The host range and biology of Cometaster pyrula; a biocontrol agent for Acacia nilotica subsp. indica in Australia

Prickly acacia, Acacia nilotica subsp. indica (Benth.) Brenan, a major weed of the Mitchell Grass Downs of northern Queensland, Australia, has been the target of biological control projects since the 1980s. The leaf-feeding caterpillar Cometaster pyrula (Hopffer) was collected from Acacia nilotica subsp. kraussiana (Benth.) Brenan during surveys in South Africa to find suitable biological control agents, recognised as a potential agent, and shipped into a quarantine facility in Australia. Cometaster pyrula has a life cycle of approximately 2 months during which time the larvae feed voraciously and reach 6 cm in length. Female moths oviposit a mean of 339 eggs. When presented with cut foliage of 77 plant species, unfed neonates survived for 7 days on only Acacia nilotica subsp. indica and Acacia nilotica subsp. kraussiana. When unfed neonates were placed on potted plants of 14 plant species, all larvae except those on Acacia nilotica subsp. indica and Acacia nilotica subsp. kraussiana died within 10 days of placement. Cometaster pyrula was considered to be highly host specific and safe to release in Australia. Permission to release C. pyrula in Australia was obtained and the insect was first released in north Queensland in October 2004. The ecoclimatic model CLIMEX indicated that coastal Queensland was climatically suitable for this insect but that inland areas were only marginally suitable.

Molecular profiling of malignant pleural mesothelioma and pulmonary fibrosis

Malignant mesothelioma (MM) is a rare, usually incurable, disease mainly caused by former exposure to asbestos. Even though MM has a strong etiological link, genetic factors may play a role, since not all cases can be linked to former asbestos exposure. This thesis focuses on lung diseases, mainly malignant mesothelioma (MM), and idiopathic pulmonary fibrosis (IPF), which resembles asbestosis. The specific asbestos-related pathways associated with malignant as well as non-malignant lung diseases, still need to be clarified. Since most patients diagnosed with MM or asbestosis/fibrosis have a dismal prognosis and few therapeutic options are available, early diagnosis and better understanding of the disease pathogenesis are of the utmost importance. The first objective of this thesis was to identify asbestos specific differentially expressed genes. This was approached by using high-resolution gene expression arrays, and three different human lung cell lines, as well as with three different bioinformatics approaches. Since the first study aimed to elucidate potential early changes, the second study was used to screen DNA copy number changes in MM tumour samples. This was performed using genome wide microarrays for identification of DNA copy number changes characterstic for MM. Study III focused on the role of gremlin in the regulation of bone morphogenetic protein (BMPs) in IPF. Further studies were conducted in asbestos-exposed cell cultures as well as in an asbestos-induced mouse model. Furthermore, GATA-6 was studied in MM and metastatic pleural adenocarcinoma. The GATA transcription factors are important during embryonic development, but their role in cancer is still unclear. GATA-6 is a co-factor/target of thyroid transcription factor 1 (TTF-1), which is used in differential diagnostics of pleural MM and adenocarcinoma. Bioinformatics probed the genes and biological processes ordered in terms of significance, clusters, and highly enriched chromosomal regions. The study revealed several already identified targets, produced new ideas about genes which are central for asbestos exposure, as well as provided supplementary data for researchers to check their own novel findings or ideas. The analysis revealed DNA copy number changes characteristic for MM tumors. The most common regions of loss were detected in 1p, 3p, 6q, 9p, 13, 14, and 22, and gains at 17q. The histological features in asbestosis and IPF are very similar, wherefore IPF can be studied in asbestos models. The BMP antagonist gremlin was up-regulated by asbestos exposure in human epithelial cell lines, which was also observed in Study I. The transforming growth factor (TGF) -β and BMP expression and signaling activities were measured from murine and human fibrotic lungs. BMP-7 signaling was down-regulated in response to up-regulation of gremlin, and restoration of BMP-7 signaling prevented progression of fibrosis in mice. Therefore, the study suggests that the restoration of BMP-7 signaling in fibrotic lung could potentially aid in the treatment of IPF patients. Study IV revealed that GATA-6 was strongly expressed in the majority of the MM cases, and correlated statistically significant with longer survival in subgroups of MM.

