Agar culture of Piscirickettsia salmonis, a serious pathogen of farmed salmonid and marine fish

Piscirickettsia salmonis, a serious bacterial pathogen of farmed marine fish, previously considered culturable only in eukaryotic cell-culture systems, was grown for the first time on agar and broth containing enhanced levels of cysteine, thus greatly increasing the potential for isolation, in vitro culture and study of this organism. Virulence towards Atlantic salmon following passage on agar media was retained in a controlled laboratory trial. Of the studied temperatures, optimal growth on agar was observed at 22 degrees C.     

The detection of hamster connexins: a comparison of expression profiles with wild-type mouse and the cancer-prone Min mouse

The open reading frames of 17 connexins from Syrian hamster (using tissues) and 16 connexins from the Chinese hamster cell line V79, were fully (Cx30, Cx31, Cx37, Cx43 and Cx45) or partially sequenced. We have also detected, and partially sequenced, seven rat connexins that previously were unavailable. The expression of connexin genes was examined in some hamster organs and cultured hamster cells, and compared with wild-type mouse and the cancer-prone Min mouse. Although the expression patterns were similar for most organs and connexins in hamster and mouse, there were also some prominent differences (Cx29 and 30.3 in testis; Cx31.1 and 32 in eye; Cx46 in brain, kidney and testis; Cx47 in kidney). This suggests that some connexins have species-specific expression profiles. In contrast, there were minimal differences in expression profiles between wild type and Min mice. Species-specific expression profiles should be considered in attempts to make animal models of human connexin-associated diseases.     

Evolutionary selection pressure and family relationships among connexin genes

We suggest an extension of connexin orthology relationships across the major vertebrate lineages. We first show that the conserved domains of mammalian connexins (encoding the N-terminus, four transmembrane domains and two extracellular loops) are subjected to a considerably more strict selection pressure than the full-length sequences or the variable domains (the intracellular loop and C-terminal tail). Therefore, the conserved domains are more useful for the study of family relationships over larger evolutionary distances. The conserved domains of connexins were collected from chicken, Xenopus tropicalis, zebrafish, pufferfish, green spotted pufferfish, Ciona intestinalis and Halocynthia pyriformis (two tunicates). A total of 305 connexin sequences were included in this analysis. Phylogenetic trees were constructed, from which the orthologies and the presumed evolutionary relationships between the sequences were deduced. The tunicate connexins studied had the closest, but still distant, relationships to vertebrate connexin 36, 39.2, 43.4, 45 and 47. The main structure in the connexin family known from mammals pre-dates the divergence of bony fishes, but some additional losses and gains of connexin sequences have occurred in the evolutionary lineages of subsequent vertebrates. Thus, the connexin gene family probably originated in the early evolution of chordates, and underwent major restructuring with regard to gene and subfamily structures (including the number of genes in each subfamily) during early vertebrate evolution.     

Morphological transformation and catalase activity of syrian hamster embryo cells treated with hepatic peroxisome proliferators,TPA and nickel sulphate

The abilities of the hepatic peroxisome proliferators (HPPs) clofibrate, di(2-ethylhexyl)phthalate (DEHP), mono(2-ethylhexyl)- phthalate (MEHP), 2,4-dichlorophenoxy acetic acid (2,4-D), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) and tiadenol to induce morphological transformation and to increase the catalase activity of Syrian hamster embryo (SHE) cells were studied. DEHP, MEHP, clofibrate and tiadenol induced morphological transformation of SHE cells and increased the catalase activity. DEHP was more potent than clofibrate and tiadenol in both inducing catalase and morphological transformation, while MEHP seemed more potent than DEHP in inducing catalase, but not morphological transformation, 2,4,5-T and 2,4-D did not induce morphological transformation, but 2,4,5-T was more potent than clofibrate in increasing the catalase activity. These results show that several HPPs induce morphological transformation of SHE cells and an increase in the catalase activity. There is, however, no direct connection between these two parameters, as seen from the results of 2,4,5-T. The tumor promoter TPA, and the metal salt nickel sulphate, induced morphological transformation of SHE cells without any appreciable increase in the catalase activity. These results further corroborate the dissociation between induction of morphological transformation and the increase in catalase activity.Abbreviations Clofibrate ethyl-2-(p-chlorophenox) isobutyrate - 2,4-D 2,4-dichlorophenoxy acetic acid - DEHP di(2-ethylhexyl)phthalate - HPP hepatic peroxisome proliferator - MEHP mono(2-ethylhexyl)phthalate - SHE Syrian hamster embryo - 2,4,5-T 2,4,5-trichlorophenoxy acetic acid - tiadenol di(hydroxyethylthio)-1,10-decane     

Sequence- and position-dependent tagging protects extracellular-regulated kinase 3 protein from 26S proteasome-mediated degradation

