Chlorophyllin protects HEp-2 cells from nuclear fragmentation induced by poliovirus

AIMS: Chlorophyllin (CHLN) is a synthetic derivative of chlorophyll that possesses antimutagenic activity against several environmental contaminants. In the present study, CHLN was assayed for its capacity to prevent nuclear fragmentation (NF) in HEp-2 cells infected with poliovirus. METHODS AND RESULTS: CHLN was assayed at concentrations of 0.5 and 2.5 microg ml(-1), and NF was monitored using the comet assay and acridine orange staining. We demonstrated that CHLN reduced the percentage of NF in poliovirus-infected HEp-2 cells, when cells were treated with drug before infection or exposed continuously to drug. However, the highest degree of protection was achieved when the virus was exposed to CHLN before infection followed by protocol where infected cultures were continuously exposed to the drug after infection. CONCLUSIONS: It is suggested that CHLN primarily binds to the virus which inhibits cell penetration, thereby maintaining nuclear integrity. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering that CHLN has several beneficial properties and no significant toxic effects in humans and animals, it would be an ideal candidate drug to test for antiviral activity.     

Glucose and glutamine utilization by rat lymphocytes, monocytes and neutrophils in culture: a comparative study

Glucose and glutamine utilization and production of glutamate and lactate were determined for up to 48 h in lymphocytes, monocytes and neutrophils cultured in medium rich in metabolites and vitamins. Glucose was utilized by the three cell types in culture in the following order: neutrophils > monocytes > lymphocytes, whereas lactate was produced in the order: monocytes > neutrophils > lymphocytes. The consumption of glucose followed the activity of glucose-6-phosphate dehydrogenase but it was not related to hexokinase activity. Glutamine was consumed by the three leukocyte types in culture as follows: neutrophils > lymphocytes > or = monocytes. The consumption of glutamine was not fully related to the activity of phosphate-dependent glutaminase. The production of glutamate was not remarkably different among the three cell types. For comparison, glutamine and glucose utilization and glutamate and lactate production were also evaluated using 1-h incubated leukocytes. Under this condition, only glucose or glutamine was added to the medium. Glucose was utilized as follows: neutrophils > monocytes > lymphocytes, whereas lactate was produced in the following order: monocytes > or = neutrophils > lymphocytes. Glutamine was consumed as follows: neutrophils > lymphocytes > monocytes, whereas glutamate was produced as follows: neutrophils > or = monocytes = lymphocytes. The ratio of the amount of glucose/glutamine consumed by 1-h incubated cells was 0.5 for neutrophils, 1.5 for monocytes, and 0.3 for lymphocytes. However, the three cell types cultured for 48 h utilized glucose to a much higher degree than glutamine. The ratio of the amount of glucose/glutamine utilized by the cultured cells was 8.9 for neutrophils, 16.4 for monocytes, and 6.7 for lymphocytes. These observations support the proposition that glutamine is required in much higher amounts than glucose to accomplish the total metabolic requirement of leukocytes. Under conditions closer to physiological when the availability of a variety of metabolites and vitamins is not restricted, glucose is the preferred substrate for lymphocytes, monocytes and neutrophils.     

Structural composition and differential anticoagulant activities of dermatan sulfates from the skin of four species of rays, Dasyatis americana, Dasyatis gutatta, Aetobatus narinari and Potamotrygon motoro

We compared the disaccharide composition of dermatan sulfate (DS) purified from the ventral skin of three species of rays from the Brazilian seacoast, Dasyatis americana, Dasyatis gutatta, Aetobatus narinari and of Potamotrygon motoro, a fresh water species that habits the Amazon River. DS obtained from the four species were composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfate groups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positions C-2 of the uronic acid and at position C-4 or C-6 of GalNAc. However, DS from the skin of P. motoro presented a very low content of the disulfated disaccharides. The anticoagulant actions of ray skin DS, measured by both APTT clotting and HCII-mediated inhibition of thrombin assays, were compared to that of mammalian DS. DS from D. americana had both high APTT and HCII activities, whereas DS from D. gutatta showed activity profiles similar to those of mammalian DS. In contrast, DS from both A. narinari and P. motoro had no measurable activity in the APTT assay. Thus, the anticoagulant activity of ray skin DS is not merely a consequence of their charge density. We speculate that the differences among the anticoagulant activities of these three DS may be related to both different composition and arrangements of the disulfated disaccharide units within their polysaccharide chains.     