Intracellular effects of atmospheric-pressure plasmas on melanoma cancer cells

Gas discharge plasmas formed at atmospheric pressure and near room temperature have recently been shown as a promising tool for cancer treatment. The mechanism of the plasma action is attributed to generation of reactive oxygen and nitrogen species, electric fields, charges, and photons. The relative importance of different modes of action of atmospheric-pressure plasmas depends on the process parameters and specific treatment objects. Hence, an in-depth understanding of biological mechanisms that underpin plasma-induced death in cancer cells is required to optimise plasma processing conditions. Here, the intracellular factors involved in the observed anti-cancer activity in melanoma Mel007 cells are studied, focusing on the effect of the plasma treatment dose on the expression of tumour suppressor protein TP73. Over-expression of TP73 causes cell growth arrest and/or apoptosis, and hence can potentially be targeted to enhance killing efficacy and selectivity of the plasma treatment. It is shown that the plasma treatment induces dose-dependent up-regulation of TP73 gene expression, resulting in significantly elevated levels of TP73 RNA and protein in plasma-treated melanoma cells. Silencing of TP73 expression by means of RNA interference inhibited the anticancer effects of the plasma, similar to the effect of caspase inhibitor z-VAD or ROS scavenger N-acetyl cysteine. These results confirm the role of TP73 protein in dose-dependent regulation of anticancer activity of atmospheric-pressure plasmas.

Testing the efficacy of low volume herbicide applications on Chromolaena odorata

Siam Weed (Chromoleana odorata) is the target of an eradication program in north Queensland; however some infestations occur on ground inaccessible to high volume, ground based herbicide spray equipment. Four foliar herbicides were applied to dense infestations of mature Siam Weed in March 2009, near Townsville, north Queensland. Low volume, high concentration solutions containing 40 g L-1 a.i. glyphosate, 1.2 g L-1 a.i metsulfuron-methyl, 10 g L-1 a.i. fluroxypyr + 0.7 g L-1 a.i. aminopyralid and 15 g L-1 a.i. triclopyr + 5 g L-1 a.i. picloram + 0.4 g L-1 a.i. aminopyralid were applied using a 5 L backpack and hand gun (or splatter gun). Relatively small amounts (approximately 24-28 mL) of the high concentration solutions were applied to each bush and assessments of the replicated treated and untreated control plots were conducted 76, 207 and 356 days after treatment. These assessments demonstrated that the fluroxypyr and triclopyr based herbicides controlled 96 to 100% of plants. The metsulfuron-methyl and glyphosate based herbicides controlled 40 and 57% of plants respectively 12 months after treatment, when 3% of untreated control plants were dead. The trial demonstrated that this application method and either of two herbicides provides an additional tool for controlling Siam weed in remote areas, which are inaccessible to traditional higher volume foliar herbicide applications. Lower volume herbicide solutions reduce the volume of water and thus the effort needed to effectively treat less accessible infestations.

The plasticity of NBS resistance genes in sorghum is driven by multiple evolutionary processes

Background Increased disease resistance is a key target of cereal breeding programs, with disease outbreaks continuing to threaten global food production, particularly in Africa. Of the disease resistance gene families, the nucleotide-binding site plus leucine-rich repeat (NBS-LRR) family is the most prevalent and ancient and is also one of the largest gene families known in plants. The sequence diversity in NBS-encoding genes was explored in sorghum, a critical food staple in Africa, with comparisons to rice and maize and with comparisons to fungal pathogen resistance QTL. Results In sorghum, NBS-encoding genes had significantly higher diversity in comparison to non NBS-encoding genes and were significantly enriched in regions of the genome under purifying and balancing selection, both through domestication and improvement. Ancestral genes, pre-dating species divergence, were more abundant in regions with signatures of selection than in regions not under selection. Sorghum NBS-encoding genes were also significantly enriched in the regions of the genome containing fungal pathogen disease resistance QTL; with the diversity of the NBS-encoding genes influenced by the type of co-locating biotic stress resistance QTL. Conclusions NBS-encoding genes are under strong selection pressure in sorghum, through the contrasting evolutionary processes of purifying and balancing selection. Such contrasting evolutionary processes have impacted ancestral genes more than species-specific genes. Fungal disease resistance hot-spots in the genome, with resistance against multiple pathogens, provides further insight into the mechanisms that cereals use in the “arms race” with rapidly evolving pathogens in addition to providing plant breeders with selection targets for fast-tracking the development of high performing varieties with more durable pathogen resistance.

