Two Modes of Interaction of the Single-stranded DNA-binding Protein of Bacteriophage T7 with the DNA Polymerase-Thioredoxin Complex

The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD. gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions.     

Long-term survival of Legionella pneumophila serogroup 1 under low-nutrient conditions and associated morphological changes

Abstract Extended survival of Legionella pneumophila , using both a clinical and an environmental isolate, was studied in drinking water, creek water, and estuarine water microcosms. Legionella populations were monitored by acridine orange direct counts (AODC) and viable count on buffered charcoal yeast extract agar amended with alpha-ketoglutarate (BCYEα). Initial colony counts of the clinical isolate in drinking and creek water microcosms were 2 × 10⁸ cfu/ml and, after incubation for 1.5 years, the plate counts decreased to 3 × 10⁶ cfu/ml. The AODC counts, however, did not change significantly. The clinical isolate in estuarine water decreased in plate counts to 10² (cfu/ml) over the same period. After incubation for 1.5 years at 15°C in the microcosms, Legionella plate counts of creek and drinking water decreased by two logs. Direct microscopic examination of aliquots removed from all microcosms revealed the presence of small bacilli, large bacilli and rare filamentous cells. The environmental isolate demonstrated only one colony morphology upon culture on BCYEα. Interestingly, after four months incubation in the microcosm, upon plating the clinical isolate on BCYEα, two distinct colony types were evident. Examination by immunofluorescent staining employing a monoclonal antibody against L. pneumophila revealed both bacillus and filamentous forms. The total cellular proteins of both morphotypes were examined by sodium dodecyl sulfate polyacrylyamide gel electrophoresis (SDS-PAGE), demonstrating identical protein patterns. Those Legionella cells remaining culturable during 1.5 years of incubation grew rapidly when transferred to BCYEα. Incubation was continued and it was found that some strains of L. pneumophila serogroup 1 can remain viable for longer than 2.4 years under low-nutrient conditions.     

Novel semisynthetic derivatives of betulin and betulinic acid with cytotoxic activity

A series of new imidazole carboxylic esters (carbamates) and N-acylimidazole derivatives of betulin and betulinic acid (14–29) have been synthesized. The new compounds were screened for in vitro cytotoxicity activity against human cancer cell lines HepG2, Jurkat and HeLa. A number of compounds have shown IC₅₀ values lower than 2 μM against the cancer cell lines tested and the vast majority has shown a better cytotoxicity profile than betulinic acid, including the betulin derivatives. N-Acylimidazole derivatives 26 and 27 (IC₅₀ 0.8 and 1.7 μM in HepG2 cells) and the C-3 carbamate derivative 16 (IC₅₀ 2.0 μM in HepG2 cells) were the most promising compounds. Based on the observed cytotoxicity, structure–activity relationships have been established.     

Insects Represent a Link between Food Animal Farms and the Urban Environment for Antibiotic Resistance Traits

Antibiotic-resistant bacterial infections result in higher patient mortality rates, prolonged hospitalizations, and increased health care costs. Extensive use of antibiotics as growth promoters in the animal industry represents great pressure for evolution and selection of antibiotic-resistant bacteria on farms. Despite growing evidence showing that antibiotic use and bacterial resistance in food animals correlate with resistance in human pathogens, the proof for direct transmission of antibiotic resistance is difficult to provide. In this review, we make a case that insects commonly associated with food animals likely represent a direct and important link between animal farms and urban communities for antibiotic resistance traits. Houseflies and cockroaches have been shown to carry multidrug-resistant clonal lineages of bacteria identical to those found in animal manure. Furthermore, several studies have demonstrated proliferation of bacteria and horizontal transfer of resistance genes in the insect digestive tract as well as transmission of resistant bacteria by insects to new substrates. We propose that insect management should be an integral part of pre- and postharvest food safety strategies to minimize spread of zoonotic pathogens and antibiotic resistance traits from animal farms. Furthermore, the insect link between the agricultural and urban environment presents an additional argument for adopting prudent use of antibiotics in the food animal industry.     

Antioxidant metabolite levels in ozone-sensitive and tolerant genotypes of snap bean

Ozone-sensitive and tolerant genotypes of snap bean ( Phaseolus vulgaris L.) were compared for differences in leaf ascorbic acid (vitamin C), glutathione and α -tocopherol (vitamin E) content to determine whether antioxidant levels were related to ozone tolerance. Seven genotypes were grown in pots under field conditions during the months of June and July. Open top chambers were used to establish either a charcoal filtered (CF) air control (36 nmol mol⁻¹ ozone) or a treatment where CF air was supplemented with ozone from 8:00 to 20:00 h with a daily 12 h mean of 77 nmol mol⁻¹. Fully expanded leaves were analyzed for ascorbic acid, chlorophyll, glutathione, guaiacol peroxidase (EC 1.11.1.7) and α -tocopherol. Leaf ascorbic acid was the only variable identified as a potential factor in ozone tolerance. Tolerant genotypes contained more ascorbic acid than sensitive lines, but the differences were not always statistically significant. Genetic differences in glutathione and α -tocopherol were also observed, but no relationship with ozone tolerance was found. Guaiacol peroxidase activity and leaf α -tocopherol content increased in all genotypes following a one week ozone exposure, indicative of a general ozone stress response. Ozone had little effect on the other variables tested. Overall, ozone sensitive and tolerant plants were not clearly distinguished by differences in leaf antioxidant content. The evidence suggests that screening for ozone tolerance based on antioxidant content is not a reliable approach.     

