Electron immunohistochemical analysis of localization of neutral Mn2+-dependent DNAase. I. Synthesis of ferritin and colloidal gold conjugates with monospecific antibodies against neutral Mn2+-dependent DNAase

Rabbit antibodies against a neutral Mn(2+)-dependent rat liver DNAse were obtained, whose specificity towards DNAse was ascertained by suppression of the enzyme activity both in vitro system and immunoblotting assays. Procedures of synthesis of ferritin and colloidal gold conjugates with antibodies are described. The biological activity of the conjugates proved to be similar to that of the original antibodies.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Mechanism of action of DNA-hydrolyzing antibodies to DNA from blood of patients with systemic lupus erythematosus

Four fractions of IgG antibodies to native DNA (nDNA) were obtained from blood of patients with systemic lupus erythematosus (SLE). These antibodies displayed a thermostable DNA-hydrolyzing activity and were different in affinity for DNA-cellulose and sorption on DEAE-cellulose. DNA-hydrolyzing antibodies to nDNA are metal-dependent endonucleases, cause mainly single-strand breaks in DNA, and are active over a wide range of pH. By atomic-force microscopy, three-dimensional images of DNA complexes with DNA-hydrolyzing antibodies to nDNA were obtained with nanometer resolution, and a nonprocessive action mechanism was shown for the DNase activity of antibodies to nDNA.     

Relation between DNA replication and neutral Mn 2+-dependent DNAse activity in chromatin

In cultures of primary murine fibroblasts the 10% serum stimulates the replicative synthesis of DNA inhibited by aphidicolin and araC (cytosine arabinoside). Using direct immunofluorescence analysis, it was shown that antibodies penetrate inside the cells and after 4 hours are pooled in the nuclei, where they remain for another 20 hours. The substitution of antibodies against chromatin DNAase by bovine serum albumin of normal serum gamma-globulins does not interfere with the DNA synthesis induction.     

Chemical characterization and anti-inflammatory effect of rauvolfian,a pectic polysaccharide of <Emphasis Type="Italic">Rauvolfia</Emphasis> callus

The pectic polysaccharide named rauvolfian RS was obtained from the dried callus of Rauvolfia serpentina L. by extraction with 0.7% aqueous ammonium oxalate. Crude rauvolfian RS was purified using membrane ultrafiltration to yield the purified rauvolfian RSP in addition to glucan as admixture from the callus, with molecular weights 300 and 100–300 kD, respectively. A peroral pretreatment of mice with the crude and purified samples of rauvolfian (RS and RSP) was found to decrease colonic macroscopic scores, the total area of damage, and tissue myelope roxidase activity in colons as compared with a colitis group. RS and RSP were shown to stimulate production of mucus by colons of the colitis mice. RSP appeared to be an active constituent of the parent RS. The glucan failed to possess anti-inflammatory activity. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 7, pp. 955–962.     

Genetic diversity of chloroquine-resistant Plasmodium vivax parasites from the western Brazilian Amazon

The molecular basis of Plasmodium vivax chloroquine (CQ) resistance is still unknown. Elucidating the molecular background of parasites that are sensitive or resistant to CQ will help to identify and monitor the spread of resistance. By genotyping a panel of molecular markers, we demonstrate a similar genetic variability between in vitro CQ-resistant and sensitive phenotypes of P. vivax parasites. However, our studies identified two loci (MS8 and MSP1-B10) that could be used to discriminate between both CQ-susceptible phenotypes among P. vivax isolates in vitro. These preliminary data suggest that microsatellites may be used to identify and to monitor the spread of P. vivax-resistance around the world.     

Quantitation of cytokine-stimulated migration of endothelium and epithelium by a new assay using microcarrier beads

Recent studies have identified a group of cytokines which appear to be cell-specific regulators of mobility in nonleukocytic mammalian cells. One example is scatter factor (SF), a soluble protein(s) produced by cultured fibroblasts and vascular smooth muscle cells which causes spreading and separation ("scattering") of tight, cohesive colonies of epithelial cells. Studies of SF action have been limited because the degree of scattering is difficult to quantitate and because scattering assays cannot be used to study potential target cells that do not form tight, cohesive colonies. We developed a simple, quantitative assay of SF-stimulated mobility based on migration of target cells off microcarrier beads onto plastic culture surfaces in 24-well plates. We showed that crude and partially purified SF derived from ras-transformed 3T3 cells stimulates migration of both epithelial and vascular endothelial cells but not of producer or nonproducer fibroblasts. Scatter and migration-stimulating activities copurified on cation exchange chromatography; and the degree of stimulation was closely correlated with scattering titer regardless of SF purity. Migration of endothelial cells from beads, while extremely sensitive to SF, was not affected by serum concentration (1 to 10%), various purified growth factors, or fibronectin. Both scattering and migration from beads were blocked by cycloheximide (0.1 microgram/ml) during assay incubation, suggesting that these processes require protein synthesis. The microcarrier bead assay may be a useful quantitative tool to study the biochemical mechanisms of SF-stimulated cell migration.     