Biological control of prickly acacia: Current research and future prospects

Prickly acacia, Vachellia nilotica subsp. indica (syn. Acacia nilotica subsp. indica) (Fabaceae), a major weed in the natural grasslands of western Queensland, has been a target of biological control since the 1980s with limited success to date. Surveys in India, based on genetic and climate matching, identified five insects and two rust pathogens as potential agents. Host-specificity tests were conducted for the insects in India and under quarantine conditions in Australia, and for the rust pathogens under quarantine conditions at CABI in the UK. In no-choice tests, the brown leaf-webber, Phycita sp. A, (Lepidoptera: Pyralidae) completed development on 17 non-target plant species. Though the moth showed a clear preference for prickly acacia in oviposition choice trials screening of additional test-plant species was terminated in view of the potential non-target risk. The scale insect Anomalococcus indicus (Hemiptera: Lecanodiaspididae) developed into mature gravid females on 13 out of 58 non-target plant species tested. In the majority of cases very few female scales matured but development was comparable to that on prickly acacia on four of the non-target species. In multiple choice tests, the scale insect showed a significant preference for the target weed over non-target species tested. In a paired-choice trial under field conditions in India, crawler establishment occurred only on prickly acacia and not on the non-target species tested. Further choice trials are to be conducted under natural field conditions in India. A colony of the green leaf-webber Phycita sp. B has been established in quarantine facilities in Australia and host-specificity testing has commenced. The gall-rust Ravenelia acaciae-arabicae and the leaf-rust Ravenelia evansii (Puccineales: Raveneliaceae) both infected and produced viable urediniospores on Vachellia sutherlandii (Fabaceae), a non-target Australian native plant species. Hence, no further testing with the two rust species was pursued. Inoculation trials using the gall mite Aceria liopeltus (Acari: Eriophyidae) from V. nilotica subsp. kraussiana in South Africa resulted in no gall induction on V. nilotica subsp. indica. Future research will focus on the leaf-weevil Dereodus denticollis (Coleoptera: Curculionidae) and the leaf-beetle Pachnephorus sp. (Coleoptera: Chrysomelidae) under quarantine conditions in Australia. Native range surveys for additional potential biological control agents will also be pursued in northern and western Africa.

Lake sediment research as a part of lake management - case studies and implications from southern Finland