Evaluation of Antifungal Activity of Medicinal Plant Extracts Against Oral <Emphasis Type="Italic">Candida albicans</Emphasis> and Proteinases

Proteinases produced by Candida albicans are one kind of virulence factor expressed that contribute to adherence and invasion of host tissue. Proteinase inhibitor of human immunodeficiency virus in experimental candidiasis suggested reduction in fungal infection, and medicinal plants could be a source of alternative agent to prevent diseases. In this study, we investigated the production of proteinases by C. albicans from clinical isolates and the action of plant extracts against strains of C. albicans and its synthesized proteinases, comparing with antifungal fluconazole and amphotericin B and proteinase inhibitors pepstatin A, amprenavir, and ritonavir. The results reported here showed that these extracts have a certain kind of action and that the search for new antifungal agents could be found at the plants.     

Prevalence of cardiovascular disease risk factors by habitat: a study on adult Asian Indians in West Bengal, India

The present community based cross-sectional study was aimed to investigate whether or not increasing prevalence of cardiovascular disease (CVD) risk factors in adult Asian Indian population are associated with increasing urbanization. The 'urban group' was comprised of 224 individuals including 122 males and 102 females being inhabitants of Kolkata (erstwhile Calcutta) under the Kolkata Metropolitan Development Authority (KMDA) area. The 'rural group' comprised 224 individuals including 135 males and 89 females and was living in a village council located about 80 kilometers from Kolkata. Therefore, a total of 448 adult (> or = 30 years) individuals (257 males and 191 females) participated in the study. Anthropometric measures, lipids profiles, fasting blood glucose and blood pressure measures were taken from participants. Obesity and body composition measures were subsequently calculated from the anthropometric measures. Accepted cut-offs were used to define metabolic syndrome (MS), lipids abnormalities, increased adiposity and high blood pressure in the study. It was found that 58.7% participants were engaged in sedentary work which includes 60.7% males and 56% females. It was further observed that the prevalence of high blood pressure was as high as 70.6% in urban females compared to 55.1% in rural females. However, the prevalence of low HDLc was remarkably high in females ofboth rural and urban areas. The prevalence ofMS was significantly higher in urban females (57.8%) than in their rural counterparts (34.8%). It seems reasonable to argue that people with changing lifestyles due to growing urbanization are associated with adverse CVD risk factors irrespective of their habitat (rural vs. urban). This in turn warranted a comprehensive risk stratification protocol at the national level for the effective management of CVD risk factors in this part of the world.     

Microtubule rearrangements during mitosis in multinucleate cells

The peroxidase-antiperoxidase (PAP) method for the detection of polymerized tubulin has been used to study the microtubule rearrangements during mitosis in PtK1 and HeLa multinucleate cells obtained by polyethyleneglycol (PEG)-mediated fusion. We demonstrate here that the transition of the microtubular cytoskeleton from interphase to mitosis is an inducible event and independent of the factor(s) responsible for chromatin condensation and nuclear envelope breakdown. However, for the induction of the microtubule rearrangements nuclear envelope breakdown is required. At midprophase, cytoskeletal microtubule rearrangements start for multinucleate PtK1 cells, whereas in HeLa cells such changes are delayed, and a more abrupt transition is observed here. After complete nuclear envelope breakdown (prometaphase) mitotic asters and spindles but no cytoplasmic (interphase) microtubuli can be observed in both systems. Metaphase is characterized by an interaction between the different mitotic poles which show the form of bipolar spindles, but individual separated mitotic poles far removed from the chromatin can also be seen.     

Abnormally spliced messenger RNA in erythroid cells from patients with β+ thalassemia and monkey cells expressing a cloned β+-thalassemic gene

The reduced β-globin synthesis characterizing the β⁺ thalassemia phenotype has been shown to be caused by anomalous processing within the small Intervening sequence (IVS1) of the β-globin mRNA precursor. The β-globin gene from such patients contains a single base substitution within IVS1, located 22 bp from the 3′ junction between IVS1 and exon 2, creating an alternative splice site within IVS1 and resulting in retention of the 3′-terminal 19 bases of IVS1. We have identified this abnormally spliced mRNA in the reticulocyte RNA of two patients with β⁺ thalassemia, by S1 nuclease mapping and primer-extension analysis. Moreover, a cloned β⁺-thalassemic gene preferentially generated the anomalously spliced RNA when expressed In monkey kidney cells. The anomalously spliced RNA constituted approximately 80%–90%, and normal β RNA approximately 10%–20%, of the total β mRNA. In contrast, the small amount of β mRNA present in reticulocytes from such patients consisted predominantly of normal β mRNA. These results suggest that the reduced amount of normally functioning β mRNA present in such patients results from preferential processing at the alternative splice site, with subsequent Instability, reduced nuclear processing and/or inadequate cytoplasmic transport of the abnormal RNA species.