Characterization of the interactions between fibronectin and the Borrelia burgdorferi lipoprotein, BBK32

The findings presented in this dissertation detail the complex interaction between BBK32 and fibronectin and describe novel consequences of the interaction. BBK32 is a fibronectin-binding protein on Borrelia burgdorferi, the causative agent of Lyme disease. We found that BBK32 contains multiple fibronectin-binding motifs, recognizing the fibronectin N-terminal domain (NTD) and the gelatin binding domain (GBD) in an anti-parallel order, where corresponding sites in BBK32 and fibronectin are aligned so that there is a one-to-one interaction between the proteins. While characterizing this interaction, we discovered that binding of BBK32 to the GBD inhibits the migration stimulating factor's (MSF) motogenic activity. In the presence of BBK32, endothelial cells do not migrate in response to increasing concentrations of MSF or the GBD. MSF is found under wound healing conditions, and inhibition of its activity may allow the tick-transmitted spirochetes to delay wound healing and to establish an infection. ^ Biophysical structural studies, designed to identify a mechanism of interaction, revealed that BBK32 binding to the NTD leads to the unfolding of plasma fibronectin, which exposes α5β1 integrin recognition motifs. Binding assays demonstrate that the BBK32-NTD interaction enhances the plasma fibronectin-α5β1 integrin interaction, which may allow B. burgdorferi to invade host cells, and thereby evade the host immune system. ^ We also determined that BBK32 binds fibronectin F3 modules, which leads to plasma fibronectin aggregation and induction of superfibronectin. The resulting superfibronectin is conformationally distinct from plasma and cellular fibronectin, and can inhibit endothelial cell proliferation. BBK32's active superfibronectin-forming motif has been located to a region between residues 160 and 175, which contains two sequence motifs that are also found in anastellin, the only other known superfibronectin-inducing protein. ^ A potential consequence of BBK32-induced superfibronectin formation was identified. BBK32-induced superfibronectin formation results in the exposure of α4β1 integrin recognition sequences in fibronectin. The α4β1 integrin is required for leukocyte transendothelial cell migration. BBK32-induced superfibronectin inhibits this activity. The inhibition of leukocyte recruitment to the infection site may slow the activity of the host immune system, and permit the spirochetes to establish an infection. ^

2-methylthio-N$\sp6$-dimethylallyl modification of adenosine 37 of tRNAs in {\it Escherichia coli\/} K-12

The hypermodified, hydrophobic 2-methylthio-N$\sp6$-(dimethylallyl)-adenosine (ms${2{\cdot}6}\atop1$A) residue occurs $3\sp\prime$ to the anticodon in tRNA species that read codons beginning with U. The first step (i$\sp6$A37 formation) of this modification is catalyzed by dimethylallyl diphosphate:tRNA dimethyallyltransferase (EC 2.5.1.8), which is the product of the miaA gene. Subsequent steps were proposed to be catalyzed by MiaB and MiaC enzymes to complete the ms${2{\cdot}6}\atop1$A37 modification. The study of functions of the ms${2{\cdot}6}\atop1$A37 is very important because this modified base is one of the best candidates for a role in global control in response to environmental stress. This dissertation describes the further delineation of functions of the ms${2{\cdot}6}\atop1$A37 modification in E. coli K-12 cells. This work provides significant information on functions of tRNA modifications in E. coli cells to adapt to stressful environmental conditions. Three hypotheses were tested in this work.^ The first hypothesis tested was that non-optimal translation processes cause increased spontaneous mutagenesis by the induction of SOS response in starving cells. To test this hypothesis, I measured spontaneous mutation rates of wild type cells and various mutant strains which are defective in tRNA modification, SOS response, or oxidative damage repair. I found that the miaA mutation acts as a mutator that increased Lac$\sp+$ reversion rates and Trp$\sp+$ reversion frequencies of the wild-type cells in starving conditions. However, the lexA3(Ind)(which abolishes the induction of SOS response) mutation abolished the mutator phenotype of the miaA mutant. The recA430 mutation, not other identified SOS genes, decreased the Lac$\sp+$ reversion to a less extent than that of the lexA3(Ind) mutation. These results suggest that RecA together with another unidentified SOS gene product are responsible for the process.^ The second hypothesis tested was that MiaA protein binds to full-length tRNA$\sp{\rm Phe}$ molecules in form of a protein dimer. To test this hypothesis, three versions of the MiaA protein and seven species of tRNA substrates were purified. Binding studies by gel mobility shift assays, filter binding assays and gel filtration shift assays support the hypothesis that MiaA protein binds to full-length tRNA$\sp{\rm Phe}$ as a protein dimer but as a monomer to the anticodon stem-and-loop. These results were further supported by using steady state enzyme kinetic studies.^ The third hypothesis tested in this work was that the miaB gene in E. coli exists and is clonable. The miaB::Tn10dCm insertion mutation of Salmonella typhimurium was transduced to E. coli K-12 cells by using P$\sb1$ and P$\sb{22}$ bacteriophages. The insertion was confirmed by HPLC analyses of nucleotide profiles of miaB mutants of E. coli. The insertion mutation was cloned and DNA sequences adjacent to the transposon were sequenced. These DNA sequences were 86% identical to the f474 gene at 14.97 min chromosome of E. coli. The f474 gene was then cloned by PCR from the wild-type chromosome of E. coli. The recombinant plasmid complemented the mutant phenotype of the miaB mutant of E. coli. These results support the hypothesis that the miaB gene of E. coli exists and is clonable. In summary, functions of the ms${2{\cdot}6}\atop1$A37 modification in E. coli cells are further delineated in this work in perspectives of adaptation to stressful environmental conditions and protein:tRNA interaction. (Abstract shortened by UMI.) ^