In Finland one of the most important current issues in the environmental management is the quality of surface waters. The increasing social importance of lakes and water systems has generated wide-ranging interest in lake restoration and management, concerning especially lakes suffering from eutrophication, but also from other environmental impacts. Most of the factors deteriorating the water quality in Finnish lakes are connected to human activities. Especially since the 1940's, the intensified farming practices and conduction of sewage waters from scattered settlements, cottages and industry have affected the lakes, which simultaneously have developed in to recreational areas for a growing number of people. Therefore, this study was focused on small lakes, which are human impacted, located close to settlement areas and have a significant value for local population. The aim of this thesis was to obtain information from lake sediment records for on-going lake restoration activities and to prove that a well planned, properly focused lake sediment study is an essential part of the work related to evaluation, target consideration and restoration of Finnish lakes. Altogether 11 lakes were studied. The study of Lake Kaljasjärvi was related to the gradual eutrophication of the lake. In lakes Ormajärvi, Suolijärvi, Lehee, Pyhäjärvi and Iso-Roine the main focus was on sediment mapping, as well as on the long term changes of the sedimentation, which were compared to Lake Pääjärvi. In Lake Hormajärvi the role of different kind of sedimentation environments in the eutrophication development of the lake's two basins were compared. Lake Orijärvi has not been eutrophied, but the ore exploitation and related acid main drainage from the catchment area have influenced the lake drastically and the changes caused by metal load were investigated. The twin lakes Etujärvi and Takajärvi are slightly eutrophied, but also suffer problems associated with the erosion of the substantial peat accumulations covering the fringe areas of the lakes. These peat accumulations are related to Holocene water level changes, which were investigated. The methods used were chosen case-specifically for each lake. In general, acoustic soundings of the lakes, detailed description of the nature of the sediment and determinations of the physical properties of the sediment, such as water content, loss on ignition and magnetic susceptibility were used, as was grain size analysis. A wide set of chemical analyses was also used. Diatom and chrysophycean cyst analyses were applied, and the diatom inferred total phosphorus content was reconstructed. The results of these studies prove, that the ideal lake sediment study, as a part of a lake management project, should be two-phased. In the first phase, thoroughgoing mapping of sedimentation patterns should be carried out by soundings and adequate corings. The actual sampling, based on the preliminary results, must include at least one long core from the main sedimentation basin for the determining the natural background state of the lake. The recent, artificially impacted development of the lake can then be determined by short-core and surface sediment studies. The sampling must be focused on the basis of the sediment mapping again, and it should represent all different sedimentation environments and bottom dynamic zones, considering the inlets and outlets, as well as the effects of possible point loaders of the lake. In practice, the budget of the lake management projects of is usually limited and only the most essential work and analyses can be carried out. The set of chemical and biological analyses and dating methods must therefore been thoroughly considered and adapted to the specific management problem. The results show also, that information obtained from a properly performed sediment study enhances the planning of the restoration, makes possible to define the target of the remediation activities and improves the cost-efficiency of the project.

Patriarkkana ikuisuuden performanssissa : Maskuliinisuuden ja miehen ruumiillisuuden rakentuminen mormonien Duties and Blessings of the Priesthood -opetuskäsikirjassa

The material I analyze for my master's thesis is a teaching manual used by the Mormons (the Church of Jesus Christ of Latter-day Saints), called "Duties and Blessings of the Priesthood". This work includes numerous lesson plans, each one with a separate topic. The manual is intended especially for teaches, but can also be used for individual study. The main target of my research is to find out how men and their bodies are constructed in the manual. Prescriptive texts together with narrative stories and illustrations create a multifaceted picture of Mormon notions of masculinity and corporeality. I approach my research material from a constructivist perspective. I build my interpretative reading upon Critical Discourse Analysis. I am especially interested in how the manual interprets and understands connections between gender, embodiment and religion. I understand gender in Judith Butler's terms, as a performance of styled and repeated gestures. Some of the discussions I raise in my work draw upon the disciplines of Critical Men's Studies and Sociology of Religion. In Mormonism, gender is thought to be an elementary part of human ontology. It is an eternal trait inherited from God the Father (and God the Mother). The place of men in Mormon cosmology is determined by their double role as patriarchs, fathers and priests. The main objective of mortal life is to gain salvation together with one's family. The personal goal of a Mormon man is to one day become a god. Patriarchs are responsible for the spiritual and material well-being of their family. The head of a household should be gentle and loving, but still an unconditional authority. In the manual, a Mormon man is depicted as a successor of mythical and exemplary men of sacred history. The perfect and sinless body of Jesus Christ serves as an ideal for the male body. Mormon masculinity is also defined by priesthood - the holy power of God - which is given to practically all male Mormons. Through the priesthood, a Mormon man serves as the governor of God on Earth. The Mormon priest has the authority to bind the immanent and the transcendent worlds together with gestures, poses and motions performed with his body. In Mormonism, the body also symbolizes a temple or a space where the sacred meets the profane. Because the priesthood borne by a man is holy, he has to treat his body accordingly. The body is valuable in itself, without it one cannot be saved. Men are forbidden of polluting their bodies by using stimulants or by having sexual relations out of wedlock. A priesthood holder must uphold healthy habits, dress neatly, and conduct himself in a temperate manner. He must also be outgoing and attentive. The manual suggests that a man's goodness or wickedness can be perceived from his external appearance. The Church of Jesus Christ of Latter-day Saints is a hierarchical and man-led organisation. The ideals of gender and corporeality are set by a homogenous priesthood leadership that consists mainly of white heterosexual American men. The larger Mormon community can control individual men by sanctioning. Growing as a Mormon man happens under the guidance of one's reference group.

