The induction of adipose conversion by bezafibrate in 3T3-L1 cells. Synergism with dibutyryl-cAMP

The induction of adipose conversion in 3T3-L1 cells by bezafibrate (Brandes, R., Hertz, R. Arad R., Naishtat S., Weil, S. and Bar-Tana, J. (1987) Life Sci., 40, 935-941) was enhanced by dibutyryl-cAMP as well as forskolin, theophylline or isobutylmethylxanthine added to the incubation medium together with the bezafibrate inducer. The synergistic effect of bezafibrate and dibutyryl-cAMP resulted in enhancing the expression of late markers of adipose conversion, e.g., lipid accumulation or glycerol-3-phosphate dehydrogenase activity and its mRNA. This enhanced expression of late markers was reflected in shortening the time period required for their first appearance as well as increasing their yield during the course of adipose conversion. By following the accumulation of glutamine synthetase mRNA serving as an early marker for adipose conversion, the synergistic effect of bezafibrate and dibutyryl-cAMP was already evident as early as 5 h following their addition to confluent 3T3-L1 cells. Hence, the induction of adipose conversion by bezafibrate in 3T3-L1 cells appears to involve an early event which is rate-limited by the availability of intracellular cAMP.     

Amphiphilic corroles bind tightly to human serum albumin

Amphiphilic 2,17-bis-sulfonato-5,10,15(trispentafluorophenyl)corrole (2) and its Ga and Mn complexes (2-Ga and 2-Mn) form tightly bound noncovalent conjugates with human serum albumin (HSA). Protein-induced changes in the electronic absorption, emission, and circular dichroism spectra of these corroles, as well as results obtained from HPLC profiles of the conjugates and selective fluorescence quenching of the single HSA tryptophan, are interpreted in terms of multiple corrole:HSA binding sites. High-affinity binding sites, close to the unique tryptophan, are fully occupied at very low concentrations. At biologically relevant HSA concentrations (2-3 orders of magnitude larger than those employed in our studies), all corroles (2, 2-Ga, and 2-Mn) may be considered as fully conjugated.     

Translesion replication by DNA polymerase beta is modulated by sequence context and stimulated by fork-like flap structures in DNA

Mutations in the human genome are clustered in hot-spot regions, suggesting that some sequences are more prone to accumulate mutations than others. These regions are therefore more likely to lead to the development of cancer. Several pathways leading to the creation of mutations may be influenced by the DNA sequence, including sensitivity to DNA damaging agents, and repair mechanisms. We have analyzed sequence context effects on translesion replication, the error-prone repair of single-stranded DNA regions carrying lesions. By using synthetic oligonucleotides containing systematic variations of sequences flanking a synthetic abasic site, we show that translesion replication by the repair polymerase DNA polymerase beta is stimulated to a moderate extent by low stacking levels of the template nucleotides downstream of the lesion, combined with homopolymeric runs flanking the lesion both upstream and downstream. A strong stimulation of translesion replication by DNA polymerase beta was seen when fork-like flap structures were introduced into the DNA substrate downstream of the lesion. Unlike for gapped substrates, this stimulation was independent of the presence of a phosphate group at the 5' terminus of the flap. These results suggest that DNA polymerase beta may participate in cellular DNA transactions involving higher order structures. The significance of these results for in vivo translesion replication is discussed.     

Superantigen antagonist protects against lethal shock and defines a new domain for T-cell activation

Superantigens trigger an excessive cellular immune response, leading to toxic shock. We have designed a peptide antagonist that inhibits superantigen-induced expression of human genes for interleukin-2, gamma interferon and tumor necrosis factor-b, which are cytokines that mediate shock. The peptide shows homology to a b-strand-hinge-a-helix domain that is structurally conserved in superantigens, yet is remote from known binding sites for the major histocompatibility class II molecule and T-cell receptor. Superantigens depend on this domain for T-cell activation. The peptide protected mice against lethal challenge with staphylococcal and streptococcal superantigens. Moreover, it rescued mice undergoing toxic shock. Surviving mice rapidly developed protective antibodies against superantigen that rendered them resistant to further lethal challenges, even with different superantigens. Thus, the lethal effect of superantigens can be blocked with a peptide antagonist that inhibits their action at the beginning of the toxicity cascade, before activation of T cells takes place.     

Dissection of the GTPase mechanism of Ras protein by MD analysis of Ras mutants

Controlling the hydrolysis rate of GTP bound to the p21ras protein is crucial for the delicate timing of many biological processes. A few mechanisms were suggested for the hydrolysis of GTP. To gain more insight into the individual elementary events of GTP hydrolysis, we carried out molecular dynamic analysis of wild-type p21ras and some of its mutants. It was recently shown that Ras-related proteins and mutants generally follow a linear free energy relationship (LFER) relating the rate of reaction to the pK(a) of the gamma-phosphate group of the bound GTP, indicating that proton transfer from the attacking water to the GTP is the first elementary event in the GTPase mechanism. However, some exceptions were observed. Thus, the Gly12 --> Aspartic p21ras (G12D) mutant had a very low GTPase activity although its pK(a) was very close to that of the wild-type ras. Here we compared the molecular dynamics (MD) of wild-type Ras and G12D, showing that in the mutant the catalytic water molecule is displaced to a position where proton transfer to GTP is unfavorable. These results suggest that the mechanism of GTPase is indeed composed of an initial proton abstraction from water by the GTP, followed by a nucleophilic attack of the hydroxide ion on the gamma-phosphorus of GTP.     