Analysis of VirB4, a membrane-associated ATPase subunit essential for T -complex transport in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a plant pathogen with the unique ability to export oncogenic DNA-protein complexes (T-complexes) to susceptible plant cells and cause crown gall tumors. Delivery of the T-complexes across the bacterial membranes requires eleven VirB proteins and VirD4, which are postulated to form a transmembrane transporter. This thesis examines the subcellular localization and oligomeric structure of the 87-kDa VirB4 protein, which is one of three essential ATPases proposed to energize T-complex transport and/or assembly. Results of subcellular localization studies showed that VirB4 is tightly associated with the cytoplasmic membrane, suggesting that it is a membrane-spanning protein. The membrane topology of VirB4 was determined by using a nested deletion strategy to generate random fusions between virB4 and the periplasmically-active alkaline phosphatase, $\sp\prime phoA$. Analysis of PhoA and complementary $\beta$-galactosidase reporter fusions identified two putative periplasmically-exposed regions in VirB4. A periplasmic exposure of one of these regions was further confirmed by protease susceptibility assays using A. tumefaciens spheroplasts. To gain insight into the structure of the transporter, the topological configurations of other VirB proteins were also examined. Results from hydropathy analyses, subcellular localization, protease susceptibility, and PhoA reporter fusion studies support a model that all of the VirB proteins localize at one or both of the bacterial membranes. Immunoprecipitation and Co$\sp{2+}$ affinity chromatography studies demonstrated that native VirB4 (87-kDa) and a functional N-terminally tagged HIS-VirB4 derivative (89-kDa) interact and that the interaction is independent of other VirB proteins. A $\lambda$ cI repressor fusion assay supplied further evidence for VirB4 dimer formation. A VirB4 dimerization domain was localized to the N-terminal third of the protein, as judged by: (i) transdominance of an allele that codes for this region of VirB4; (ii) co-retention of a His-tagged N-terminal truncation derivative and native VirB4 on Co$\sp{2+}$ affinity columns; and (iii) dimer formation of the N-terminal third of VirB4 fused to the cI repressor protein. Taken together, these findings are consistent with a model that VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains and that VirB4 assembles as homodimers via an N-terminal dimerization domain. Dimer formation is postulated to be essential for stabilization of VirB4 monomers during T-complex transporter assembly. ^

A molecular basis for polyketide biosynthesis in marine dinoflagellates with emphasis on Karenia brevis

Polyketides derived from dinoflagellates are among the most complex and unique structures identified to date. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). No studies of the biosynthesis of dinoflagellate derived polyketides at the genomic level have been reported to date. Nine strains representing seven different species of dinoflagellates were screened for the presence of type I and type II polyketide synthases (PKS) by PCR and RT-PCR. Seven of the nine strains yielded products that were homologous with known and putative type I polyketide synthases. In each case, the presence of a PKS gene was correlated with the presence of bacteria in the cultures as identified by amplification of the bacterial 16S rRNA gene. However, residual phylogenetic signals, resistance to methylation sensitive restriction enzymes and the lack of hybridization to bacterial isolates support a dinoflagellate origin for most of these genes. ^ A more detailed analysis of Karenia brevis, a toxic marine dinoflagellate endemic to the Gulf of Mexico, also supports the hypothesis that dinoflagellates have polyketide synthase genes. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. PKS encoding genes amplified from K. brevis culture were found to be similar to PKS genes from the closely related protist, Cryptosporidium parvum. This suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. This dissertation reports the localization of these PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate. ^