Inhibition of Human Two-Pore Domain K+ Channel TREK1 by Local Anesthetic Lidocaine: Negative Cooperativity and Half-of-Sites Saturation Kinetics

TWIK-related K+ channel TREK1, a background leak K+ channel, has been strongly implicated as the target of several general and local anesthetics. Here, using the whole-cell and single-channel patch-clamp technique, we investigated the effect of lidocaine, a local anesthetic, on the human (h) TREK1 channel heterologously expressed in human embryonic kidney 293 cells by an adenoviral-mediated expression system. Lidocaine, at clinical concentrations, produced reversible, concentration-dependent inhibition of hTREK1 current, with IC50 value of 180 mu M, by reducing the single-channel open probability and stabilizing the closed state. We have identified a strategically placed unique aromatic couplet (Tyr352 and Phe355) in the vicinity of the protein kinase A phosphorylation site, Ser348, in the C-terminal domain (CTD) of hTREK1, that is critical for the action of lidocaine. Furthermore, the phosphorylation state of Ser348 was found to have a regulatory role in lidocaine-mediated inhibition of hTREK1. It is interesting that we observed strong intersubunit negative cooperativity (Hill coefficient = 0.49) and half-of-sites saturation binding stoichiometry (half-reaction order) for the binding of lidocaine to hTREK1. Studies with the heterodimer of wild-type (wt)-hTREK1 and Delta 119 C-terminal deletion mutant (hTREK1(wt)-Delta 119) revealed that single CTD of hTREK1 was capable of mediating partial inhibition by lidocaine, but complete inhibition necessitates the cooperative interaction between both the CTDs upon binding of lidocaine. Based on our observations, we propose a model that explains the unique kinetics and provides a plausible paradigm for the inhibitory action of lidocaine on hTREK1.

Environmental effects of thermal and radioactive discharges from nuclear power plants in the boreal brackish-water conditions of the northern Baltic Sea

