Mutant Thermotoga neapolitana DNA polymerase I: altered catalytic properties for non-templated nucleotide addition and incorporation of correct nucleotides

Thermotoga neapolitana (Tne) DNA polymerase belongs to the DNA polymerase I (Pol I) family. The O-helix region of these polymerases is involved in dNTP binding and also plays a role in binding primer–template during DNA synthesis. Here we report that mutations in the O-helix region of Tne DNA polymerase (Arg722 to His, Tyr or Lys) almost completely abolished the enzyme’s ability to catalyze the template-independent addition of a single base at the 3′-end of newly synthesized DNA in vitro. The mutations did not significantly affect the DNA polymerase catalytic activity and reduced base misinsertions 5- to 50-fold. The same Arg722 mutations dramatically increased the ability of the enzyme’s 3′→5′ exonuclease to remove mispaired 3′ bases in a primer extension assay. These mutant DNA polymerases can be used to accurately amplify target DNA in vitro for gene cloning and genotyping analysis.     

Processing of nucleopeptides mimicking the topoisomerase I-DNA covalent complex by tyrosyl-DNA phosphodiesterase

Tyrosyl-DNA phosphodiesterase-1 (Tdp1) is the only known enzyme to remove tyrosine from complexes in which the amino acid is linked to the 3′-end of DNA fragments. Such complexes can be produced following DNA processing by topoisomerase I, and recent studies in yeast have demonstrated the importance of TDP1 for cell survival following topoisomerase I-mediated DNA damage. In the present study, we used synthetic oligodeoxynucleotide–peptide conjugates (nucleopeptides) and recombinant yeast Tdp1 to investigate the molecular determinants for Tdp1 activity. We find that Tdp1 can process nucleopeptides with up to 13 amino acid residues but is poorly active with a 70 kDa fragment of topoisomerase I covalently linked to a suicide DNA substrate. Furthermore, Tdp1 was more effective with nucleopeptides with one to four amino acids than 15 amino acids. Tdp1 was also more effective with nucleopeptides containing 15 nt than with homolog nucleopeptides containing 4 nt. These results suggest that DNA binding contributes to the activity of Tdp1 and that Tdp1 would be most effective after topoisomerase I has been proteolyzed in vivo.     

DNA sequence dependent and independent conformational changes in multipartite operator recognition by lambda-repressor

Binding of regulatory proteins to multipartite DNA binding sites often occurs with protein-protein interaction, resulting in cooperative binding. The operators of bacteriophage lambda have several pairs of repressor binding sites (O(R)1-O(R)2, O(R)2-O(R)3, O(L)1-O(L)2, and O(L)2-O(L)3) separated by a variable number of base pairs, and thus, bacteriophage lambda is a model system for studying multipartite operator recognition by DNA-binding proteins. Near-UV circular dichroism spectra show that the DNA is distorted in O(R)1-O(R)2 and O(L)2-O(L)3 but much less so in O(R)2-O(R)3. Upon titration of lambda-repressor with single-operator sites O(R)1, O(R)2, and O(R)3, it was observed that the tryptophan fluorescence quenches to different degrees, suggesting different conformations of the protein in the three DNA-protein complexes. Acrylamide quenching of tryptophan fluorescence of lambda-repressor bound to these single operators also shows different Stern-Volmer constants, supporting the above conclusions. Titration of lambda-repressor with oligonucleotides containing pairs of operator sites also causes different degrees of fluorescence quenching. In particular, fluorescence quenching induced by O(R)1-O(R)2 binding is less than the quenching induced by either of the single operators alone, suggesting additional conformational changes upon establishment of protein-protein contact. Stern-Volmer constants obtained from acrylamide quenching of tryptophan fluorescence of lambda-repressor bound cooperatively to pairs of operator sites are different from those of the single-operator-site-bound repressors. For example, O(R)2-O(R)3-bound repressor has significantly higher acrylamide quenchable components than either of the O(R)2- or O(R)3-bound proteins, again suggesting additional conformational changes upon establishment of protein-protein contact. We conclude that the strategy of recognition of multipartite operator by lambda-repressor is complex and varied, involving conformational changes in both DNA and protein that are determined by the separation of the binding sites as well as the nucleic acid sequence.     