The inducing NO-vasodilation by chemical reduction of coordinated nitrite ion in cis-[Ru(NO(2))L(bpy)(2)](+) complex

The synthesis of [Ru(NO(2)) L(bpy)(2)](+) (bpy = 2,2'-bipyridine and L = pyridine (py) and pyrazine (pz)) can be accomplished by addition of [Ru(NO) L(bpy) 2](PF(6))(3) to aqueous solutions of physiological pH. The electrochemical processes of [Ru(NO2) L(bpy) 2]+ in aqueous solution were studied by cyclic voltammetry and differential pulse voltammetry. The anodic scan shows a peak around 1.00 V vs. Ag/AgCl attributed to the oxidation process centered on the metal ion. However, in the cathodic scan a second peak around-0.60 V vs. Ag/AgCl was observed and attributed to the reduction process centered on the nitrite ligand. The controlled reduction potential electrolysis at-0.80 V vs. Ag/AgCl shows NO release characteristics as judged by NO measurement with a NO-sensor. This assumption was confirmed by ESI/MS(+) and spectroelectrochemical experiment where cis-[Ru(bpy)(2)L(H(2)O)](2+) was obtained as a product of the reduction of cis-[Ru(II)(NO(2)) L(bpy)(2)](+). The vasorelaxation observed in denuded aortic rings pre-contracted with 0.1 mu mol L(-1) phenylephrine responded with relaxation in the presence of cis-[RuII(NO2) L(bpy) 2]+. The potential of rat aorta cells to metabolize cis-[RuII(NO(2)) L(bpy)(2)](+) was also followed by confocal analysis. The obtained results suggest that NO release happens by reduction of cis-[RuII(NO(2)) L(bpy)(2)](+) inside the cell. The maximum vasorelaxation was achieved with 1 x 10(-5) mol L(-1) of cis-[RuII(NO(2)) L(bpy)(2)](+) complex.

Studies on the Biology of the Clam Marcia opima Gmelin from kayamkulam Lake

Division of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology

STUDIES ON THE BIOLOGY OF THE WEDGE CLAM DONAX INCARNATUS (GMELIN) FROM THE MALIPPURAM BEACH OF KERALA

The present investigations have considerably enhanced the existing knowledge on the biology and distribution/availability pattern of D.incarnatus in the Malippuram region. The species occurs in good concentration during October - March/April, and disappears from the area during late premonsoon and monsoon months. Recolonising the area in September, it grows fast in the subsequent months. The life span of the species is estimated to be about an year. Studies on the reproductive biology of the species have revealed that there are two spawning peaks, the major peak in February - March and minor peak, in December. The salinity regime of the area influences the reproductive activity. These observations form the original contribution in the thesis. The information on variation in water content, protein,glycogen and lipid levels in relation to reproductive cycle has helped to a better understanding of the gametogenic activity and spawning of the species. Similarly, the findings on salinity tolerance and filtration rate have shown that small sized clams exhibit greater tolerance range than larger clams, and grow at a faster rate with active metabolism. It is hoped that these information would considerably add to the present knowledge of the basic facts which are relevant to the improvement and management of the clam fishery of this region.

Biology of Flat Toadfish,Colletteichthys dussumieri(Valenciennes,1837) of Cochin Estuary

Dept.of Marine Biology,Microbiology and Biochemistry,Cochin University of Science and Technology

Biology, population characteristics and fishery of the speckled shrimp Metapenaeus monoceros (Fabricius, 1798) along the Kerala coast