During recent decades, thermal and radioactive discharges from nuclear power plants into the aquatic environment have become the subject of lively debate as an ecological concern. The target of this thesis was to summarize the large quantity of results obtained in extensive monitoring programmes and studies carried out in recipient sea areas off the Finnish nuclear power plants at Loviisa and Olkiluoto during more than four decades. The Loviisa NPP is located on the coast of the Gulf of Finland and Olkiluoto NPP on that of the Bothnian Sea. The state of the Gulf of Finland is clearly more eutrophic; the nutrient concentrations in the surface water are about 1½ 2 times higher at Loviisa than at Olkiluoto, and the total phosphorus concentrations still increased in both areas (even doubled at Loviisa) between the early 1970s and 2000. Thus, it is a challenge to distinguish the local effects of thermal discharges from the general eutrophication process of the Gulf of Finland. The salinity is generally low in the brackish-water conditions of the northern Baltic Sea, being however about 1 higher at Olkiluoto than at Loviisa (the salinity of surface water varying at the latter from near to 0 in early spring to 4 6 in late autumn). Thus, many marine and fresh-water organisms live in the Loviisa area close to their limit of existence, which makes the biota sensitive to any additional stress. The characteristics of the discharge areas of the two sites differ from each other in many respects: the discharge area at Loviisa is a semi-enclosed bay in the inner archipelago, where the exchange of water is limited, while the discharge area at Olkiluoto is more open, and the exchange of water with the open Bothnian Sea is more effective. The effects of the cooling water discharged from the power plants on the temperatures in the sea were most obvious in winter. The formation of a permanent ice cover in the discharge areas has been delayed in early winter, and the break-up of the ice occurs earlier in spring. The prolonging of the growing season and the disturbance of the overwintering time, in conditions where the biota has adjusted to a distinct rest period in winter, have been the most significant biological effects of the thermal pollution. The soft-bottom macrofauna at Loviisa has deteriorated to the point of almost total extinction at many sampling stations during the past 40 years. A similar decline has been reported for the whole eastern Gulf of Finland. However, the local eutrophication process seems to have contributed into the decline of the zoobenthos in the discharge area at Loviisa. Thermal discharges have increased the production of organic matter, which again has led to more organic bottom deposits. These have in turn increased the tendency of the isolated deeps to a depletion of oxygen, and this has further caused strong remobilization of phosphorus from the bottom sediments. Phytoplankton primary production and primary production capacity doubled in the whole area between the late 1960s and the late 1990s, but started to decrease a little at the beginning of this century. The focus of the production shifted from spring to mid- and late summer. The general rise in the level of primary production was mainly due to the increase in nutrient concentrations over the whole Gulf of Finland, but the thermal discharge contributed to a stronger increase of production in the discharge area compared to that in the intake area. The eutrophication of littoral vegetation in the discharge area has been the most obvious, unambiguous and significant biological effect of the heated water. Myriophyllum spicatum, Potamogeton perfoliatus and Potamogeton pectinatus, and vigorous growths of numerous filamentous algae as their epiphytes have strongly increased in the vicinity of the cooling water outlet, where they have formed dense populations in the littoral zone in late summer. However, the strongest increase of phytobenthos has extended only to a distance of about 1 km from the outlet, i.e., the changes in vegetation have been largest in those areas that remain ice-free in winter. Similar trends were also discernible at Olkiluoto, but to a clearly smaller extent, which was due to the definitely weaker level of background eutrophy and nutrient concentrations in the Bothnian Sea, and the differing local hydrographical and biological factors prevailing in the Olkiluoto area. The level of primary production has also increased at Olkiluoto, but has remained at a clearly lower level than at Loviisa. In spite of the analogous changes observed in the macrozoobenthos, the benthic fauna has remained strong and diversified in the Olkiluoto area. Small amounts of local discharge nuclides were regularly detected in environmental samples taken from the discharge areas: tritium in seawater samples, and activation products, such as 60Co, 58Co, 54Mn, 110mAg, 51Cr, in suspended particulate matter, bottom sediments and in several indicator organisms (e.g., periphyton and Fucus vesiculosus) that effectively accumulate radioactive substances from the medium. The tritium discharges and the consequent detection frequency and concentrations of tritium in seawater were higher at Loviisa, but the concentrations of the activation products were higher at Olkiluoto, where traces of local discharge nuclides were also observed over a clearly wider area, due to the better exchange of water than at Loviisa, where local discharge nuclides were only detected outside Hästholmsfjärden Bay quite rarely and in smaller amounts. At the farthest, an insignificant trace amount (0.2 Bq kg-1 d.w.) of 60Co originating from Olkiluoto was detected in Fucus at a distance of 137 km from the power plant. Discharge nuclides from the local nuclear power plants were almost exclusively detected at the lower trophic levels of the ecosystems. Traces of local discharge nuclides were very seldom detected in fish, and even then only in very low quantities. As a consequence of the reduced discharges, the concentrations of local discharge nuclides in the environment have decreased noticeably in recent years at both Loviisa and Olkiluoto. Although the concentrations in environmental samples, and above all, the discharge data, are presented as seemingly large numbers, the radiation doses caused by them to the population and to the biota are very low, practically insignificant. The effects of the thermal discharges have been more significant, at least to the wildlife in the discharge areas of the cooling water, although the area of impact has been relatively small. The results show that the nutrient level and the exchange of water in the discharge area of a nuclear power plant are of crucial importance.

