Protein purification and gene isolation of chlamysin, a cold-active lysozyme-like enzyme with antibacterial activity

An antibacterial approximately 11 kDa protein designated chlamysin was isolated from viscera of the marine bivalve Chlamys islandica. Chlamysin inhibited the growth of all Gram-positive and Gram-negative bacteria tested. The isolated protein was highly efficient in hydrolyzing Micrococcus luteus cells only at low pH (4.5-6.2) and at low temperature (4-35 degrees C). No significant loss of enzyme activity was observed after 30 days storage at room temperature or after heating to 70 degrees C for 15 min, suggesting relatively high protein structure stability. Sequence-analyzed fragments of the protein revealed data which guided the isolation of the cDNA gene, encoding a 137 amino acid chlamysin precursor in scallops. The deduced protein contains a high portion of cysteine, serine and histidine residues and has a predicted isoelectric point below 7. The chlamysin protein was found to have sequence homology to an isopeptidase and to a recently published bivalve lysozyme.     

Fish microsporidia: fine structural diversity and phylogeny

Structural diversity of fish microsporidian life cycle stages and of the host-parasite interface is reviewed. In the infected cell of the fish host, microsporidia may either cause serious degradation of the cytoplasm and demise of the cell, or they may elicit host cell hypertrophy, producing a parasite-hypertrophic host cell complex, the xenoma. The structure of the xenoma and of its cell wall may differ according to the genus of the parasite, and seems to express properties of the parasite rather than those of the host. In merogony, the parasite cell surface interacts with the host cell in diverse ways, the most conspicuous being the production of thick envelopes of different types. Sporogony stages reveal different types of walls or membranes encasing the sporoblasts and later the spores and these envelopes may be of host or parasite origin. Nucleospora differs from all other fish microsporidia by its unique process of sporogony. Except for the formation of conspicuous xenomas, there are no essentially different structures in fish-infecting microsporidia compared with microsporidia from other hosts. Although the structures associated with the development of fish microsporidia cannot be attributed importance in tracing the phylogeny, they are relevant for practical determination and assessing the relation to the host. The possibility of the existence of an intermediate host is discussed. Higher-level classification of Microsporidia is briefly discussed and structure and evolutionary rates in microsporidian rDNA are reviewed. Discussion of rDNA molecular phylogeny of fish-infecting microsporidia is followed by classification of these parasites. Most form a rather cohesive clade. Outside this clade is the genus Nucleospora, separated at least at the level of Order. Within the main clade, however, there are six species infecting hosts other than fish. Based on data available for analysis, a tentative classification of fish-infecting microsporidia into five groups is proposed. Morphologically defined groups represent families, others are referred to as clades. Group 1, represented by family Pleistophoridae, includes Pleistophora, Ovipleistophora and Heterosporis; Vavraia and Trachipleistophora infect non-fish hosts. Group 2, represented by family Glugeidae, is restricted to genus Glugea and Tuzetia weidneri from crustaceans. Group 3 comprises three clades: Loma and a hyperparasitic microsporidian from a myxosporean; Ichthyosporidium and Pseudoloma clade and the Loma acerinae clade. For the latter species a new genus has to be established. Group 4 contains two families, Spragueidae with the genus Spraguea and Tetramicridae with genera Microgemma and Tetramicra, and the Kabatana and Microsporidium seriolae clade. Group 5 is represented by the family Enterocytozoonidae with the genus Nucleospora and mammal-infecting genus Enterocytozoon.     

DNA base excision repair of uracil residues in reconstituted nucleosome core particles

The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase beta and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase beta on nucleosome cores.     

Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory beta-subunit of CK2. The interaction between FGF-1 and CK2 alpha and beta was characterized by surface plasmon resonance, giving K(D) values of 0.4 +/- 0.3 and 1.2 +/- 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2 alpha are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2 beta. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2 alpha was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed.     

A global non-conjugative Tet C plasmid,pRAS3, from Aeromonas salmonicida

Two 11.8 kb non-conjugative, but mobilizable R plasmids designated pRAS3.1 and pRAS3.2 were isolated from Aeromonas salmonicida subspecies salmonicida and atypical A. salmonicida, respectively. Differences between the plasmids were of minor extent and they are considered as being variants of the same plasmid, pRAS3. The genes repA, repB, mobA, mobC, mobD, and mobE were organized similar to corresponding genes in the small, mobilizable plasmid pTF-FC2 isolated from Acidithiobacillus ferrooxidans (previously Thiobacillus ferrooxidans). The nucleotide identity between these genes from pRAS3.1 and pTF-FC2 ranged from 89.5 to 98.2%. The tetA(C), tetR(C), and approximately 960 base pairs adjacent to tetR(C) were highly similar to the nucleotide sequence in pSC101. Plasmid pRAS3 was also found in a Scottish A. salmonicida strain, and appears to be identical to the R plasmid pJA8102-2 isolated from A. salmonicida in Japan.     