Crustaceans comprising numerous edible species of prawns, lobsters and crabs inhabiting different ecosystem form significant portion of the aquatic food resources of the world. Among the crustaceans, prawns are the most commercially exploited group and hold premier rank by virtue of their importance as an esteemed food of gourmet and on account of their high export value. Met-ape-naeus manoceras (Fabricius, 1798) which is known IS,Speckled shrimp’ (FAD name) and ‘Brown shrimp’ ( common nameused in the industry) is one of the commercially important marine penaeid prawns of India. During 1995, M. monaceros catch constituted 7.5 Z of the all India marine penaeid prawn landings. M. monoceros attains a maximum length of about 200 mm and has high export potential.Thus realising the growing importance of M. monoceros in the capture fisheries, it was felt, that it would be ideal to carry out detailed study on this species for rational exploitation and management of its fishery. Hence, the present work entitled, “Biology, population characteristics and fishery of the speckled shrimp Hetapenaeus monoceros (Fabricius, 1798) along Kerala coast“ was undertaken by the author. The thesis is laid out in seven chapters comprising TAXONOMY, FOOD AND FEEDING HABITS, AGE AND GROWTH, REPRODUCTION,LENGTH-WEIGHT RELATIONSHIP, FISHERY and POPULATION DYNAMICS

Studies on The Eco Biology of The Indian Pearl Oyster, Pinctada Fucata (Gould)

The present work aims to study induced maturation of the pearl oyster for induced spawning experiments. The work on larval development was done with a view to developing techniques for the artificial rearing of commercially important pearl oyster P fucata, and also to elucidate the principles and problems of tropical bivalve larvae in general for detailed investigations in the future. The present study is designed to probe into the details of the basic aspects of the biology related to the hatchery technology of Pinctada fucata and the understanding of the factors which influence induction of maturation, spawning, larval rearing and spat settlement. This would go a long way in the upgradation of hatchery technology of the Indian Pearl oyster Pinctada fucata fora commercial level seed production..

An evaluation of a Lake Houston tributary, Cypress Creek, for contamination and water quality

Introduction. Lake Houston serves as a reservoir for both recreational and drinking water for residents of Houston, Texas, and the metropolitan area. The Texas Commission on Environmental Quality (TCEQ) expressed concerns about the water quality and increasing amounts of pathogenic bacteria in Lake Houston (3). The objective of this investigation is to evaluate water quality for the presence of bacteria, nitrates, nitrites, carbon, phosphorus, dissolved oxygen, pH, turbidity, suspended solids, dissolved solids, and chlorine in Cypress Creek. The aims of this project are to analyze samples of water from Cypress Creek and to render a quantitative and graphical representation of the results. The collected information will allow for a better understanding of the aqueous environment in Cypress Creek.^ Methods. Water samples were collected in August 2009 and analyzed in the field and at UTSPH laboratory by spectrophotometry and other methods. Mapping software was utilized to develop novel maps of the sample sites using coordinates attained with the Global Positioning System (GPS). Sample sites and concentrations were mapped using Geographic Information System (GIS) software and correlated with permitted outfalls and other land use characteristic.^ Results. All areas sampled were positive for the presence of total coliform and Escherichia coli (E. coli). The presences of other water contaminants varied at each location in Cypress Creek but were under the maximum allowable limits designated by the Texas Commission on Environmental Quality. However, dissolved oxygen concentrations were elevated above the TCEQ limit of 5.0 mg/L at majority of the sites. One site had near-limit concentration of nitrates at 9.8 mg/L. Land use above this site included farm land, agricultural land, golf course, parks, residential neighborhoods, and nine permitted TCEQ effluent discharge sites within 0.5 miles upstream.^ Significance. Lake Houston and its tributary, Cypress Creek, are used as recreational waters where individuals may become exposed to microbial contamination. Lake Houston also is the source of drinking water for much of Houston/Harris and Galveston Counties. This research identified the presence of microbial contaminates in Cypress Creek above TCEQ regulatory requirements. Other water quality variables measured were in line with TCEQ regulations except for near-limit for nitrate at sample site #10, at Jarvis and Timberlake in Cypress Texas.^

Fermentation of Prawn Shell Waste and Its Application of its Product as Dietary Ingredient for Penaeus Indicus (H. Milne Edwards)

This work envisages the fermentation of prawn shell waste into a more nutritious product with simpler components for application as a feed ingredient in aquaculture. This product would be a rich source of protein along with chitin, minerals, vitamins and N-acetyl glucosamine. A brief description of the various processing (chemical and bioprocess) methods employed for chitin, chitosan and single sell protein preparations from shell waste. It deals with the isolation of micro flora associated with prawn shell degradation. It describes the methods adopted for fermentation of prawn shell degradation and fermentation of prawn shell waste with the selected highly chitinoclastic strains. The comparison of SSF and SmF for each selected strain in terms of enrichment of protein, lipid and carbohydrate in the fermented product was done. Detailed analysis of product quality is discussed. The feed for mulation and feeding experiment explained in detail. Statistical analysis of various biogrowth parameters was done with Duncan’s multiple range test. Very briefly explains 28 days of feeding experiment. A method for the complete utilization of shell waste explains with the help of experiments.

