NOPR Collection:

Phospholipase C from two bacterial strains acts differently on pure phospholipids and membrane bound glycosylphosphatidylinositol (GPI) anchors

Fri, 01 Apr 2005 00:00:00 GMT

Title: Phospholipase C from two bacterial strains acts differently on pure phospholipids and membrane bound glycosylphosphatidylinositol (GPI) anchors Authors: Rastogi, Arshi; Hutchinson, Tarun E; Pereira, Ben M J Abstract: Phospholipase C (PLC) was purified to homogeneity from the culture filtrate of Bacillus cereus (65-fold, 540 U/mg protein) and B. thuringiensis (76-fold, 306 U/mg protein) by conventional techniques of enzyme purification. The purified enzymes have the molecular mass of 34 kDa and 38 kDa respectively, as determined by SDS-PAGE. Both the PLCs exhibited identical sensitivity to pH, temperature, cations, anions and inhibitors like glutathione and p-chloromercuribenzoate. PLC-Bc showed a preference for phosphatidylinositol, while PLC-Bt favoured phosphatidylcholine as the substrate. Although both the enzymes were able to hydrolyze pure phosphatidylinositol, distinct differences were observed in their activity on phosphatidylinositol-anchored membrane proteins. PLC-Bc cleaved and released alkaline phosphatase, a GPI-anchored marker enzyme from microsomal membranes to a greater extent, than PLC-Bt. Experiments with sperm membranes, followed by SOS-PAGE revealed that the pattern of proteins released from their GPI-anchors by PLC-Bc and PLC-Bt were dissimilar. Although some proteins were cleaved in common by both PLCs, some others including a prominent 57 kDa protein were resistant to PLC-Bt, but sensitive to cleavage by PLC-Bc. The type of modification in the GPI anchor, special environment on membranes, and relative charge of host plasma membrane to the charge of PLC may be the factors that are responsible for the differential action of two enzymes. Page(s): 92-99

Temporal expression patterns of timeless in vg and cry^b mutants of Drosophila melanogaster

Fri, 01 Apr 2005 00:00:00 GMT

Title: Temporal expression patterns of timeless in vg and cry^b mutants of Drosophila melanogaster Authors: Suthakar, G; Subramanian, P; Manivasagam, T Abstract: The temporal expression patterns of timeless (tim) in Drosophila melanogaster at various time points were studied in intestine and salivary gland of wild type (WT), vestigial (vg) and cryptochrome-absent (cry^b) mutants under 12 hr:12 hr white light:darkness (LD) and 12 hr:12 hr blue light (450 nm):darkness (BD) conditions. At ZT 06 and ZT 10, tim expression was almost nil and at ZT 18 and ZT 22, the expression was most pronounced in WT and mutants, when compared to other time points. As vg flies have greatly reduced wings, their gross locomotor activity was poorer and levels of tim expression were also least than WT flies. The weaker expression of tim in cry^b flies suggested the significant role of blue light photoreceptor cryptochrome for a stronger synchronization of circadian clock. The expression patterns of rim in the salivary gland of larvae further suggested the presence of peripheral oscillators during the developmental stages. Page(s): 87-91

Variant of mitochondrial ribosomal protein s28 (mrps28) gene is differentially expressed in response to radiation in a cervical carcinoma derived cell line

Fri, 01 Apr 2005 00:00:00 GMT

Title: Variant of mitochondrial ribosomal protein s28 (mrps28) gene is differentially expressed in response to radiation in a cervical carcinoma derived cell line Authors: Gopal, G; Rajkumar, T Abstract: Gene expression before and after radiation treatment (10 Gy) in a cervical carcinoma derived cell line SiHa was studied. Differential display performed on non-irradiated and irradiated cells (10 Gy) using a combination of 3 anchor primers and 16 arbitrary primers yielded 20 differentially expressed fragments. Differential expression of one of the fragments differentially expressed In the irradiated sample was also confirmed in Northern blots. Cloning and sequencing of the fragment revealed it to be similar to a splice variant of the MRPS28 gene. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using primers designed to amplify the splice variant and confirmed the expression. In conclusion, the study shows that the splice variant kDec03 of the MRPS28 gene is differentially expressed in response to radiation in SiHa cells. Page(s): 81-86

Effects of metal ions and an inhibitor on the fluorescence and activity of acutolysin A from Agkistrodon acutus venom

Fri, 01 Apr 2005 00:00:00 GMT

Title: Effects of metal ions and an inhibitor on the fluorescence and activity of acutolysin A from Agkistrodon acutus venom Authors: Liu, Xianghu; Xu, Xiaolong; Chen, Jiexia; Liu, Wenqi; Liu, Qingliang Abstract: Acutolysin A, a protein isolated from the venom of Chinese Five-pace snake (Agkistrodon acutus) has shown marked hemorrhagic and proteolytic activities. In the present study, the effects of metal ions and an inhibitor EDTA on the fluorescence and function of autolysin A have been studied, by following fluorescence and activity measurements. Acutolysin A contains a Ca²⁺-binding site, which provides it with important structural stability, and a Zn²⁺-binding site, which is essential for its enzymatic activities. The removal of metal ions in acutolysin A by incubation with EDTA results in irreversible inhibition and complete denaturation, and a marked decrease in its fluorescence intensity. The fluorescence intensity of acutolysin A is also decreased in the presence of Cu²⁺, Co²⁺, Mn²⁺ or Mg²⁺, but does not change in the presence of Ca²⁺, Cd²⁺, or Tb³⁺. Caseinolytic activity of acutolysin A is enhanced by Co²⁺, Ca²⁺ and Mg²⁺, but is partly inhibited by Cu²⁺, Mn²⁺ and Tb³⁺, and completely inhibited by Cd²⁺. Both Zn²⁺ and Co²⁺ recover the loss of activity of the protein caused by Cd²⁺. Page(s): 100-105

NOPR Collection:

Phospholipase C from two bacterial strains acts differently on pure phospholipids and membrane bound glycosylphosphatidylinositol (GPI) anchors

Temporal expression patterns of timeless in vg and cryb mutants of Drosophila melanogaster

Variant of mitochondrial ribosomal protein s28 (mrps28) gene is differentially expressed in response to radiation in a cervical carcinoma derived cell line

Effects of metal ions and an inhibitor on the fluorescence and activity of acutolysin A from Agkistrodon acutus venom

Temporal expression patterns of timeless in vg and cry^b mutants of Drosophila melanogaster