The Danish Registry for Plastic Surgery of the Breast: establishment of a nationwide registry for prospective follow-up,quality assessment,and investigation of breast surgery

Although numerous epidemiologic studies have examined the long-term safety of silicone breast implants during the past decade, there is a relative lack of surveillance data on short-term health effects and complications following cosmetic surgery of the breast. The Danish Registry for Plastic Surgery of the Breast, established in May of 1999, provides plastic surgeons with a nationwide system for the collection of preoperative, perioperative, and postoperative data on women undergoing breast implantation, breast reduction, or mastopexy. The purpose of the Registry is to examine short-term and, eventually, long-term local complications and possible health effects, and to contribute to an ongoing evaluation of surgical results and surveillance of the products. Furthermore, the Registry will allow the identification of new areas for research into cosmetic and reconstructive breast surgery. Women accepting registration in the Danish Registry for Plastic Surgery of the Breast complete a self-administered questionnaire focusing on medical history and demographic and behavioral factors. Preoperative blood samples are drawn for storage. Surgical data, postoperative results, and complications are registered following surgery and at postoperative visits. Currently, registration has been initiated at 24 private and public clinics, representing more than 80 percent of the plastic surgery clinics in Denmark. As of November of 2001, a total of 1472 women with breast implants and 560 women with breast reduction were included in the Registry. These figures are expected to increase annually by 1000 women undergoing breast implantation and 500 women undergoing breast reduction or mastopexy. The authors present their experience of establishing the first nationwide comprehensive clinical-epidemiologic database and biological bank for cosmetic and reconstructive surgery procedures.     

Seed 1-cysteine peroxiredoxin antioxidants are not involved in dormancy, but contribute to inhibition of germination during stress

Peroxiredoxins (Prx) are thiol-dependent antioxidants containing one (1-cysteine [-Cys]) or two (2-Cys) conserved Cys residues that protect lipids, enzymes, and DNA against reactive oxygen species. In plants, the 1-Cys Prxs are highly expressed during late seed development, and the expression pattern is dormancy related in mature seeds. We have expressed the Arabidopsis 1-Cys Prx AtPER1 in Escherichia coli and show that this protein has antioxidant activity in vitro and protects E. coli in vivo against the toxic oxidant cumene hydroperoxide. Although some 1-Cys Prxs are targeted to the nucleus, a green fluorescent protein-AtPER1 fusion protein was also localized to the cytoplasm in an onion epidermis subcellular localization assay. It has been proposed that seed Prxs are involved in maintenance of dormancy and/or protect the embryo and aleurone layer surviving desiccation against damage caused by reactive oxygen species. These hypotheses were tested using transgenic Arabidopsis lines overexpressing the barley (Hordeum vulgare) 1-Cys PER1 protein and lines with reduced levels of AtPER1 due to antisensing or RNA interference. We found no correlation between Prx levels and the duration of the after-ripening period required before germination. Thus, Prxs are unlikely to contribute to maintenance of dormancy. RNA interference lines almost devoid of AtPER1 protein developed and germinated normally under standard growth room conditions. However, seeds from lines overexpressing PER1 were less inclined to germinate than wild-type seeds in the presence of NaCl, mannitol, and methyl viologen, suggesting that Prx can sense harsh environmental surroundings and play a part in the inhibition of germination under unfavorable conditions.     

Genetic relationship between Mongolian and Norwegian horses?

Human populations of Central Asian origin have contributed genetic material to northern European populations. It is likely that migrating humans carried livestock to ensure food and ease transportation. Thus, eastern genes could also have dispersed to northern European livestock populations. Using microsatellite data, we here report that the essentially different genetic distances DA and (deltamu)2 and their corresponding phylogenetic trees show close associations between the Mongolian native horse and northern European horse breeds. The genetic distances between the northern European breeds and Standardbred/Thoroughbred, representing a southern-derived source of horses, were notably larger. We suggest that contribution of genetic material from eastern horses to northern European populations is likely to have occurred.     

Diseases, prophylaxis and treatment of the Atlantic halibut Hippoglossus hippoglossus: a review

After substantial investments in research, the Atlantic halibut Hippoglossus hippoglossus is now being cultivated commercially in Norway, Iceland, Scotland and Canada. As with other domesticated species, disease problems have been experienced. This review summarizes the current state of knowledge of diseases of the Atlantic halibut, and their diagnosis, prophylaxis and treatment. In economic terms, the most important losses have been suffered at the larval and juvenile stages. The most important infections are caused by nodaviruses, causative agents of Viral Encephalopathy and Retinopathy (VER), which are the major reason why Norway's production of halibut fry has been level since 1995. An aquatic birnavirus, Infectious Pancreatic Necrosis Virus, is also an important agent of mortality. Vibrio anguillarum, Flexibacter ovolyticus and atypical Aeromonas salmonicida are the major bacterial pathogens. The protozoan parasites recorded include Ichthyobodo sp., the microsporidium Enterocytozoon sp., Trichodina hippoglossi, and the metazoan pathogens include myxozoans, helminths, Entobdella hippoglossi, Lepeophtheirus hippoglossi and other parasitic copepods. Experimental vaccines have been tested against V anguillarum and atypical A. salmonicida, with good results. A recombinant vaccine against nodaviruses is under development. A few trials have been carried out on non-specific immunostimulants, but no such treatment is currently available. A number of efficacy and pharmacokinetic trials with various antibacterial agents have also been published.     

RNA: versatility in form and function

RNA performs a remarkable range of functions in all cells. In addition to its central role in information transfer from DNA to protein, it is essential for functions as diverse as RNA processing, chromosome end-maintenance and dosage compensation. The versatility of RNA derives from its unique ability to use direct readout via base-pairing for sequence specific targeting (or templating) in combination with its capacity to form elaborate three dimensional structures. Such structures can perform catalysis or serve as protein recognition surfaces. In this short review, we attempt to give a flavor for the diversity of functional RNAs in the cell and highlight, using selected examples, two quite distinct activities, catalysis and sequence specific targeting. Within each section, we discuss how the lessons we have learned from these systems may apply to other, less well understood, RNAs.