Dibutyryl c-AMP as an inducer of sporidia formation: Biochemical and antigenic changes during morphological differentiation of Karnal bunt (<Emphasis Type="Italic">Tilletia indica</Emphasis>) pathogen in axenic culture

Effect of dibutyryl adenosine 3′,5′-cyclic monophosphate (dbc-AMP), an analogue of c-AMP, was investigated on growth and morphological differentiation ofTilletia indica. Exponential growth was observed up to 21 days in both presence and absence of dbc-AMP; however, increasing concentration of dbc-AMP was deleterious to mycelial growth in liquid culture. A slow increase of mycelial biomass up to 21 days and decline at 30 days in the presence of 2.5 mM dbc-AMP was observed, therefore, this concentration was chosen in subsequent investigations. The inhibitory influence of dbc-AMP was further substantiated by decrease in soluble protein. The fungus on exposure to dbc-AMP experienced morphological differentiation from vegetative mycelial phase to sporogenous mycelial phase, and was induced to produce filiform sporidia. Use of quantitative ELISA further suggested that sporidia formation took more than 21 days in the presence of dbc-AMP. Variations of proteins during different stages ofT. indica grown in the presence and absence of dbc-AMP suggested the expression of stage-specific proteins or differential expression of proteins induced by dbc-AMP. The changes in expression of cell surface antigens as evidenced from decrease and increase binding of anti-mycelial and anti-sporidial antibodies in dbc-AMP treated culture by ELISA was further interpreted on the basis of morphological differentiation from mycelial to sporidial phase     

A soluble VE-cadherin fragment forms 2D arrays of dimers upon binding to a lipid monolayer

A high concentration of cadherin molecules at cell-cell adhesion sites is believed to be essential for generating strong intercellular junctions. In order to determine the interactions of cadherin domains involved in the early stages of lateral cluster formation on the cell surface, a recombinant fragment encompassing the first four domains of human VE-cadherin with a His-tag at the C terminus (VE-EC1-4-His) was produced. Two-dimensional crystals of VE-EC1-4-His were formed at the air-water interface using conventional lipids modified to contain a Ni(2+)-chelating group, which provides a specific site for interaction with the polyhistidine tag. The VE-EC1-4-His was monomeric at the concentration employed for crystal formation; however, the crystals exhibited a p2 symmetry and the presence of cis-dimer interactions between symmetry-related molecules. The VE-EC1-4-His molecules in the crystalline array have a remarkably compact conformation in contrast to the elongated "string of pearls" conformation seen in the hexameric assembly of VE-EC1-4-His in solution, and as seen in the crystal structure of C-cadherin. These results indicate that VE-cadherin can exist in at least two oligomeric states with different interactions between domains and can adopt highly different conformational states. We suggest that the compact cis-dimeric state may occur on isolated cells and that the compact form may serve to protect the molecule from degradation. As previously proposed we suppose that the trans-hexameric form is involved in intercellular adhesion.     

A cancer chemopreventive agent silibinin, targets mitogenic and survival signaling in prostate cancer

There are many epigenetic variables that affect the biological responses of autocrine, paracrine and endocrine regulatory molecules, which determine the growth and development of different cancers including prostate cancer (PCA). One of the focuses of the current cancer chemoprevention studies is the search for non-toxic chemopreventive agents that inhibit mitogenic and cell survival signaling in cancer cells. In general, advanced stage cancer cells harbor many constitutively active mitogenic signaling and anti-apoptotic mechanisms, which make them less dependent on external growth factors as well as resistant to chemotherapeutic agents. In this regard, silibinin (a naturally occurring flavanone) has shown the pleiotropic anticancer effects in different cancer cells. Our extensive studies with PCA have shown that inhibition of mitogenic and cell survival signaling, such as epidermal growth factor receptor, insulin-like growth factor receptor type I and nuclear factor kappa B signaling are the most likely molecular targets of silibinin's efficacy in PCA. We have observed that silibinin inhibits prostate tumor growth in animal models without any apparent signs of toxicity. At the same time, silibinin is also physiologically available in different organs of the body including plasma and prostate, which is generally required for the pharmacological dosing and translational mechanistic studies of the compound. There are substantial amount of data to support the inhibitory effect of silibinin on mitogenic and cell survival signaling in PCA, which are reviewed in the present communication.     

Silibinin modulates UVB-induced apoptosis via mitochondrial proteins, caspases activation, and mitogen-activated protein kinase signaling in human epidermoid carcinoma A431 cells

Several recent studies by us have shown the strong chemopreventive efficacy of silibinin against both ultraviolet B (UVB) radiation and chemical carcinogen-induced tumorigenesis in mouse skin models. The molecular mechanisms underlying silibinin protective efficacy, however, are not completely known. Here, we examined the effect of silibinin on UVB-caused apoptosis in human epidermoid carcinoma A431 cells. Irradiation of cells with different doses of UVB (5-100 mJ/cm2) and different time periods (0.5-24h) resulted in a dose- and time-dependent increase in apoptosis (P < 0.05-0.001). Silibinin (100-200 microM) pre-treatment, however, resulted in an increase in UVB-induced apoptosis (P < 0.05-0.001); interestingly, its post-treatment caused a decrease in UVB-induced apoptosis (P < 0.05-0.001). A similar pattern in the activation of caspases-9, -3, and -7 was observed with these silibinin treatments. Further, silibinin treatment prior to or immediately after UVB exposure altered Bcl-2, Bax, Bak, and cytochrome c levels in mitochondria and cytosol in favor of or against apoptosis, respectively. Silibinin treatment prior to UVB also increased the activation of mitogen/stress activated protein kinases Erk1/2, JNK, and p38 kinase as compared to its post-treatment. Together, for the first time, our results demonstrate the role of mitochondrial apoptotic machinery and MAPK signaling cascade in silibinin-caused increase as well as protection in UVB-induced apoptosis in A431 cells, and suggest that similar mechanisms might be involved in preventive efficacy of silibinin against UVB-induced skin tumorigenesis.     

