Interactions of Meso-tetra-(4-N-oxyethylpyridyl) Porphyrin,Its 3-N Analog and Their Metallocomplexes with Duplex DNA

Abstract

Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.     

Intestinal microflora of autistic children

Autistic behavior is often accompanied by numerous disturbing symptoms on the part of gastrointestinal system, such as abdominal pain, constipation or diarrhea. These problems are often connected with deregulation of physiological microflora in intestine. The aim of this study was to determine differences in intestinal microflora of autistic and healthy children. Strains of Clostridium spp. and enterococci were isolated more frequently from stool samples of autistic children and rarely lactobacilli. Quantitative differences were observed maliny among staphylococci, Candida spp. and Clostridium perfringens. Monitoring and stabilization of intestinal microflora and knowledge about role of particular strains in etiology of autistic disorders can increase the chances for appropriate therapy.     

Lethal factor of Clostridium histolyticum kills cells by apoptosis

In this study, for the first time, Clostridium histolyticum lethal factor was purified by ammonium sulfate precipitation of culture broth, centrifugation through an Amicon filter device and hydrophobic interaction chromatography. The purified lethal factor was devoid of proteolytic activity. At a concentration of 150 ng mL(-1) the lethal factor killed 50% of HeLa cells within 24 h of exposure. Abrogation of actin filaments, activation of caspases, fragmentation of nuclear DNA as well as ultrastructural changes indicated that the cell death occurred by apoptosis. The apoptotic action of the lethal factor is in agreement with changes induced in animal tissues by administration of C. histolyticum culture medium.     

Vacuolating,lethal, collagenase and clostripain activities of six reference ATCC <Emphasis Type="Italic">Clostridium histolyticum</Emphasis> strains

We have previously demonstrated that C. histolyticum reference strain ATCC 19401 produces not only lethal factor but also hitherto unrecognized vacuolating toxin. The aim of this study was to compare vacuolating and lethal activities of six reference C. histolyticum strains (ATCC 6282, 8034, 17859, 17860, 19401 and 25770) and to determine whether production of vacuolating toxin is strain-dependent and how the amounts of both toxins produced by the same strain are related to each other and also to protease, collagenase and clostripain activities. All strains produced vacuolating and lethal toxins as well as collagenase, clostripain and proteases, but with different yield. Strain ATCC 19401 demonstrated considerable vacuolating and lethal activities and low activity of collagenase, clostripain and proteases. In other strains such relationship was not evident. Positive correlations were observed in collagenase and clostripain activities of all studied C. histolyticum strains (r = 0.71). Positive correlations were detected also in vacuolating activities of studied strains and clostripain (r = 0.62) and collagenase (r = 0.87) production, and this effect was statistically significant (P < 0.05). Negative non-significant correlations were detected: (a) in lethal activities of studied strains and clostripain, or collagenase activities, (b) in vacuolating activities and protease production.     

The blockade of endothelin A receptor protects astrocytes against hypoxic injury: common effects of BQ-123 anderythropoietin on the rejuvenation of the astrocyte population

In the present study the role of endothelin (ET) and its receptors (ETA-R and ETB-R) in cellular mechanisms underlying the resistance of astroglial cells to low oxygen level and development of hypoxia has been investigated. To define the influences of ET and its receptors on survival and on antigenic as well as morphologic differentiation of rat astroglial cells in normoxic (NC) and hypoxic culture (HC) the selective antagonists of ETA-R (BQ-123) and ETB-R (BQ-788) were used. Treatment of HC with BQ-123 caused an increase in cell number and inhibited the hypoxia-induced apoptosis by 37%. BQ-123 decreased the hypoxia-induced cytotoxicity in HC. These effects of BQ-123 were abolished in cultures simultaneously treated with BQ-123 and BQ-788. Administration of BQ-788 alone decreased the number of living cells in NC, but not in HC. The activity of caspase-3/-7 was not changed by exposure of NC and HC to BQ-788. The protection provided by BQ-123 to astroglial cells against cytotoxicity in NC and HC was similar to that of erythropoietin (EPO), a cytokine with established neuroprotective effects. The functional improvement of astroglial cells and slowing down of their differentiation under exposure to BQ-123, or EPO, or BQ-123 + EPO has been evidenced by an increased number of nestin+/glial fibrillary acidic protein-positive (GFAP+) astrocytes accompanied by decrease of nestin-/GFAP+ cells. The simultaneous treatment with BQ-123 and EPO additionally decreased the activities of caspase-3/-7 (64%) and release of LDH into the medium (94%). The benefits in the functional states of astrocytes obtained by combined treatment of HC with BQ-123 and EPO suggest a new therapeutic strategy in treatment of hypoxic brain injury.     

