Pharmacokinetic analysis of polyamide nucleic-acid-cell penetrating peptide conjugates targeted against HIV-1 transactivation response element

We have demonstrated that polyamide nucleic acids complementary to the transactivation response (TAR) element of HIV-1 LTR inhibit HIV-1 production when transfected in HIV-1 infected cells. We have further shown that anti-TAR PNA (PNA(TAR)) conjugated with cell-penetrating peptide (CPP) is rapidly taken up by cells and exhibits strong antiviral and anti-HIV-1 virucidal activities. Here, we pharmacokinetically analyzed (125)I-labeled PNA(TAR) conjugated with two CPPs: a 16-mer penetratin derived from antennapedia and a 13-mer Tat peptide derived from HIV-1 Tat. We administered the (125)I-labeled PNA(TAR)-CPP conjugates to male Balb/C mice through intraperitoneal or gavage routes. The naked (125)I-labeled PNA(TAR) was used as a control. Following a single administration of the labeled compounds, their distribution and retention in various organs were monitored at various time points. Regardless of the administration route, a significant accumulation of each PNA(TAR)-CPP conjugate was found in different mouse organs and tissues. The clearance profile of the accumulated radioactivity from different organs displayed a biphasic exponential pathway whereby part of the radioactivity cleared rapidly, but a significant portion of it was slowly released over a prolonged period. The kinetics of clearance of individual PNA(TAR)-CPP conjugates slightly varied in different organs, while the overall biphasic clearance pattern remained unaltered regardless of the administration route. Surprisingly, unconjugated naked PNA(TAR) displayed a similar distribution and clearance profile in most organs studied although extent of its uptake was lower than the PNA(TAR)-CPP conjugates.     

Screening and characterization of antifungal clusterbean (Cyamopsis tetragonoloba) rhizobacteria

About 377 guar (Cyamopsis tetragonoloba) rhizobacteria were isolated from cultivated soils of north-west India (Thar Desert) and their antifungal activity against Macrophomina phaseolina (strains of groundnut, mungbean and guar) and Fusarium oxysporum (strains of chickpea and cumin) was examined. Isolates were characterised for generic types and physiological/functional diversity. About 19% isolates representing 24% locations were inhibitory to fungal growth. Isolates 009071, 009073, 009078 and 102354 recorded maximum inhibition of pathogenic fungi on plates. Isolate 034206 gave highest %RI, 009073 showed maximum protease activity and 102354 gave highest salt tolerance. Net house and field screening results revealed that isolates 004052, 009071, 009073, 001001, 094340 and 102354 had potential for biocontrol of disease. Partial sequencing of 16S rRNA gene of 61 isolates showed that 85% of isolates belonged to genus Bacillus. Phylogenetically, however, there were four clusters in the Bacillus group comprising of Bacillus subtilis, B. cereus, B. pumilus and B. sphaericus. One isolate was identified as B. flexus, while six isolates were Bacillus spp. Four isolates were identified as Achromobacter xylosoxidans, two as Bacterium (unclassified bacteria), and one each as Ochrobactrum intermedium, Pseudomonas aeruginosa and Ralstonia sp.     

Characterization of the Entamoeba histolytica ornithine decarboxylase-like enzyme

Background

The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, is a highly regulated enzyme.

Methodology and Results

To use this enzyme as a potential drug target, the gene encoding putative ornithine decarboxylase (ODC)-like sequence was cloned from Entamoeba histolytica, a protozoan parasite causing amoebiasis. DNA sequence analysis revealed an open reading frame (ORF) of ∼1,242 bp encoding a putative protein of 413 amino acids with a calculated molecular mass of 46 kDa and a predicted isoelectric point of 5.61. The E. histolytica putative ODC-like sequence has 33% sequence identity with human ODC and 36% identity with the Datura stramonium ODC. The ORF is a single-copy gene located on a 1.9-Mb chromosome. The recombinant putative ODC protein (48 kDa) from E. histolytica was heterologously expressed in Escherichia coli. Antiserum against recombinant putative ODC protein detected a band of anticipated size ∼46 kDa in E. histolytica whole-cell lysate. Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, had no effect on the recombinant putative ODC from E. histolytica. Comparative modeling of the three-dimensional structure of E. histolytica putative ODC shows that the putative binding site for DFMO is disrupted by the substitution of three amino acids—aspartate-332, aspartate-361, and tyrosine-323—by histidine-296, phenylalanine-305, and asparagine-334, through which this inhibitor interacts with the protein. Amino acid changes in the pocket of the E. histolytica enzyme resulted in low substrate specificity for ornithine. It is possible that the enzyme has evolved a novel substrate specificity.

