Polymethoxylated flavones induce Ca(2+)-mediated apoptosis in breast cancer cells

Flavonoids, polyphenolic phytochemicals which include flavones and isoflavones, are present in the common human diet. It has been suggested that these compounds may exert anticancer activity; however, the mechanisms involved remain unknown. We have recently shown (Sergeev, 2004, Biochem Biophys Res Commun 321: 462-467) that isoflavones can activate the novel apoptotic pathway mediated by cellular Ca(2+). Here, we report that polymethoxyflavones (PMFs) derived from sweet orange (Citrus sinensis L.) inhibit growth of human breast cancer cells via Ca(2+)-dependent apoptotic mechanism. The treatment of MCF-7 breast cancer cells with 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (3'-OH-TtMF) induced a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) resulting from both depletion of the endoplasmic reticulum Ca(2+) stores and Ca(2+) influx from the extracellular space. This increase in [Ca(2+)](i) was associated with the activation of the Ca(2+)-dependent apoptotic proteases, mu-calpain and caspase-12, as evaluated with the calpain and caspase-12 peptide substrates and antibodies to active (cleaved) forms of the enzymes. Corresponding non-hydroxylated PMFs, 3,5,6,7,8,3',4'-heptamethoxyflavone (HpMF) and 5,6,7,3',4'-pentamethoxyflavone (PtMF), were dramatically less active in inducing Ca(2+)-mediated apoptosis. Our results strongly suggest that the cellular Ca(2+) modulating activity of flavonoids underlies their apoptotic mechanism and that hydroxylation of PMFs is critical for their ability to induce an increase in [Ca(2+)](i) and, thus, activate Ca(2+)-dependent apoptotic proteases.     

Concepts of classic and modern biogeography: Contribution of Russian entomologists

The main stages and ways of development of insect biogeography in Russia are discussed. Three general research programs (studies of ranges, zones, and regions) are described. The role of the regional approach in biogeography is characterized. Some problems related to studies of insect ranges are evaluated, and the need for creating practical range maps is emphasized. The relationships between the insect distribution patterns and the life zones are discussed. The specific features of zonation in insect biogeography are noted. Special attention is paid to establishing the zones with unified population distribution patterns within a species range. The role of geozoological investigations and opportunities of modern information technologies are characterized relative to evaluation of general patterns of insect assemblage distribution. The problems of regionalization in insect biogeography are discussed. Three main approaches (intuitive, a priori, and a posteriori) are determined. Some advantages of using population and assemblage distribution patterns in regionalization are emphasized. The role and possibilities of insect biogeography studies for in the field of reconstruction of past and long-term forecasting are characterized.     

Interpreting the effect of vaccination on steady state infection in animals challenged with Simian immunodeficiency virus

A representative vaccinated macaque challenged with SIVmac251 establishes a persistent infection with a lower virus load, higher CTL frequencies, and much higher helper cell frequencies, than a representative control animal. The reasons for the difference are not fully understood. Here we interpret this effect using a mathematical model we developed recently to explain results of various experiments on virus and CTL dynamics in SIV-infected macaques and HIV-infected humans. The model includes two types of cytotoxic lymphocytes (CTLs) regulated by antigen-activated helper cells and directly by infected cells, respectively, and predicts the existence of two steady states with different viremia, helper cell and CTL levels. Depending on the initial level of CTL memory cells and helper cells, a representative animal ends up in either the high-virus state or the low-virus state, which accounts for the observed differences between the two animal groups. Viremia in the low-virus state is proportional to the antigen sensitivity threshold of helper cells. Estimating the infectivity ratio of activated and resting CD4 T cells at 200-300, the correct range for the critical memory cell percentage and the viremia peak suppression is predicted. However, the model does not explain why viremia in the “low-virus state” is surprisingly high , relative to vaccinated animals infected with SHIV, and broadly distributed among challenged animals. We conclude that the model needs an update explaining extremely low sensitivity of uninfected helper cells to antigen in vaccinated animals.     