Evaluation of blood cultures of children hospitalized in Upper Silesian Health Center of Child and Mother in Katowice

Blood cultures (1613) taken from children hospitalized in 13 wards of Upper Silesian Health Center of Child and Mother were studied using Bact/Alert 240 monitoring system (bioMerieux). Around 17.7% of studied cultures were positive: 285 microorganisms were isolated. Gram-positive cocci dominated: 32.3% were strains of MRCNS Gram-negative rods, mainlyEnterobacteriaceae were isolated in 18.6% of cases, non-fermenters--in 12.9%, yeasts (mainly C. albicans)--in 10.5%. More frequently blood cultures were positive in Intensive Care Unit (37.5%).     

Further similarities between astrocytes and perisinusoidal stellate cells of liver (Ito cells): Colocalization of desmin and glial fibrillary acidic protein in astroglial primary cultures

The colocalization of desmin and glial fibrillary acidic protein (GFAP) in astrocytes was inferred from previous studies demonstrating a unique antigenic composition comprising GFAP, desmin and vimentin in perisinusoidal stellate cells (PSC) of liver which share several features with astrocytes. In the present study the colocalization of GFAP and desmin was investigated by double-immunolabeling experiments in 12-day-old rat astroglial primary cultures with antiserum against GFAP and two commercial monoclonal antibodies against desmin, antibodies of clone DEU-10 and clone DEB-5. These antibodies selectively decorated the perisinusoidal stellate cells (PSC) of liver for which desmin is known to be a marker. The results obtained with astroglial cells demonstrate that both GFAP and desmin are coexpressed in morphologically different types, process-bearing and process-lacking astrocytes. The expression of desmin was apparently more pronounced in process-lacking astrocytes and was considerably lower in process-bearing ones. In process-lacking astrocytes, in contrast to filamentous cytoplasmic staining for GFAP, the immunoreactivity for desmin was non-filamentous and was irregularly spread in the perinuclear cytoplasm of the cells, while in process-bearing astrocytes the pattern of staining for desmin was similar to that of GFAP. The variability in the intensity and pattern of staining for desmin in astrocytes might be due to transitional stages of differentiation for part of the cells. This interpretation was supported by the presence of GFAP in the cells weakly expressing smooth muscle alpha-actin and the absence of GFAP in the cells enriched with microfilaments.     

Copper (II) Ions Affect <Emphasis Type="Italic">Escherichia coli</Emphasis> Membrane Vesicles’ SH-Groups and a Disulfide-Dithiol Interchange Between Membrane Proteins

The SH-groups in Escherichia coli membrane vesicles, prepared from cells grown in fermentation conditions on glucose at slightly alkaline pH, have a role in the F0F1-ATPase operation. The changes in the number of these groups by ATP are observed under certain conditions. In this study, copper ions (Cu2+) in concentration of 0.1 mM were shown to increase the number of SH-groups in 1.5- to 1.6-fold independent from K+ ions, and the suppression of the increased level of SH-groups by ATP was determined for Cu2+ in the presence of K+. Moreover, the increase in the number of SH-groups by Cu2+ was absent as well as the inhibition in ATP-dependent increasing SH-groups number by Cu2+ lacked when vesicles were treated with N-ethylmaleimide (NEM), specific thiol-reagent. Such an effect was not observed with zinc (Zn2+), cobalt (Co2+), or Cu+ ions. The increased level of SH-groups was observed in the hycE or hyfR mutants with defects in hydrogenases 3 or 4, whereas the ATP-dependent increase in the number of these groups was determined in hycE not in hyfR mutants. Both changes in SH-groups number disappeared in the atp or hyc mutants deleted for the F0F1-ATPase or hydrogenase 3 (no activity of hydrogenase 4 was detected in the hyc mutant used). A direct effect of Cu2+ but not Cu+ on the F0F1-ATPase is suggested to lead to conformational changes or damaging consequences, increasing accessible SH-groups number and disturbing disulfide-dithiol interchange within a protein-protein complex, where this ATPase works with K+ uptake system or hydrogenase 4 (Hyd-4); breaks in disulfides are not ruled out.     