Isolation and characterization of eight pregnancy-associated glycoproteins present at high levels in the ovine placenta between day 60 and day 100 of gestation

Pregnancy-associated glycoproteins (PAG), structurally related to aspartic proteinases, are expressed in the outer epithelial cell layer (chorion/trophectoderm) of the ungulate placenta. The aim of the present study was to isolate as many PAG molecules as possible from placentae collected between day 60 and day 100 of gestation and to characterize their amino-terminal amino-acid sequences. Three heterologous radioimmunoassays were used to monitor PAG immunoreactivity throughout the isolation procedures. Sequential use of DEAE-cellulose, gel filtration, and CM ceramic chromatographies led to the isolation of several fractions rich in PAG immunoreactivity. The fractions with a large amount of proteins were also purified by chromatofocusing. The analysis of immunoreactive fractions by SDS-PAGE, Western blotting and two-dimensional electrophoresis followed by amino-terminal microsequencing on PVDF membranes allowed to identify eight different ovPAG with apparent molecular masses ranging from 55 to 66 kDa and isoelectric points from 4.0 to 6.8. The N-terminal sequences were determined and their comparison to those previously identified revealed that four of them are identical to those encoded by previously known cDNA, while the additional four sequences appear to be novel since they have not yet been described.     

Effect of angiotensin II and losartan on the phagocytic activity of peritoneal macrophages from Balb/C mice

Angiotensin II (AII), a product of rennin-angiotensin system, exerts an important role on the function of immune system cells. In this study, the effect of AII on the phagocytic activity of mouse peritoneal macrophages was assessed. Mice peritoneal macrophages were cultured for 48 h and the influence of different concentrations of AII (10(-14) to 10(-7) M) and/or losartan, 10(-16) to 10(-6) M), an AT1 angiotensin receptor antagonist, on phagocytic activity and superoxide anion production was determined. Dimethylthiazoldiphenyltetrazolium bromide reduction and the nucleic acid content were used to assess the cvtotoxicity of losartan. A stimulatory effect on phagocytic activity (P < 0.05) was observed with 10(-13) M and 10(-12 M) AII concentrations. The addition of losartan (up to10(-14) M) to the cell cultures blocked (P < 0.001) the phagocytosis indicating the involvement of AT1 receptors. In contrast, superoxide anion production was not affected by AII or losartan. The existence of AT1 and AT2 receptors in peritoneal macrophages was demonstrated by immunofluorescence microscopy. These results support the hypothesis that AII receptors can modulate murine macrophage activity and phagocytosis, and suggest that AII may have a therapeutic role as an immunomodulatory agent in modifying the host resistance to infection.     

Respiratory Chains from Aerobic Thermophilic Prokaryotes

Thermophiles are organisms that grow optimally above 50 degrees C and up to approximately 120 degrees C. These extreme conditions must have led to specific characteristics of the cellular components. In this paper we extensively analyze the types of respiratory complexes from thermophilic aerobic prokaryotes. The different membrane-bound complexes so far characterized are described, and the genomic data available for thermophilic archaea and bacteria are analyzed. It is observed that no specific characteristics can be associated to thermophilicity as the different types of complexes I-IV are present randomly in thermophilic aerobic organisms, as well as in mesophiles. Rather, the extensive genomic analyses indicate that the differences concerning the several complexes are related to the organism phylogeny, i.e., to evolution and lateral gene transfer events.     

Removal of chromium by mucilaginous seeds of Ocimum basilicum

Polysaccharides bound to bacteria or in isolated form have been shown to bind heavy metals. A limitation of this technology can be overcome by immobilization. In view of this Ocimum basilicum seeds which swell upon wetting could serve as natural immobilized source of agriculturally-based polysaccharides. The seeds consist of an inner hard core and a pectinous fibrillar outer layer. Pretreating the seeds with acid, alkali, periodate or boiling in water was found to alter the metal binding capacity. Of the various treatments given, seeds boiled in water were found to be superior in terms of mechanical stability and exhibited fairly optimal Cr(VI) uptake kinetics. The maximum adsorption capacity as calculated from the Langmuir isotherm was 205 mg Cr/g dry seeds. Biosorption of Cr(VI) was found to be pH dependent with maximum uptake at pH 1.5 wherein sorption was not affected by the presence of other metal ions such as Cd(2+), Cu(2+), Ca(2+) and Na(+). Seeds were used in a packed bed reactor for the continuous removal of Cr(VI). Thus O. basilicum seeds may have application as a potential bioresource in tropical countries such as India where they are widely available.     

Synthesis and pharmacological evaluation of prenylated and benzylated pterocarpans against snake venom

Edunol (3), a pterocarpan isolated from Harpalyce brasiliana, a plant used in the northeast of Brazil against snakebites, was obtained by synthesis and showed antimyotoxic, antiproteolytic and PLA2 inhibitor properties. These proprieties could be improved through the synthesis of a bioisoster (5), where the prenyl group was replaced by the benzyl group.     

Identification of a C-terminal poly(A)-binding protein (PABP)-PABP interaction domain: role in cooperative binding to poly (A) and efficient cap distal translational repression

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.