Programmed cell death in bacteria

Programmed death (PDC) of individual cells is a genetically controlled biological process related to the development of multicellular organisms. It proceeds in most cases as apoptosis characterized by DNA degradation and breakdown of dying cells to apoptotic bodies, and ending by their phagocytosis by macrophages or by the surrounding tissue. Unlike apoptosis, necrosis is a genetically unregulated sudden death of a group of cells caused by a severe damage of membranes and other cell components. In bacteria, programmed cell death is mostly related to population development. This holds mainly for sporulation of bacilli where the process is best understood at the morphological, physiological and genetic level. Sporulation of bacilli begins by an asymmetric division of the nongrowing cell into two parts—the mother and the forespore compartment, whose fate is different. Whereas the smaller compartment develops into the spore, the function of the larger is twofold. It participates in the spore development mainly by forming spore coast but it also synthesizes or activates the autolytic apparatus which lyzes the sporangium cell wall at the end of the process. Some phases of the development of myxobacteria and streptomycetes also have characteristic features of programmed death. Unlike sporulation of bacilli, the autolysis of a portion of population of myxobacteria or hyphae of streptomycetes proceeds in the middle of their developmental cycle. Extensive turnover of cell membranes in growing myxobacteria results in the formation of a fatty acid mixture—theautocide—which kills a smaller or greater portion of the myxobacterial population. The dead cells are digested by extracellular enzymes released by myxobacteria and the digest is used as nutrient for completion of the developmental cycle of the remaining living population. Similar events take place also during the formation of aerial mycelium in streptomycetes. Here the autolysis of a portion of vegetative (substrate) mycelium supplies amino acids for the formation of aerial mycelium. The recently discovered programmed death of plasmid-free descendants of a plasmid-bearing population of different bacteria is based on the loss of control of toxin activity by its antidote. Both substances are encoded by plasmid DNA and the loss of the plasmid results in an “enforced suicide” of the host cell because the effective concentration of the antidote decreases more rapidly than that of the toxin. The mechanisms of this suicide can vary. In addition to the above mentioned kinds of programmed death, other events of developmentally regulated death of prokaryotes probably exist. Some bacteria contain “death genes” in their chromosome which trigger cell death at the onset of the stationary phase. The physiological function of this kind of suicide is not known. However, most nonsporulating bacteria developed a strategy of surviving at the nongrowing stage by transforming the growing cell to a more resistant dormant (cryptobiological) form.     

Spores of microorganisms

Calcium-dipicolinate (Ca-DPA)-rich and Ca-DPA-deficientBacillus cereus spores were incubated in a synthetic medium with germination stimulants and in bactopeptone medium with a fairly high calcium ion concentration. In the complex medium the germination of Ca-DPA-rich spores was completely blocked at a concentration of 0.5m CaCl₂, whereas the complete blockage of germination in the synthetic medium required higher concentrations (0.6–0.8m) of calcium chloride. Ca-DPA-deficient spores germinated more slowly and less completely in the synthetic medium than in the bactopeptone medium. The germination of these spores took place, however, even at higher calcium ion concentrations (0.6–0.8m). On the contrary, lower calcium chloride concentrations (0.1–0.4m) accelerated the germination of these spores in the synthetic medium and the final percentage of phase-dark and stainable spores was higher. “H-forms” of the Ca-DPA-rich and Ca-DPA-deficient spores prepared by acid titration germinated in both media. The germination of the latter spores being slower and proceeding less completely. “H-forms” germinated completely or partially in media with a high concentration of calcium chloride. The percentage of germinated spores, however, was strongly influenced by the concentration of this cation, especially the “H-forms” of Ca-DPA-deficient spores. Moreover, the germination of Ca-DPA-deficient spores in this medium was affected by the length of previous storage and, in the case of “H-forms” by the pH at which they were titrated. It was assumed that the increased permeability of calcium into the calciumundersaturated spore periphery in Ca-DPA-deficient and in “H-forms” of spores of both types co-determines (in the presence of germinants) the germinability of bacterial spores.     

The determination of the nature of the interaction of neutral Mn2+-dependent DNAse with DNA by immunoelectron microscopy

The interaction of neutral DNAase with native and denatured DNA was shown by immunoelectron microscopy method with the help of colloidal gold. The neutral DNAase of the rat liver nuclear chromatin is absorbed both to denatured DNA, in which the denatured regions are arranged at 5'-3' ends, and to DNA in which these regions are distributed along the whole molecule.