RNA Virus Population Diversity,an Optimum for Maximal Fitness and Virulence

The ability of an RNA virus to exist as a population of genetically distinct variants permits the virus to overcome events during infections that would otherwise limit virus multiplication or drive the population to extinction. Viral genetic diversity is created by the ribonucleotide misincorporation frequency of the viral RNA-dependent RNA polymerase (RdRp). We have identified a poliovirus (PV) RdRp derivative (H273R) possessing a mutator phenotype. GMP misincorporation efficiency for H273R RdRp in vitro was increased by 2–3-fold that manifested in a 2–3-fold increase in the diversity of the H273R PV population in cells. Circular sequencing analysis indicated that some mutations were RdRp-independent. Consistent with the population genetics theory, H273R PV was driven to extinction more easily than WT in cell culture. Furthermore, we observed a substantial reduction in H273R PV virulence, measured as the ability to cause paralysis in the cPVR mouse model. Reduced virulence correlated with the inability of H273R PV to sustain replication in tissues/organs in which WT persists. Despite the attenuated phenotype, H273R PV was capable of replicating in mice to levels sufficient to induce a protective immune response, even when the infecting dose used was insufficient to elicit any visual signs of infection. We conclude that optimal RdRp fidelity is a virulence determinant that can be targeted for viral attenuation or antiviral therapies, and we suggest that the RdRp may not be the only source of mutations in a RNA virus genome.     

The role of chromatic and achromatic signals for fruit detection by birds

Fruit color changes during ripening are typically viewed asan adaptation to increase signal efficacy to seed dispersers.Plants can increase signal efficacy by enhancing chromatic (wavelengthrelated) and/or achromatic (intensity related) contrasts betweenfruit and background. To assess how these contrasts determinethe detectability of fruit signals, we conducted 2 experimentswith free-flying crows (Corvus ossifragus) under seminaturalconditions in a 2025 m² aviary. Crows searched first for artificialred and black fruits and detected red fruits from a larger distance.Because artificial red fruits had higher chromatic and lowerachromatic contrasts against foliage than artificial black fruits,crows apparently prioritized chromatic contrasts. Thus, thecommon change in fruit color from red to black during ripeningdoes not increase signal efficacy to crows. In a second trial,crows searched for UV-reflecting and black blueberries (Vaccinummyrtillus) against backgrounds of foliage and sand. Againstfoliage, UV-reflecting berries had higher chromatic and achromaticcontrasts than black berries, and crows detected them from alarger distance. Against sand, UV-reflecting berries had lowachromatic contrasts and black berries low chromatic contrasts.Crows detected both fruit types equally, suggesting that theyused chromatic contrasts to detect UV-reflecting berries andachromatic contrasts to detect black berries. Birds prioritizedchromatic contrasts when searching for artificial red fruitsin foliage but not when searching for blueberries on sand. Wesuggest that the relative importance of chromatic and achromaticcontrasts is contingent on the chromatic and achromatic varianceof the background. Models of signal perception can be improvedby incorporating background-specific effects.     

CHROMOSOME PULVERIZATION IN HUMAN BINUCLEATE CELLS FOLLOWING COLCEMID TREATMENT

Under the influence of Colcemid, a substantial number of binucleate human cells from a line infected with herpes-like virus was found to possess pulverized chromosomes. Although this abnormality was also detected in untreated binucleate cells, the increase in the number of pulverized cells after the addition of Colcemid was too striking to be explained by accumulation of spontaneously occurring cells in response to the mitotic inhibition by Colcemid. Furthermore, the induction of pulverization may be dependent upon Colcemid concentration. These findings imply an involvement of Colcemid in the mechanism of pulverization induction in the system studied. When tritiated thymidine was added to the culture medium simultaneously with Colcemid, the majority of binucleate cells with an intact and a pulverized chromosome set incorporated this isotope into the pulverized set only. This obviously suggests that the nuclei in the binucleate cell are asynchronous in DNA synthesis, and that this asynchrony is intimately related to the induction of the pulverization phenomenon. It seems very probable that the late S phase in the late synthesizing nuclei represents a critical stage at which damage to the chromosomes most readily occurs.     

Differences between ATP-mediated cytotoxicity and cell-mediated cytotoxicity.

There has been considerable recent interest in the possible role of extracellular ATP in cell-mediated cytotoxicity. To investigate the similarities and differences between ATP-mediated lysis and CTL-mediated lysis, we studied in detail the ATP-mediated lysis of EL-4 cells, which are highly susceptible to lysis by extracellular ATP. ATP-mediated lysis was time and concentration dependent; most lysis occurred within 4 to 6 h of incubation. The kinetics of ATP- and cell-mediated lysis were similar. However, in contrast to CTL-mediated lysis, ATP-mediated lysis of EL-4 cells did not appear to be accompanied by characteristic chromosomal degradation (apoptosis). In order to compare these cytotoxic processes in greater detail, ATP-resistant clones were derived by growing EL-4 cells continuously in the presence of high concentrations of ATP. These cloned EL-4 lines showed marked resistance to ATP-mediated cytotoxicity across a wide range of concentrations but were as susceptible as the parent EL-4 cells to cell-mediated cytotoxicity by an alloreactive effector population from a MLC. Thus, there appear to be a number of differences between ATP-mediated and cell-mediated cytotoxicity in this system; most notable is the identification of cell lines that are resistant to ATP but susceptible to CTL-mediated lysis.     