Conclusion

To our knowledge this is the first report on the molecular characterization of putative ODC-like sequence from E. histolytica. Computer modeling revealed that three of the critical residues required for binding of DFMO to the ODC enzyme are substituted in E. histolytica, resulting in the likely loss of interactions between the enzyme and DFMO.     

Calcium-binding protein 1 of Entamoeba histolytica transiently associates with phagocytic cups in a calcium-independent manner

EhCaBP1, a calcium-binding protein of the parasite Entamoeba histolytica, is known to participate in cellular processes involving actin filaments. This may be due to its direct interaction with actin. In order to understand the kinetics of EhCaBP1 in such processes, its movement was studied in living cells expressing GFP-EhCaBP1. The results showed that EhCaBP1 accumulated at phagocytic cups and pseudopods transiently. The time taken for appearance and disappearance of EhCaBP1 was found to be around 12 s. Site-directed mutagenesis was used to generate an EhCaBP1 mutant with reduced Ca(2+)- and G-actin binding ability without any defect in its ability to bind F-actin. The overexpression of this mutant EhCaBP1 in the E. histolytica trophozoites resulted in the impairment of erythrophagocytosis, uptake of bacterial cells, killing of target cells but not fluid-phase pinocytosis. However, the mutant protein was still found to transiently localize with F-actin at the phagocytic cups and pseudopods. The mutant protein displayed reduced ability to activate endogenous kinase(s) suggesting that phagosome formation may require Ca(2+)-EhCaBP1 transducing downstream signalling but initiation of phagocytosis may be independent of its intrinsic ability to bind Ca(2+). The results suggest a dynamic association of EhCaBP1 with F-actin-mediated processes.     

The bacteriophage lambda DNA replication protein P inhibits the oriC DNA- and ATP-binding functions of the DNA replication initiator protein DnaA of Escherichia coli

Under the condition of expression of lambda P protein at lethal level, the oriC DNA-binding activity is significantly affected in wild-type E. coli but not in the rpl mutant. In purified system, the lambda P protein inhibits the binding of both oriC DNA and ATP to the wild-type DnaA protein but not to the rpl DnaA protein. We conclude that the lambda P protein inhibits the binding of oriC DNA and ATP to the wild-type DnaA protein, which causes the inhibition of host DNA synthesis initiation that ultimately leads to bacterial death. A possible beneficial effect of this interaction of lambda P protein with E. coli DNA initiator protein DnaA for phage DNA replication has been proposed.     

Comet assay responses in human lymphocytes are not influenced by the menstrual cycle: a study in healthy Indian females

The single-cell gel electrophoresis or Comet assay measures qualitative and quantitative DNA damage in single cells. Its simplicity and non-invasive nature has made it widely accepted for the monitoring of human genotoxicity, employing peripheral blood lymphocytes. Factors, such as gender, age, and dietary and smoking habits are known to affect the Comet assay responses in lymphocytes. However, there is no information regarding the influence of the menstrual cycle on the results of the assay in lymphocytes of females. A study was therefore undertaken among 18 healthy Indian female volunteers to assess the effect of the menstrual cycle on Comet assay responses. During a complete menstrual cycle, only minor changes were observed in the basal levels of DNA damage in the lymphocytes as evident by Comet parameters, such as tail length (microm), tail DNA (%) and Olive tail moment (arbitrary units). To assess the effect of the estrogen 17beta-estradiol (at physiological concentrations of 0.5, 1.0 and 2.0 nM) on the Comet assay responses, an in vitro study was conducted in the human lymphocyte cell line JM-1 and the breast cancer cell line MCF-7. As was evident from the Comet parameters, a significant (p < 0.01) concentration-dependent increase in the level of DNA damage was observed in the MCF-7 cells while no significant change was found in the JM-1 cells. The results indicate that the menstrual cycle does not influence the Comet assay responses in lymphocytes; hence, these can serve as a model for monitoring genotoxicity in females.