Phylogeography and Adaptive Divergence of Three-spined Stickleback Populations

Genetic studies on phylogeography and adaptive divergence in Northern Hemisphere fish species such as three-spined stickleback (Gasterosteus aculeatus) provide an excellent opportunity to investigate genetic mechanisms underlying population differentiation. According to the theory, the process of population differentiation results from a complex interplay between random and deterministic processes as well historical factors. The main scope in this thesis was to study how historical factors like the Pleistocene ice ages have shaped the patterns molecular diversity in three-spined stickleback populations in Europe and how this information could be utilized in the conservation genetic context. Furthermore, identifying footprints of natural selection at the DNA level might be used in identifying genes involved in evolutionary change. Overall, the results from phylogeographic studies indicate that the three-spined stickleback has colonized the Atlantic basin relatively recently but constitutes three major evolutionary lineages in Europe. In addition, the colonization of freshwater appears to result from multiple and independent invasions by the marine conspecifics. Molecular data together with morphology suggest that the most divergent freshwater populations are located in the Balkan Peninsula and these populations deserve a special conservation genetic status without warranting further taxonomical classification. In order to investigate the adaptive divergence in Fennoscandian three-spined stickleback populations several approaches were used. First, sequence variability in the Eda-gene, coding for the number of lateral plates, was concordant with the previously observed global pattern. Full plated allele is in high frequencies among marine populations whereas low plated allele dominates in the freshwater populations. Second, a microsatellite based genome scan identified both indications of balancing and directional selection in the three-spined stickleback genome, i.e. loci with unusually similar or unusually different allele frequencies over populations. The directionally selected loci were mainly associated with the adaptation to freshwater. A follow up study conducting a more detailed analysis in a chromosome region containing a putatively selected gene locus identified a fairly large genomic region affected by natural selection. However, this region contained several gene predictions, all of which might be the actual target of natural selection. All in all, the phylogeographic and adaptive divergence studies indicate that most of the genetic divergence has occurred in the freshwater populations whereas the marine populations have remained relatively uniform.

Comparative sequence analysis and expression in E-coli of the subgroup I-specific antigen VP6 from a G2 serotype human rotavirus IS2

VP6, the intermediate capsid protein of the virion, specifies subgroup specificity of rotavirus, It is also the most conserved, both at nucleotide and amino acid levels, among group A rotaviruses and is the target of choice for rotavirus detection, In this study we report the sequence of the subgroup I (SGI)-specific VP6 from the serotype G2 strain IS2 isolated from a child suffering from acute diarrhoea in Bangalore ana its comparison with the published VP6 sequences. Interestingly, IS2 gene 6 shared highest homology with that from bovine UK strain and the protein contained substitutions by lysine at amino acid positions 97 and 134, In contrast, the amino acids Met and Glu/Asp at these respective positions are highly conserved in all the other group A rotaviruses sequenced so far, These observations have obvious implications for the evolution of serotype G2 and G2-like strains circulating in India, The SGI VP6, of a human rotavirus, possessing epitopes that are conformationally similar to those found in the native protein in the virion, was successfully expressed in E. coli and purified for the first time by single-step affinity chromatography.

