase-related protein (PKRP) from Plasmodium berghei mediates infection in the midgut and salivary glands of the mosquito

The completion of the Plasmodium (malaria) life cycle in the mosquito requires the parasite to traverse first the midgut and later the salivary gland epithelium. We have identified a putative kinase-related protein (PKRP) that is predicted to be an atypical protein kinase, which is conserved across many species of Plasmodium. The pkrp gene encodes a RNA of about 5300 nucleotides that is expressed as a 90 kDa protein in sporozoites. Targeted disruption of the pkrp gene in Plasmodium berghei, a rodent model of malaria, compromises the ability of parasites to infect different tissues within the mosquito host. Early infection of mosquito midgut is reduced by 58-71%, midgut oocyst production is reduced by 50-90% and those sporozoites that are produced are defective in their ability to invade mosquito salivary glands. Midgut sporozoites are not morphologically different from wild-type parasites by electron microscopy. Some sporozoites that emerged from oocysts were attached to the salivary glands but most were found circulating in the mosquito hemocoel. Our findings indicate that a signalling pathway involving PbPKRP regulates the level of Plasmodium infection in the mosquito midgut and salivary glands.     

Production and processing of a recombinant Fasciola hepatica cathepsin B-like enzyme (FhcatB1) reveals potential processing mechanisms in the parasite

The liver fluke, Fasciola hepatica, apparently uses a number of cysteine proteases during its life cycle, most likely for feeding, immune evasion and invasion of tissues. A cathepsin B-like enzyme (herein referred to as FhcatB1) appears to be a major enzyme secreted by the invasive, newly excysted juvenile flukes of this parasite. To examine the processing mechanisms for this enzyme, a recombinant form was expressed in Pichia pastoris and purified to yield a homogenous pool of the enzyme. The purified enzyme could be autoactivated at low pH via a bi-molecular mechanism, a process that was greatly accelerated by the presence of large, negatively charged molecules such as dextran sulfate. The enzyme could also apparently be processed to the correct size by an asparaginyl endopeptidase via cleavage in an unusual insertion N-terminal to the normal cleavage site used to yield the active form of the enzyme. Thus, there appear to be a number of ways in which this enzyme can be processed to its optimally active form prior to secretion by F. hepatica.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.     

Biogenesis of mitochondria. The effects of altered membrane lipid composition on cation transport by mitochondria of Saccharomyces cerevisiae

1. The fatty acid composition of the membrane lipids of a fatty acid desaturase mutant of Saccharomyces cerevisiae was manipulated by growing the organism in a medium containing defined fatty acid supplements. 2. Mitochondria were obtained whose fatty acids contain between 20% and 80% unsaturated fatty acids. 3. Mitochondria with high proportions of unsaturated fatty acids in their lipids have coupled oxidative phosphorylation with normal P/O ratios, accumulate K(+) ions in the presence of valinomycin and an energy source, and eject protons in an energy-dependent fashion. 4. If the unsaturated fatty acid content of the mitochondrial fatty acids is lowered to 20%, the mitochondria simultaneously lose active cation transport and the ability to couple phosphorylation to respiration. 5. The loss of energy-linked reactions is accompanied by an increased passive permeability of the mitochondria to protons. 6. Free fatty acids uncouple oxidative phosphorylation in yeast mitochondria and the effect is reversed by bovine serum albumin. 7. The free fatty acid contents of yeast mitochondria are unaffected by depletion of unsaturated fatty acids, and free fatty acids are not responsible for the uncoupling of oxidative phosphorylation in organelles depleted in unsaturated fatty acids. 8. It is suggested that the loss of energy-linked reactions in yeast mitochondria that are depleted in unsaturated fatty acids is a consequence of the increased passive permeability to protons, and is caused by a change in the physical properties of the lipid phase of the inner mitochondrial membrane.     

Vaccination with a Plasmodium chabaudi adami multivalent DNA vaccine cross-protects A/J mice against challenge with P. c. adami DK and virulent Plasmodium chabaudi chabaudi AS parasites

A current goal of malaria vaccine research is the development of vaccines that will cross-protect against multiple strains of malaria. In the present study, the breadth of cross-reactivity induced by a 30K multivalent DNA vaccine has been evaluated in susceptible A/J mice (H-2a) against infection with the Plasmodium chabaudi adami DK strain and a virulent parasite subspecies, Plasmodium chabaudi chabaudi AS. Immunized A/J mice were significantly protected against infection with both P. c. adami DK (31–40% reduction in cumulative parasitemia) and P. c. chabaudi AS parasites, where a 30–39% reduction in cumulative parasitemia as well as enhanced survival was observed. The 30K vaccine-induced specific IFN-γ production by splenocytes in response to native antigens from both P. c. chabaudi AS and P. c. adami DK. Specific antibodies reacting with surface antigens expressed on P. c. adami DS and P. c. chabaudi AS infected red blood cells, and with opsonizing properties, were detected. These results suggest that multivalent vaccines encoding conserved antigens can feasibly induce immune cross-reactivity that span Plasmodium strains and subspecies and can protect hosts of distinct major histocompatibility complex haplotypes.