INTRACELLULAR OSMOREGULATION IN THE ESTUARINE MOLLUSC VILLORITA CYPRINOIDES VAR. COCHINENSIS (Mollusca: Bivalvia) Hanley

The present investigation is dedicated to understanding various mechanisms of salinity tolerance in the estuarine clam V. cyprinoides var. cochinensis. Even though V. cyprinoids var. cochinensis and V. cyprinoides are found to coexist in the same area, V. cyprinoids is reported to tolerate higher salinities than variety cochinenesis. Variations in the salinity of sea water may affect the aquatic organisms through specific gravity control and variations in osmotic pressure. The specific gravity of most soft tissues is close to that of normal seawater. Many bottom living forms, both attached and motile, have very high specific gravities eg.villorita cyprinoids. Villorita spp. Occurs abundantly in the reaches of the estuary and backwaters of Kerala. In both marine and estuarine forms, it is observed that mantle employs a lesser quantity of amino acids compared to adductor and foot. The regulation of cell volume is not carried out equally in all types of tissues. The capability of salinity tolerance is an aggregate of both the capabilities of extra cellular anisosmotic and intracellular isosmotic regulations in osmoconforming animals. The ultimate aim of water regulation is to regulate the cell volume.T here are slight changes occur in cell volume even in osmoregulators. These studies can also help in revealing the changes brought about in the cellular organelles like lysosomes, which were found to have a role in the osmoregulatory process. The osmoregulatory machinery of estuarine animals is more streamlined for a successful life in the estuarine regime.

Ecological and Biochemical Studies On Cyanobacteria of Cochin Estuary and Their Application as Source of Antioxidants

The main objectives of the present investigation were to evaluate the qualitative and quantitative distribution of natural cyanobacterial population and their ecobiological properties along the Cochin estuary and their application in aquaculture systems as a nutritional supplement due to their nutrient-rich biochemical composition and antioxidant potential. This thesis presents a detailed account of the distribution of cyanobacteria in Cochin estuary, an assessment of physico-chemical parameters and the nutrients of the study site, an evaluation of the effect of physico-chemical parameters on cyanobacterial distribution and abundance, isolation, identification and culturing of cyanobacteria, the biochemical composition an productivity of cyanobacteria, and an evaluation of the potential of the selected cyanobacteria as antioxidants against ethanol induced lipid peroxidation. The pH, salinity and nutritional requirements were optimized for low-cost production of the selected cyanobacterial strains. The present study provides an insight into the distribution, abundance, diversity and ecology of cyanobacteria of Cochin estuary. From the results, it is evident that the ecological conditions of Cochin estuary support a rich cyanobacterial growth.

Biochemical Investigations on the Stability of Biological Membranes

In this project, an attempt has been made to study the stability of erythrocyte and lysosomal membranes biochemically. Erythrocytes were chosen for the study because of their ready availability and relative simplicity. Biological membranes forming closed boundaries between compartments of varying composition consist mainly of proteins and lipids. They are asymmetric, fluid structures that are thermodynamically stable and metabolically active. Normal cellular function begins with normal membrane structure and any variation in it may upset the normal functions. The degree of fluidity of a membrane depends on the chain length of its lipids and degree of unsaturation of constituent fatty acids. In response to environmental changes, many cells can regulate composition of their membranes to maintain the overall semi fluid environment necessary for many membrane associated functions. The assembly and Maintenance of membrane structures in cells is a dynamic process. The components are not only synthesized and inserted into a growing membrane but are also continuously degraded at a slower rate. This turnover process varies with each individual molecule.Lysosomes are important in the catabolic processes occurring in the cell. Lysosomes contain hydrolytic enzymes and are stable under normal conditions. In certain pathological conditions, the lysosomal membrane may rupture, releasing the hydrolytic enzymes into the cell and digestion of cell takes place as a whole. This is very dangerous. In normal life processes of multi cellular organisms, lysosomes rupture following the death of a cell and it may have some value as a built in mechanism for selfremoval of dead cells.An attempt has also been made in this project towards developing lysosome membrane stability as an index of fish spoilage during storage. Different membranes within the cell and between cells have different compositions as reflected in the ratio of protein to lipid. The difference is not surprising given the very different functions of membranes