Screening of compounds toxicity against human Monocytic cell line-THP-1 by flow cytometry

The worldwide rapid increase in bacterial resistance to numerous antibiotics requires on-going development of new drugs to enter the market. As the development of new antibiotics is lengthy and costly, early monitoring of compound’s toxicity is essential in the development of novel agents. Our interest is in a rapid, simple, high throughput screening method to assess cytotoxicity induced by potential agents. Some intracellular pathogens, such as Mycobacterium tuberculosis primary site of infection is human alveolar macrophages. Thus, evaluation of candidate drugs for macrophage toxicity is crucial. Protocols for high throughput drug toxicity screening of macrophages using flow cytometry are lacking in the literature. For this application we modified a preexisting technique, propidium iodide (PI) exclusion staining and utilized it for rapid toxicity tests. Samples were prepared in 96 well plates and analyzed by flow cytometry, which allowed for rapid, inexpensive and precise assessment of compound’s toxicity associated with cell death. Published: October 1, 2004.     

Characterization of a novel murine retrovirus mixture that facilitates hematopoiesis

A new virus previously arose in BALB/c females mated repeatedly to C57BL/6 (B6) males and then injected with fixed, activated B6 male spleen cells (V. S. Ter-Grigorov, O. Krifuks, E. Liubashevsky, A. Nyska, Z. Trainin, and V. Toder, Nat. Med. 3:37-41, 1997). In the present study, BALB/cJ mice inoculated with virus-containing plasma from affected mice developed splenomegaly, which was caused by increased numbers of Sca-1(+) Lin(-) hematopoietic stem cells (HSC) and their differentiated progeny. Biological and molecular analyses of a new virus revealed a mixture of murine leukemia viruses (MuLVs). These MuLVs comprised ecotropic and mink lung cell focus-forming (MCF) virus classes and are termed Rauscher-like MuLVs because they bear numerous similarities to the ecotropic and MCF viruses of the Rauscher MuLV complex but do not include a spleen focus-forming virus. The ecotropic virus component alone transferred some disease characteristics, while MCF virus alone did not. Thus, we have described a novel virus mixture, termed Rauscher-like MuLV, that causes an increase in hematopoiesis due to activation of pluripotent HSC. Experiments using mice and a protocol that replicated the pregnancy and immunization strategy of the original experiment demonstrated that endogenous BALB/c mouse ecotropic and xenotropic MuLVs are activated by these treatments. Emv1 was expressed in the spleens of multiparous mice but not in those of virgin mice, and Bxv1Emv1-pseudotyped MuLVs were recovered following injection of fixed, activated B6 cells. Thus, multiple pregnancies and allostimuli appear to have provided the signals required for activation of and recombination among endogenous viruses and could have resulted in generation of the Rauscher-like MuLV mixture.     

Regulated phase transitions of bacterial chromatin: a non-enzymatic pathway for generic DNA protection

The enhanced stress resistance exhibited by starved bacteria represents a central facet of virulence, since nutrient depletion is regularly encountered by pathogens in their natural in vivo and ex vivo environments. Here we explore the notion that the regular stress responses, which are mediated by enzymatically catalyzed chemical transactions and promote endurance during the logarithmic growth phase, can no longer be effectively induced during starvation. We show that survival of bacteria in nutrient-depleted habitats is promoted by a novel strategy: finely tuned and fully reversible intracellular phase transitions. These non-enzymatic transactions, detected and studied in bacteria as well as in defined in vitro systems, result in DNA sequestration and generic protection within tightly packed and highly ordered assemblies. Since this physical mode of defense is uniquely independent of enzymatic activity or de novo protein synthesis, and consequently does not require energy consumption, it promotes virulence by enabling long-term bacterial endurance and enhancing antibiotic resistance in adverse habitats.     

Antiviral effect of red microalgal polysaccharides on Herpes simplex and Varicella zoster viruses

The cell-wall sulphated polysaccharide of the red microalga Porphyridium sp. has impressive antiviral activity against Herpessimplex viruses types 1 and 2 (HSV 1, 2) and Varicella zoster virus(VZV). Treatment of cells with 1 g mL^-1 polysaccharideresulted in 50% inhibition of HSV-infection as measured by the plaqueassay. Inhibition of the production of new virus particles was also shownwhen pre-infected cell cultures were treated with the polysaccharide. Inaddition, there was indirect evidence for a strong interaction between thepolysaccharide and HSV and a weak interaction with the cell surface.Depending on the concentration, the polysaccharide completely inhibitedor slowed down the development of the cytopathic effect in HSV or VZVpreinfected cells, but did not show any cytotoxic effects on Vero cells evenwhen a concentration as high as 250 g mL^-1 was used. Itseems therefore that the polysaccharide is able to inhibit viral infection bypreventing adsorption of virus into the host cells and/or by inhibiting theproduction of new viral particles inside the host cells. Thus, this alga seems tobe a good candidate for the development of an antiviral drug.     

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens^1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region^4,6,7. There are also reports of biocidal effect of red and near infra red⁸ as well as green light⁹.In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems¹⁰.The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log''s.The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.