Evaluation of Cervical Cancer Screening Programs in C?te d’Ivoire,Guyana, and Tanzania: Effect of HIV Status

Background

HIV infection increases a woman’s risk for cervical cancer, and cervical cancer incidence and mortality rates are higher in countries with high HIV prevalence and limited resources for screening. Visual inspection with acetic acid (VIA) allows screening and treatment of cervical lesions in a single-visit approach (SVA), but data on its performance in HIV-infected women are limited. This study’s objective was to examine cervical cancer screening using VIA/SVA in programs serving HIV-infected women.

Methods

A VIA/SVA program with cryotherapy for VIA-positive lesions was implemented in Côte d’Ivoire, Guyana, and Tanzania from 2009 to 2012. The effect of HIV status on VIA positivity and on presence of cryotherapy-eligible lesions was examined using a cross-sectional study design, with Chi-square tests for comparisons and constructed multivariate logistic regression models. A P-value of < 0.05 was significant.

Findings

VIA was performed on 34,921 women, 10% (3,580) were VIA positive; 2,508 (85%) eligible women received cryotherapy during the same visit; only 234 (52%) of those who postponed returned for treatment; 622 (17%) VIA-positive women had lesions too large to be treated with cryotherapy and were referred for excisional treatment. In multivariate analysis—controlling for HIV status, location of the screening clinic, facility location, facility type, and country—compared to HIV-uninfected/unknown women, HIV-infected women had higher odds of being VIA positive (OR 1.95, 95% CI 1.76, 2.16, P<0.0001) and of having large lesions requiring referral (OR 1.93, 95% CI 1.49, 2.51, P< 0.0001). Minor treatment complications occurred in 19 of 3,032 (0.63%) women; none required further intervention.

Conclusions

This study found that compared to HIV-uninfected/unknown women, HIV-infected women had nearly twice the odds of being VIA-positive and to require referral for large lesions. SVA was safe and resulted in significant reductions in loss to follow-up. There is increased need for excisional treatment in countries with high HIV prevalence.     

Genome Re-duplication and Irregular Segregation Occur During the Cell Cycle of Entamoeba histolytica

Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica. Cells with multiple nuclei and nuclei with heterogenous genome contents suggest that regulatory mechanisms that ensure alternation of DNA synthesis and mitosis are absent in this organism. Therefore, several endo-reduplicative cycles may occur without mitosis. The data also shows that unlike other endo-reduplicating organisms, E.histolytica does not undergo a precise number of endo-reduplicative cycles. We propose that irregular endo-reduplication and genome partitioning lead to heterogeneity in the genome content of E.histolytica trophozoites in their proliferative phase. The goal of future studies should be aimed at understanding the mechanisms that are involved in (a) accumulation of multiple genome contents in a single nucleus; (b) genome segregation in nuclei that contain multiple genome contents and (c) maintenance of genome fidelity in E. histolytica.     

Intestinal Parasitosis in Relation to Anti-Retroviral Therapy,CD4+ T-cell Count and Diarrhea in HIV Patients

Intestinal parasitic infections are one of the major causes of diarrhea in human immunodeficiency virus (HIV) seropositive individuals. Antiretroviral therapy has markedly reduced the incidence of many opportunistic infections, but parasite-related diarrhea still remains frequent and often underestimated especially in developing countries. The present hospital-based study was conducted to determine the spectrum of intestinal parasitosis in adult HIV/AIDS (acquired immunodeficiency syndrome) patients with or without diarrhea with the levels of CD4⁺ T-cell counts. A total of 400 individuals were enrolled and were screened for intestinal parasitosis. Of these study population, 200 were HIV seropositives, and the remaining 200 were HIV uninfected individuals with or without diarrhea. Intestinal parasites were identified by using microscopy as well as PCR assay. A total of 130 (32.5%) out of 400 patients were positive for any kinds of intestinal parasites. The cumulative number of parasite positive patients was 152 due to multiple infections. A significant association of Cryptosporidium (P<0.001) was detected among individuals with CD4⁺ T-cell counts less than 200 cells/μl.     

