PARl' I SYNTHESIS OF L AMINO ACID OXIDASE BY A SERINE OR GLYCINE R~RING STRAIN OF NEIJROOPORA PARl' II STUDIES CONCERNING MUIJrIPLE ELEX TROPHORErIC FORMS OF TYROOINASE IN NEIJROOPORA Thesis by Joyce[.]
Coexpression of Lifeact-TagRFP and ␤ -tubulin–GFP re- vealed distinct but coordinated recruitment of F-actin and mi- crotubules during different stages of cell polarization and tip extension during colony initiation. Previous studies using mi- crotubule-depolymerizing drugs showed that germ tube emer- gence (but not elongation) can be achieved in N. crassa without microtubules (7). Our data showing that polarization of F-actin always preceded polarization of microtubules further rein- forces the notion that germ tube emergence and elongation is a two-step process (9, 21) that first involves F-actin to establish a polarized bud and maintain tip polarity but subsequently requires microtubules for further extension. In contrast, CATs are thinner than germ tubes, show determinate growth (45), and do not require microtubules to facilitate cell fusion (46). Consistently, we observed the dynamic rearrangement of actin organization during CAT formation and fusion, suggesting a predominant role for actin in these processes. This notion is supported by findings in Ustilago maydis, where it has been demonstrated that cell-cell recognition and cell-cell fusion ex- clusively depend on F-actin during all stages of polar growth whereas microtubules are required only for long-distance growth of hyphae (19). The function of CATs is to connect cells that are less than 10 to 12 m apart, i.e., CATs do not need to extend further than 5 to 6 m. The F-actin cytoskel- eton is apparently sufficient to support this short-distance growth and to facilitate fusion. Our observations suggest that recruitment of both cytoskeletal elements occurs in a distinct but coordinated manner and might influence which protrusion is being formed and maintained at any point in time.
LAAOs form a family of proteins with various enzymatic properties, structure and biological function. Extensive studies indicate that LAAOs have promising biotechno- logical and medical applications. This enzyme is widely distributed in nature including snake venoms, insect drugs, sea hare, fungi, bacteria and algae. Unlike snake venom LAAOs which have been widely and deeply investigated to show broad bioactivities such as apoptosis, cytotoxic- ity, edema, hemolysis, hemorrhage, platelet aggregation, parasite-killing activity and antimicrobial activity, non- snake venom LAAOs need to be further studied and their functional role and application remain to be revealed. Especially, very little is known about LAAOs from ma- rine microorganism. In this study, we successfully iso- lated an LAAO-producing marine bacterium from inter- tidal zone of Dinghai sea area. Based on physiological and biochemical tests together with molecular analysis, it was designated as Pseudoalteromonas sp. R3. To arrive at a better understanding and address the commercializa- tion of LAAO from this isolate, future works are planned to clone the gene coding for this Pseudoalteromonas sp. R3 LAAO and to study its structure, biological and phy- siological roles, relationship between function and struc- ture, and mechanism of transcription in vivo.
lant and anti-bacterial properties, platelet aggregation, apoptosis and edema. These effects are attributed to the release of high amounts of H2O2, a known reactive ox- ygen species (ROS), during the reaction. According to Ande et al. (4), ROS is formed extracellularly and acts directly by altering cell membrane permeability, and is also involved in cell apoptosis. Anticoagulant proteins have a major contribution in the mechanism of blood coagulation. Thrombosis and hemostasis are the major targets of snake venom proteins and can provide po- tential for designing and developing new drugs to pre- vent/treat blood clotting disorder. Few in vitro studies have reported the possible involvement of snake ven- om proteins and their components, in inhibiting blood clot formation (5). Sakurai et al. reported that purified LAAO from A. h. blomhoffii venom possesses antico- agulant activity (6). Several venom proteins including PLA2, LAAO and proteases exhibit their enzymatic anticoagulant properties though different mechanisms. Snake venom proteins with molecular masses between 6 kDa and 35 kDa are said to prolong coagulation and inhibit blood coagulation (7). Cancer is one of the leading causes of death worldwide, and hence there is an urgent need for a better treatment for cancers. The search to cure cancer using natural resources has been in practice for long time as surgery, radiotherapy and chemotherapy do not provide adequate protection against cancer cells. Current cancer treatment methods simultaneously affect normal cells along with cancer- ous cells, causing more serious side effects. Calmette in 1993 first reported with the use of snake venom as a treatment for cancer in laboratory animals (8). Venoms from elapidae, viperidae and crotalide were found to have cytotoxic properties against B16F10 melanoma cell lines. The cytotoxicity was stronger in elapidae venom compared to viperidae and crotalide in causing cell aggregations (9). But this anticancer agent involves various side effects, that are toxic to normal cells and thus decreases its therapeutic indexes (10). This finding led the research for identifying other agents for cancer treatment from naturally available products. The aim of the present study was to further investigate the antico- agulant properties and the mechanism of cytotoxicity of the purified enzyme – L-aminoacidoxidase (Nn- LAAO) from the Indian cobra (Naja naja) venom, be- longing to the western part of the Indian subcontinent, against two human cancer cell lines, including Human colorectal carcinoma (HCT 116) and breast cancer cell lines (MDA MB-231).