Physicochemical Characterization of Berberine Chloride: A Perspective in the Development of a Solution Dosage Form for Oral Delivery

The objective of the present research was to evaluate the physicochemical characteristics of berberine chloride and to assess the complexation of drug with 2-hydroxypropyl-β-cyclodextrin (HPβCD), a first step towards solution dosage form development. The parameters such as log P value were determined experimentally and compared with predicted values. The pH-dependent aqueous solubility and stability were investigated following standard protocols at 25°C and 37°C. Drug solubility enhancement was attempted utilizing both surfactants and cyclodextrins (CDs), and the drug/CD complexation was studied employing various techniques such as differential scanning calorimetry, Fourier transform infrared, nuclear magnetic resonance, and scanning electron microscopy. The experimental log P value suggested that the compound is fairly hydrophilic. Berberine chloride was found to be very stable up to 6 months at all pH and temperature conditions tested. Aqueous solubility of the drug was temperature dependent and exhibited highest solubility of 4.05 ± 0.09 mM in phosphate buffer (pH 7.0) at 25°C, demonstrating the effect of buffer salts on drug solubility. Decreased drug solubility was observed with increasing concentrations of ionic surfactants such as sodium lauryl sulfate and cetyl trimethyl ammonium bromide. Phase solubility studies demonstrated the formation of berberine chloride–HPβCD inclusion complex with 1:1 stoichiometry, and the aqueous solubility of the drug improved almost 4.5-fold in the presence of 20% HPβCD. The complexation efficiency values indicated that the drug has at least threefold greater affinity for hydroxypropyl-β-CD compared to randomly methylated-β-CD. The characterization techniques confirmed inclusion complex formation between berberine chloride and HPβCD and demonstrated the feasibility of developing an oral solution dosage form of the drug.KEY WORDS: berberine chloride, complexation, cyclodextrin, solubility, surfactants     

Modeling Susceptibility versus Resistance in Allergic Airway Disease Reveals Regulation by Tec Kinase Itk

Murine models of allergic asthma have been used to understand the mechanisms of development and pathology in this disease. In addition, knockout mice have contributed significantly to our understanding of the roles of specific molecules and cytokines in these models. However, results can vary significantly depending on the mouse strain used in the model, and in particularly in understanding the effect of specific knockouts. For example, it can be equivocal as to whether specific gene knockouts affect the susceptibility of the mice to developing the disease, or lead to resistance. Here we used a house dust mite model of allergic airway inflammation to examine the response of two strains of mice (C57BL/6 and BALB/c) which differ in their responses in allergic airway inflammation. We demonstrate an algorithm that can facilitate the understanding of the behavior of these models with regards to susceptibility (to allergic airway inflammation) (S_aai) or resistance (R_aai) in this model. We verify that both C57BL/6 and BALB/c develop disease, but BALB/c mice have higher S_aai for development. We then use this approach to show that the absence of the Tec family kinase Itk, which regulates the production of Th2 cytokines, leads to R_aai in the C57BL/6 background, but decreases S_aai on the BALB/c background. We suggest that the use of such approaches could clarify the behavior of various knockout mice in modeling allergic asthma.     

Identification of a new cross-link and unique histidine adduct from bovine serum albumin incubated with malondialdehyde

Malondialdehyde, acetaldehyde, acrolein, and 4-hydroxynonenal are all products of fatty acid oxidation found in the fatty streaks of atherosclerotic arteries due to a lack of antioxidants and an increase in glycation products. Previously identified cross-links derived from these molecules have nearly always required more than one molecule of each type, although this is physiologically less likely than a reaction involving a single molecule. Here we provide indirect but strong evidence for a malondialdehyde-derived cross-link requiring just one malondialdehyde molecule to link arginine and lysine, giving 2-ornithinyl-4-methyl(1epsilon-lysyl)1,3-imidazole following a 4-day incubation of albumin with 8 mm malondialdehyde. This cross-link was identified as its partial degradation product Nepsilon-(2-carboxyl,2-aminoethane)-Nepsilon-methanoyl-lysine by NMR and mass spectrometry. Analysis of plasma from treated diabetic patients revealed that one patient levels had as high as 0.46%, 0.67% of their lysine/arginine residues modified by this cross-link, although others had lower levels. Alkaline hydrolysis of serum albumin also revealed two acid-labile malondialdehyde adducts of histidine in significant quantities, the isomers 4- and 2-ethylidene-histidine. These constituted up to 0.93% of the histidines in treated diabetic patients. Although collagen is readily cross-linked by malondialdehyde, none of these particular products could be found in incubations of collagen with malondialdehyde.