Models to understand the population-level impact of mixed strain M. tuberculosis infections

Over the past decade, numerous studies have identified tuberculosis patients in whom more than one distinct strain of Mycobacterium tuberculosis is present. While it has been shown that these mixed strain infections can reduce the probability of treatment success for individuals simultaneously harboring both drug-sensitive and drug-resistant strains, it is not yet known if and how this phenomenon impacts the long-term dynamics for tuberculosis within communities. Strain-specific differences in immunogenicity and associations with drug resistance suggest that a better understanding of how strains compete within hosts will be necessary to project the effects of mixed strain infections on the future burden of drug-sensitive and drug-resistant tuberculosis. In this paper, we develop a modeling framework that allows us to investigate mechanisms of strain competition within hosts and to assess the long-term effects of such competition on the ecology of strains in a population. These models permit us to systematically evaluate the importance of unknown parameters and to suggest priority areas for future experimental research. Despite the current scarcity of data to inform the values of several model parameters, we are able to draw important qualitative conclusions from this work. We find that mixed strain infections may promote the coexistence of drug-sensitive and drug-resistant strains in two ways. First, mixed strain infections allow a strain with a lower basic reproductive number to persist in a population where it would otherwise be outcompeted if has competitive advantages within a co-infected host. Second, some individuals progressing to phenotypically drug-sensitive tuberculosis from a state of mixed drug-sensitive and drug-resistant infection may retain small subpopulations of drug-resistant bacteria that can flourish once the host is treated with antibiotics. We propose that these types of mixed infections, by increasing the ability of low fitness drug-resistant strains to persist, may provide opportunities for compensatory mutations to accumulate and for relatively fit, highly drug-resistant strains of M. tuberculosis to emerge.     

Effect of cucumarioside (a triterpene glycoside from the holothurian Cucumaria japonica) on the development of an immune response in mice to corpuscular pertussis vaccine

The influence of cucumarioside, triterpene glycoside obtained from Cucumaria japonica (Echinodermata, Holoturioidea), or sea cucumbers, on the resistance of mice to Bordetella pertussis infection (with the use experimental pertussis meningoencephalitis as a model) and on the development of immune response to corpuscular pertussis vaccine was studied. The preparation under test was shown to have greatly pronounced immunomodulating properties depending on both the concentration of cucumarioside and the route of its administration, as well as on the dose of pertussis vaccine. When administered orally in a dose of 4 micrograms per mouse and intraperitoneally in doses of 0.04 and 0.0004 micrograms, cucumarioside enhanced the protective effect of corpuscular pertussis vaccine. The use of cucumarioside in a dose of 0.001 micrograms per mouse abolished the suppressive action of large doses of pertussis vaccine in the background rosette-formation test at an early period after immunization and increased number of immune rosettes formed by lymphocytes in the spleen of mice immunized with different doses of the corpuscular vaccine.     

Oculocutaneous albinism type 1: link between mutations,tyrosinase conformational stability,and enzymatic activity

Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intramelanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site‐directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities, tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants showed very low protein expression and protein yield and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactivate tyrosinase and result in OCA1B albinism.     

Functional inhibition of transitory proteins by intrabody-mediated retention in the endoplasmatic reticulum

Intrabodies are recombinantly expressed intracellular antibody fragments that can be used to specifically bind and inhibit the function of cellular proteins of interest. Intrabodies can be targeted to various cell compartments by attaching an appropriate localization peptide sequence to them. An efficient strategy with a high success rate is to anchor intrabodies in the endoplasmatic reticulum where they can inhibit transitory target proteins by binding and preventing them to reach their site of action. Intrabodies can be assembled from antibody gene fragments from various sources into dedicated expression vectors. Conventionally, antibody cDNA sequences are derived from selected hybridoma cell clones that express antibodies with the desired specificity. Alternatively, appropriate clones can be isolated by affinity selection from an antibody in vitro display library. Here an evaluation of endoplasmatic reticulum targeted intrabodies with respect to other knockdown approaches is given and the characteristics of various intrabody expression vectors are discussed. A step by step protocol is provided that was repeatedly used to construct intrabodies derived from diverse antibody isotypes producing hybridoma cell clones. The inactivation of the cell surface receptor neural cell adhesion molecule (NCAM) by a highly efficacious novel endoplasmatic reticulum-anchored intrabody is demonstrated.     

Study of the GABA-benzodiazepine receptor system of the human myometrium

A specific [3H] GABA and [14C] flunitrazepam binding sites have been identified in a membrane fraction of human myometrium. The specific binding of [14C] GABA was displaced by unlabelled GABA and bicuculline. It was shown that the binding of [3H] flunitrazepam to membrane preparations is enhanced in the presence of GABA. A similar reciprocal effect of benzodiazepines to enhance [14C] GABA binding has been demonstrated. The present results indicate that GABAA-BD receptors complexes may have a functional significance in human ovary.