Extracellular-regulated kinase 3, an atypical member of the mitogen-activated protein kinase subfamily of extracellular-regulated kinases, was originally identified in 1991. Little is known about the biochemical properties, regulation, and biological functions of this protein kinase, partially due to the unstable nature of endogenous and low ectopical expression level of the protein. Here, we report that a single C-terminal c-myc tag increases the half-life of ectopic expressed tagged extracellular-regulated kinase 3 approximately four times compared to the reported 30 min half-life time for the endogenous protein and ectopic expressed extracellular-regulated kinase 3 deprived of its c-myc tag. These findings indicate that this C-terminal tag stabilizes the extracellular-regulated kinase 3. The stabilizing effect of the C-terminal c-myc tag is observed in all cell types tested, but is position- and tag sequence-dependent as neither N-terminal c-myc tag nor C-terminal HA tag stabilize the protein. The c-myc tag on extracellular-regulated kinase 3 did not interfere with its kinase activity, nor did it abrogate its ability to interacts with its bona fide substrate mitogen-activated protein kinase-activated protein kinase 5, indicating that tagging did not alter the known biological properties of the protein. Stabilization of the tagged extracellular-regulated kinase 3 protein probably results from reduced ubiquitination. In conclusion, position and sequence specific tagging should provide an easy and useful tool to generate a more stable protein that can functionally substitute the endogenous unstable protein. A stabilized variant may facilitate studies on the biological role of the protein.     

The connexin gene family in mammals

Unannotated mammalian genome databases (dog, cow, opossum) were searched for candidate connexin genes, using sequences from annotated genomes (man, mouse). All 18 'multi-species' connexin genes, i.e., orthologs of connexin26 , 29/31.3 (duplicated in opossum), 30, 30.2/31.9, 30.3, 31, 31.1, 32, 36, 37, 39/40.1, 40, 43, 45, 44/46, 47, 50, and 57/62 , were found in dog, cow and opossum. Connexin25 and 58 have been considered specific for man, but evident orthologs of connexin25 were found in dog, cow and opossum, and orthologs of connexin58 were found in dog and cow. Moreover, a connexin43 -like sequence (approx. 80% identical to connexin43 ) was found in man, chimpanzee, dog and cow. In the three former species, the sequences were located on the X chromosome. In man, chimpanzee and cow, there were stop codons in all reading frames; these sequences are therefore judged as pseudogenes, called here Cx43pX . In the dog, the sequence contained an open reading frame for a protein of 35.7 kDa (connexin35.7). We suggest that these sequences are orthologs of connexin33 , previously considered as a rodent-specific connexin gene. Thus, connexin25 , 33 and 58 are not species-specific genes. However, the opossum may possess a candidate, connexin39.2 , without obvious orthologs in other mammals. Furthermore, pseudogenes of primate connexin31.3 and opossum connexin35 (one of the two orthologs of primate connexin31.3) were detected. These results suggest that the structure of the mammalian connexin gene family should be revised, especially with regard to the so-called 'species-specific' connexins .     

One dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) induces tumours in Min/+ mice by truncation mutations or LOH in the Apc gene

The C57BL/6J-Min/+ (multiple intestinal neoplasia) mouse has a heterozygous nonsense Apc(Min) (adenomatous polyposis coli) mutation, and numerous adenomas spontaneously develop in the intestine. Neonatal exposure of Min/+ mice to the food carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (one injection of 50mg/kg) increased the number of small intestinal tumours about three- and two-fold, respectively. The number of colonic tumours was only increased in males. We examined whether the wild-type Apc allele was affected in intestinal tumours induced by either PhIP or IQ. In spontaneously formed and in IQ-induced small intestinal and colonic tumours from these mice, the main mechanism for tumour induction was loss of wild-type Apc allele, i.e. loss of heterozygosity (LOH). In contrast to the IQ-induced (84% LOH) and spontaneously (88% LOH) formed tumours, only 55% of the PhIP-induced small intestinal tumours from males showed LOH. Tumours that apparently had retained the wild-type Apc allele were further analysed for the presence of truncated Apc proteins by the in vitro synthesised protein (IVSP) assay. Truncated Apc proteins, indicating truncation mutations in exon 15 of the Apc gene, were detected in two of the 12 PhIP-induced tumours in segment 2 (codons 686-1217), and two of five IQ-induced tumours, one in segment 2 and the other in segment 3 (codons 1099-1693). Three of these four mutations, all in segment 2 of the Apc gene, were confirmed by sequencing. The PhIP-induced mutations were detected at codon 1125 (C deletion) and 1130 (G-T transversion), and the IQ-induced mutation was at codon 956 (C-T transition). Importantly, no truncated proteins were detected in tumours from unexposed mice with apparently retained wild-type Apc allele. These results show that one injection of either PhIP or IQ induces intestinal tumours in the Min/+ mice by inactivation of the wild-type Apc allele either by causing LOH or truncation mutations.