Analysis of the amino acid sequences of plant Bowman-Birk inhibitors

Plant seeds contain a large number of protease inhibitors of animal, fungal, and bacterial origin. One of the well-studied families of these inhibitors is the Bowman-Birk family(BBI). The BBIs from dicotyledonous seeds are 8K, double-headed proteins. In contrast, the 8K inhibitors from monocotyledonous seeds are single headed. Monocots also have a 16K, double-headed inhibitor. We have determined the primary structure of a Bowman-Birk inhibitor from a dicot, horsegram, by sequential edman analysis of the intact protein and peptides derived from enzymatic and chemical cleavage. The 76-residue-long inhibitor is very similar to that ofMacrotyloma axillare. An analysis of this inhibitor along with 26 other Bowman-Birk inhibitor domains (MW 8K) available in the SWISSPROT databank revealed that the proteins from monocots and dicots belong to related but distinct families. Inhibitors from monocots show larger variation in sequence. Sequence comparison shows that a crucial disulphide which connects the amino and carboxy termini of the active site loop is lost in monocots. The loss of a reactive site in monocots seems to be correlated to this. However, it appears that this disulphide is not absolutely essential for retention of inhibitory function. Our analysis suggests that gene duplication leading to a 16K inhibitor in monocots has occurred, probably after the divergence of monocots and dicots, and also after the loss of second reactive site in monocots.

Inhibition of CYP1A2-mediated drug metabolism in vitro and in humans : With special emphasis on rofecoxib and other NSAIDs

The cytochrome P450 1A2 (CYP1A2) is one of the major metabolizing enzymes. The muscle relaxant tizanidine is a selective substrate of CYP1A2, and the non-steroidal anti-inflammatory drug (NSAID) rofecoxib was thought to modestly in-hibit it. Cases suggesting an interaction between tizanidine and rofecoxib had been reported, but the mechanism was unknown. Also other NSAIDs are often used in combination with muscle relaxants. The aims of this study were to investigate the effect of rofecoxib, several other NSAIDs and female sex steroids on CYP1A2 ac-tivity in vitro and in vivo, and to evaluate the predictability of in vivo inhibition based on in vitro data. In vitro, the effect of several NSAIDs, female sex steroids and model inhibitors on CYP1A2 activity was studied in human liver microsomes, without and with preincubation. In placebo controlled, cross-over studies healthy volunteers ingested a single dose of tizanidine after a pretreament with the inhibitor (rofecoxib, tolfenamic acid or celecoxib) or placebo. Plasma (and urine) concentrations of tizanidine and its metabolites were measured, and the pharmacodynamic effects were recorded. A caffeine test was also performed. In vitro, fluvoxamine, tolfenamic acid, mefenamic acid and rofecoxib potently in-hibited CYP1A2. Ethinylestradiol, celecoxib, desogestrel and zolmitriptan were moderate, and etodolac, ciprofloxacin, etoricoxib and gestodene were weak inhibi-tors of CYP1A2. At 100 µM, other tested NSAIDs and steroids inhibited CYP1A2 less than 35%. Rofecoxib was found to be a mechanism-based inhibitor of CYP1A2. In vivo, rofecoxib greatly increased the plasma concentrations (over ten-fold) and the pharmacodynamic effects of tizanidine. Also the metabolism of caf-feine was impaired by rofecoxib. Despite the relatively strong in vitro CYP1A2 inhibitory effects, tolfenamic acid and celecoxib did not have a significant effect on tizanidine and caffeine concentrations in humans. Competitive inhibition model and the free plasma concentration of the inhibitor predicted well the effect of fluvoxam-ine and the lack of effect of tolfenamic acid and celecoxib on tizanidine concentra-tions in humans, and mechanism-based inhibition model explained the effects of rofecoxib. However, the effects of ciprofloxacin and oral contraceptives were un-derestimated from the in vitro data. Rofecoxib is a potent mechanism-based inhibitor of CYP1A2 in vitro and in vivo. This mechanism may be involved in the adverse cardiovascular effects of rofecoxib. Tolfenamic acid and celecoxib seem to be safe in combination with tizanidine, but mefenamic acid might have some effect on tizanidine concentrations in vivo. Con-sidering the mechanism of inhibition, and using the free plasma concentration of the inhibitor, many but not all CYP1A2 interactions can be predicted from in vitro data.

Evidence for the inability of the intestinal mitochondria of the silkworm, Bombyx mori L.to oxidize fatty acids

Conditions for the preparation of mitochondria from silkworm intestines have been standardized. The inability of mitochondria to oxidize fatty acids has been demonstrated. Evidence for the absence of an inhibitor in the mitochondria has been obtained.