Surficial gains and subsoil losses of soil carbon and nitrogen during secondary forest development

Reforestation of formerly cultivated land is widely understood to accumulate above‐ and belowground detrital organic matter pools, including soil organic matter. However, during 40 years of study of reforestation in the subtropical southeastern USA, repeated observations of above‐ and belowground carbon documented that significant gains in soil organic matter (SOM) in surface soils (0–7.5 cm) were offset by significant SOM losses in subsoils (35–60 cm). Here, we extended the observation period in this long‐term experiment by an additional decade, and used soil fractionation and stable isotopes and radioisotopes to explore changes in soil organic carbon and soil nitrogen that accompanied nearly 50 years of loblolly pine secondary forest development. We observed that accumulations of mineral soil C and N from 0 to 7.5 cm were almost entirely due to accumulations of light‐fraction SOM. Meanwhile, losses of soil C and N from mineral soils at 35 to 60 cm were from SOM associated with silt and clay‐sized particles. Isotopic signatures showed relatively large accumulations of forest‐derived carbon in surface soils, and little to no accumulation of forest‐derived carbon in subsoils. We argue that the land use change from old field to secondary forest drove biogeochemical and hydrological changes throughout the soil profile that enhanced microbial activity and SOM decomposition in subsoils. However, when the pine stands aged and began to transition to mixed pines and hardwoods, demands on soil organic matter for nutrients to support aboveground growth eased due to pine mortality, and subsoil organic matter levels stabilized. This study emphasizes the importance of long‐term experiments and deep measurements when characterizing soil C and N responses to land use change and the remarkable paucity of such long‐term soil data deeper than 30 cm.     

Fluorescent cyclin-dependent kinase inhibitors block the proliferation of human breast cancer cells

Inhibitors of cyclin-dependent kinases (CDKs) are an emerging class of drugs for the treatment of cancers. CDK inhibitors are currently under evaluation in clinical trials as single agents and as sensitizers in combination with radiation therapy and chemotherapies. Drugs that target CDKs could have important inhibitory effects on cancer cell cycle progression, an extremely important mechanism in the control of cancer cell growth. Using rational drug design, we designed and synthesized fluorescent CDK inhibitors (VMY-1-101 and VMY-1-103) based on a purvalanol B scaffold. The new agents demonstrated more potent CDK inhibitory activity, enhanced induction of G2/M arrest and modest apoptosis as compared to purvalanol B. Intracellular imaging of the CDK inhibitor distribution was performed to reveal drug retention in the cytoplasm of treated breast cancer cells. In human breast cancer tissue, the compounds demonstrated increased binding as compared to the fluorophore. The new fluorescent CDK inhibitors showed undiminished activity in multidrug resistance (MDR) positive breast cancer cells, indicating that they are not a substrate for p-glycoprotein. Fluorescent CDK inhibitors offer potential as novel theranostic agents, combining therapeutic and diagnostic properties in the same molecule.     

Regeneration of plantlets from in vitro raised leaf explants of Cleisostoma racimeferum Lindl

Protocorm like bodies (PLBs), callus and shoot buds developed in culture from in vitro raised foliar explants of Cleisostoma racimeferum. Among the different basal media, better result was obtained on MS medium containing sucrose (3%) and BA (2 microM) with approximately 80% frequency after 40 days of culture. Young leaves (15 week old) produced better PLBs. Whole leaf placed vertically upside-up orientation can regenerate PLBs and shoot buds (80%). PLBs and shoot buds formed on entire surface of the leaves. Cultures on BA and NAA (2 and 2 microM respectively in combination) stimulated callus mediated regeneration (68%). The rooted plantlets regenerated within 8-10 week from PLBs and shoot buds on MS medium containing IAA and kinetin (2 microM each in combination). BA containing medium triggered multiple shoot bud formation, while NAA alone or in combination with other growth regulators was inhibitory. Incorporation of activated charcoal (0.01%) in the medium stimulated formation of repetitive PLBs and multiple shoot buds. Rooted plants were ready for harvest after 20-22 week of initiation of culture. About 65% of the potted plants survived after 3 months in the poly house.