Intestinal colonization of newborns treated in intensive care units by multiple drug resistant microorganisms

The aim of the study was to examine the digestive tract colonisation of the newborns by multiple drug resistant bacteria during hospitalization. On the day of admission, after 5 days of hospitalization and at the day of discharge swabs from the anus of the 31 newborns hospitalized in OITiPN were taken and cultured on nutrient and selective media for staphylococci, enterococci, gram negative bacilli and fungi. Susceptibility to antibiotics of bacteria was determined, with giving attention to such resistance mechanisms as: methicillin resistant staphylococci (MRS), high level aminoglycoside resistant (HLAR) and vancomycin resistant enterococci (VRE) and the production of extended spectrum beta-lactamases (type ESBL) by gram negative bacilli. On the day of admission in 7 newborns methicillin resistant staphylococci (MRSCN) were grown in 24 no multiple drug resistant bacteria were found. Among those in 23 already after 5 days of hospitalization, colonization by multiple drug resistant strains was determined: coagulase-negative methicillin resistant staphylococci (MRSCN) were found in 16 children, strains of enterococci (HLAR) in 3 newborns and gram negative ESBL (+) bacilli also in 3 cases. On the day of discharge from hospital (after 13-141 days) in 23 out of 24 newborns enteric tract colonization by multiple drug resistant strains was assessed. In the enteric tract of 3 newborns hospitalized up to 2 weeks coagulase-negative methicillin resistant staphylococci (MRSCN) and/or HLAR enterococci were found; gram negative bacilli that produce ESBL appeared in newborns hospitalized for longer than 14 days. They were isolated in 12 out of 21 newborns. Forming of the enteric tract bacterial flora of the long hospitalized newborns depends on the time of hospitalization as well as on the used therapy.     

Similar protective effects of BQ-123 and erythropoietin on survival of neural cells and generation of neurons upon hypoxic injury

Our recent study [Danielyan et al., 2005. Eur. J. Cell Biol. 84, 567-579] showed an additive protective effect of endothelin (ET) receptor A (ETA-R) blockade and erythropoietin (EPO) on the survival and rejuvenation of rat astroglial cells exposed to hypoxia. Whether the effects observed with rodent astroglial cells can be reproduced in human astrocytes and whether these effects of ETA-R blockade and EPO on astrocytes are associated with neuronal survival remained open. Therefore, in the present study, the effects of the ETA-R antagonist BQ-123 and EPO on the maintenance of the neuronal population and survival of the human fetal astroglial cell line (SV-FHAS) under normoxic and hypoxic conditions (NC and HC, respectively) were investigated. Rat brain primary cultures exposed to BQ-123 and/or EPO revealed an increase in the number of beta-III tubulin-positive neurons under NC. The hypoxia-caused loss of neurons was abolished by administration of BQ-123 or EPO. Simultaneous application of EPO and BQ-123 led to an additive protective effect on the generation of neurons under NC only. By contrast, BQ-788, the selective ETB-R antagonist, diminished the neuronal population both in NC and HC. Both under NC and HC the number of non-differentiated nestin+/GFAP- neural cells increased upon application of EPO or BQ-123. SV-FHAS responded to BQ-123 or EPO by a decrease in LDH activity in the culture medium under NC (35%) and HC (26% LDH decrease). Concomitant effects of EPO and BQ-123 were illustrated in an additional increase in the survival of human astrocytes (33% under NC and 17% under HC). These data hint at a neuroprotective therapeutic potency of ETA-R blockade, which either alone or in combination with EPO may improve the survival of astroglial and neuronal cells upon hypoxic injury.