Synaptobrevin transmembrane domain dimerization-revisited

Synaptobrevin is a membrane-spanning soluble N-ethyl maleimid-sensitive factor (NSF) attachment protein receptor (SNARE) protein of synaptic vesicles that is essential for neurotransmitter release. Various lines of evidence indicate that it exists alternatively as a monomer, as a homodimer, as a heterodimer with synaptophysin, or as a ternary complex with other SNAREs at the various stages of the synaptic vesicle cycle. Homodimerization of synaptobrevin was previously shown by different authors to depend on its single transmembrane segment, and the crucial residues forming the helix-helix interface have been mapped. Since another recent study challenged these results, we reinvestigated this issue. Here, we show that native synaptobrevin can be cross-linked in synaptic vesicle membranes to a homodimer by disulfide bond formation between cysteine residues of the transmembrane segment. Further, we demonstrate that determination of synaptobrevin transmembrane segment interactions in membranes or in detergent solution requires careful control of experimental conditions. Thus, our present results corroborate that homodimerization of synaptobrevin is mediated by its transmembrane segment.     

Determination of colchicine in Colchicum steveni and C. hierosolymitanum (Colchicaceae): comparison between two analytical methods

The amounts of colchicine present in two Jordanian species of Colchicum, namely, C. steveni Kunth and C. hierosolymitanum Feibrun (Colchicaceae), have been determined. An HPLC-UV (photodiode array) method employing gradient elution was developed and the results compared with those obtained using a simple TLC-spectrophotometric method. The levels of colchicine as measured by these methods were not significantly different (p < 0.05) indicating that the spectrophotometric method is an acceptable alternative to HPLC. With respect to C. steveni, the leaves contained the largest amount of colchicine (0.204/100 g), whilst in C. hierosolymitanum corms showed the highest colchicine content (0.126/100 g). As a source of colchicine, the two investigated species showed levels comparable with those found in C. autumnale, the traditional source of colchicine.     

Vascular endothelial growth factor stimulates preantral follicle growth in the rat ovary

The regulation of preantral follicle growth in mammals is poorly understood. The availability of an adequate vascular supply to provide endocrine and paracrine signals may be important during the early states of follicle growth as well as the later states of follicle selection and dominance. The objective of the present study was to investigate whether vascular endothelial growth factor (VEGF) plays a role in preantral follicular development in the rat ovary. Immature (age, 21 days) Sprague-Dawley rats were injected with 500 ng of VEGF in saline or 50 microg of diethylstilbestrol (DES) in oil under the bursa of one ovary. The contralateral ovary was injected with a corresponding volume of vehicle. Rats were killed 48 h later, and the ovaries were removed and analyzed histologically. Intrabursal administration of VEGF significantly increased the number of primary and small secondary, but not of large secondary, preantral follicles in the ovary, similar to the effect of DES (P < 0.05). The VEGF stimulated preantral follicle growth in a time- and dose-dependent manner. Subcutaneous DES administration increased the number of primary and secondary follicles, and both s.c. and intrabursal estrogen administration stimulated VEGF protein expression in the rat ovary. These data indicate that VEGF stimulates preantral follicular development in the rat ovary, is regulated by estrogen, and may be one of the factors that participate in the regulation of early follicle growth in the rat.     

Antagonistic effect of fluorescent pseudomonads against Macrophomina phaseolina that causes charcoal rot of groundnut

Maximum colony growth inhibition was observed due to Pseudomonas PS2 (74%) as compared to PS1 (71%) on trypticase soy agar (TSM) plates after 5 days of incubation. Light and scanning electron microscopic examination showed hyphal coiling, vacuolation, coagulation and granulation of cytoplasm resulting in lysis of hyphae of M. phaseolina by pseudomonads. Cell free culture filtrates of strains PS1 and PS2 restricted the growth of mycelium of M. phaseolina. PS1 and PS2 caused maximum colony growth inhibition by 57 and 61% respectively at 20% concentration of culture filtrate after 4 days of incubation. Volatile substances produced by PS1 and PS2 also inhibited the colony growth of M. phaseolina by 25 and 32%, respectively. Inhibitory effect of volatile substances, however, decreased with advancing in incubation period. Colony growth of M. phaseolina was significantly decreased by PS1 and PS2 as compared to control both in iron- sufficient and iron-deficient conditions. PS2 showed higher antagonistic activity than PS1, as evidenced by pronounced colony growth inhibition.     

Internet--implications for the future of phytopharmacological research

Modern information technologies and world wide communications through the Internet play a significant role in medicinal plant research across the globe. The phenomenal growth in Internet usage is largely due to the success of World Wide Web. Various useful websites and databases on phytopharmacology are already in the "Net" and many more are being added constantly. The future of phytopharmacological research is handling the existing information in proper way. In this review of the Internet, compilation of important websites is expected to stimulate, instruct and update academicians and researchers involved in phytopharmacological research.