Screening for microbial inhibitors against signal transduction in Eukaryotes and Mycobacterium

Puah , Seok Hwa (2006) Screening for microbial inhibitors against signal transduction in Eukaryotes and Mycobacterium. Masters thesis, Universiti Malaysia Sabah.

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Abstract

This study involves the search for microbial inhibitors that disrupt Ras/Raf-1 interaction in the yeast two-hybrid system by the expression of HIS3 and lacZ reporter genes. It is a continuation of the previous study where a Streptomyces, H7372 was identified as a possible inhibitor of Ras/Raf-1 protein-protein interaction. H7372 caused larger inhibition zone on histidine minus plates and reduce l3-galactosidase activity. Radicicol was also positive in this screening. Radicicol and geldanamycin are two inhibitors of Ras/Raf protein-protein interaction by acting on HSP90 causing the degradation of Raf. H7372 also inhibited the growth of Mycobacterium smegmatis. Raf-1 is a serine-threonine kinase. Mycobacterium also possessed eukaryotic-like serine-threonine kinases. This creates the opportunity to investigate the possibility that the inhibitor of Raf-1 also iflhibit the serine-threonine kinases of Mycobacterium. The first part of this study concerned isolating more actinomycetes for screening and the second part was to study H7372 in detail. Eighty actinomycete isolates were obtained by two selective isolation methods using soil samples from three forests in Sabah. No Ras/Raf-1 protein-protein interaction inhibitor was found. One false positive H 11337 was identified. The extract was positive in the HIS3 reporter gene but negative in the lacZ reporter gene. Crude freeze-dried extract of H7372 reduced p-ERK1/2 level in MCF-7 cells under insulin stimulation at 250µg/ml and 500µg/mi. A purified fraction, H7372PRE was isolated by cold precipitation method and produced one single peak at 26.03 min retention time by reversed-phase HPLC. H7372PRE inhibited both HIS3 and lacZ reporter expression. H7372PRE reduced the level of p-ERK1/2 at 75µg/ml and 100µg/ml and increased the level of p-MEK1/2 at 75Iµg/ml and 100µg/ml. HPLC fraction of H7372PRE reduced the level of p-ERK1/2 at 50µg/ml. Crude freeze-dried extract of H7372 decreased JNKlSAPK1 c, MAPKAP1 a and PKCa kinase activity to 54-63% of the original activity. The binding to HSP90 was not detected by surface plasmon resonance. The crude extract and fraction of H7372 consistently reduced p-ERK level, indicating its intervention to the MAPK pathway, possibly through disruption of Ras/Raf-1 interaction through HSP90. H7372PRE did not inhibit the growth of M. smegmatis. H9318 (Penilicium sp.) did not inhibit M. smegmatis but it's semi-purified fraction, H9318_S1 that inhibited PP2A and PP1 in vitro inhibited M. smegmatis, indicating that the inhibition was possibly through the eukaryotic-like phosphatase of Mycobacterium. This study demonstrated that the actinomycetes from forests in Sabah possess interesting compounds that affected the signal transduction pathways in eukaryotes and Mycobacterium.

Item Type:Thesis (Masters)
Uncontrolled Keywords:microbial inhibitor, hybrid system, interaction, actinomycetes, Myccobacterium, Eukaryotes
Subjects:Q Science > QR Microbiology
Divisions:SCHOOL > School of Science and Technology
ID Code:6500
Deposited By:IR Admin
Deposited On:09 Jul 2013 11:24
Last Modified:09 Jul 2013 11:24

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