Phenotypic characterisation of porcine counterparts of human NK cell populations--implications for pre-clinical studies

The phenotype of cryopreserved crossbred porcine PBMC, with special emphasis on NK cell related surface markers, was characterised. The phenotype expressed prior to stimulation with rHu IL2 and rHu IL12 was related to the in vitro cytotoxic capacity estimated in a 4-h or 21-h 51Cr-release assay. PBMC incubated in growth medium without addition of cytokines were used to investigate the spontaneous cytotoxic capacity. The results of this study suggest that crossbred porcine PBMC comprise a specific subset expressing the phenotype CD2+CD3-CD4-CD8+SWC3-CD16+CD21-. No spontaneous cytotoxicity of the PBMC could be estimated, but the expression of CD16 seems to be a basic marker of the cytokine induced cytotoxic activity. This study suggests that cryopreserved porcine PBMC can be used when possible influences on the porcine NK cell system are investigated in relation to disease models conducted experimentally in crossbred pigs.     

Characterization of rhesus macaque natural killer activity against a rhesus-derived target cell line at the single-cell level

Natural killer (NK) cells and NK cell activities in the rhesus macaque have been incompletely characterized. Using a recently developed rhesus NK target cell line with down-regulated MHC-I (B116Lo) as stimulators and FACS-sorted cells as effectors in a 4-h [51Cr]-release assay we showed that the CD3-CD8lo subpopulation is the primary effector population for NK cell-mediated cytolysis. The majority of these cells co-express CD16, CD11b, NKG2D, and NKp46. To evaluate functional activity at the individual cell level, we employed intracellular cytokine staining and a flow cytometric assay for degranulation, based on cell surface CD107a expression. Flow cytometric analysis revealed that a greater proportion of NK cells degranulated than produced cytokines in response to B116Lo stimulation; the frequency of CD107a-expressing cells within the total NK cell population ranging from 5 to 39%. Somewhat surprisingly, we did not find a significant correlation between lysis, measured by [51Cr]-release assay, and the size of the degranulating NK cell population, implying that additional mechanisms may regulate lytic activity. Use of these approaches should facilitate an improved understanding of NK activity in the rhesus macaque.     

Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating

Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.     

Combined ACTH and glucocorticoid treatment improves survival and organ maturation in premature newborn calves

Glucocorticoids play an important role in prenatal organ maturation in many species. In humans, maternal treatment with synthetic glucocorticoids improves neonatal adaptation of prematurely born infants. In cows, pre-term calf survival is improved following a single maternal glucocorticoid administration. We hypothesized that stimulation of endogenous cortisol secretion by adrenocorticotropin (ACTH) treatment combined with maternal dexamethasone treatment, would be even more efficient in stimulating organ maturation in the prematurely delivered calf. Three groups of premature calves were delivered by caesarian section at 90% of gestation length from dams which were either untreated or injected with dexamethasone before delivery, combined with either prenatal or postnatal ACTH treatment to the calf. During the first 24h after birth, thermoregulation, blood chemistry, liver values and organ weights were recorded. In the untreated calves, survival was significantly correlated with blood oxygenation, sodium and calcium levels at the moment of birth. There were marked maturational effects of the treatments on body temperature regulation, blood acid-base status, oxygenation, glucose, insulin, IGF-1 levels, weight of the heart, liver, gastrointestinal tract and thymus weight. For many of the measured metabolic, endocrine and organ weight parameters, the intrauterine ACTH treatment was associated with improved values relative to the postnatal ACTH treatment, which appeared to have no immediate effect on calf viability. In conclusion, the premature calf delivered by caesarian section at 90% of gestation length showed blood chemistry, metabolic, endocrine and organ growth characteristics that indicated severe prematurity. However, the maturation of organ function in newborn premature calves following maternal glucocorticoid injections was further enhanced if is was preceded by intra-fetal injections of ACTH.     

Characterization of the novel Fusobacterium nucleatum plasmid pKH9 and evidence of an addiction system

Fusobacterium nucleatum is an important oral anaerobic pathogen involved in periodontal and systemic infections. Studies of the molecular mechanisms involved in fusobacterial virulence and adhesion have been limited by lack of systems for efficient genetic manipulation. Plasmids were isolated from eight strains of F. nucleatum. The smallest plasmid, pKH9 (4,975 bp), was characterized and used to create new vectors for fusobacterial genetic manipulation. DNA sequence analysis of pKH9 revealed an open reading frame (ORF) encoding a putative autonomous rolling circle replication protein (Rep), an ORF predicted to encode a protein homologous to members of the FtsK/SpoIIIE cell division-DNA segregation protein family, and an operon encoding a putative toxin-antitoxin plasmid addiction system (txf-axf). Deletion analysis localized the pKH9 replication region in a 0.96-kbp fragment. The pKH9 rep gene is not present in this fragment, suggesting that pKH9 can replicate in fusobacteria independently of the Rep protein. A pKH9-based, compact Escherichia coli-F. nucleatum shuttle plasmid was constructed and found to be compatible with a previously described pFN1-based fusobacterial shuttle plasmid. Deletion of the pKH9 putative addiction system (txf-axf) reduced plasmid stability in fusobacteria, indicating its addiction properties and suggesting it to be the first plasmid addiction system described for fusobacteria. pKH9, its genetic elements, and its shuttle plasmid derivatives can serve as useful tools for investigating fusobacterial properties important in biofilm ecology and pathogenesis.     

Patterns of nitrogen accumulation and cycling in riparian floodplain ecosystems along the Green and Yampa rivers

Patterns of nitrogen (N) accumulation and turnover in riparian systems in semi-arid regions are poorly understood, particularly in those ecosystems that lack substantial inputs from nitrogen fixing vegetation. We investigated sources and fluxes of N in chronosequences of riparian forests along the regulated Green River and the free-flowing Yampa River in semi-arid northwestern Colorado. Both rivers lack significant inputs from N-fixing vegetation. Total soil nitrogen increased through time along both rivers, at a rate of about 7.8 g N m^–2 year^–1 for years 10–70, and 2.7 g N m^–2year^–1 from years 70–170. We found that the concentration of N in freshly deposited sediments could account for most of the soil N that accumulated in these floodplain soils. Available N (measured by ion exchange resin bags) increased with age along both rivers, more than doubling in 150 years. In contrast to the similar levels of total soil N along these rivers, N turnover rates, annual N mineralization, net nitrification rates, resin-N, and foliar N were all 2–4 times higher along the Green River than the Yampa River. N mineralization and net nitrification rates generally increased through time to steady or slightly declining rates along the Yampa River. Along the Green River, rates of mineralization and nitrification were highest in the youngest age class. The high levels of available N and N turnover in young sites are not characteristic of riparian chronosequences and could be related to changes in hydrology or plant community composition associated with the regulation of the Green River.     

The effect of pH on the oxidation of bovine serum albumin by hypervalent myoglobin species

Bovine serum albumin (BSA) was used as a probe for the oxidation of proteins by hypervalent myoglobin species in solutions with pH from 5.3 to 7.7. The reaction between perferrylmyoglobin, *MbFe(IV)=O, and BSA was studied by activating metmyoglobin with equimolar amounts of hydrogen peroxide in the presence of BSA. A minor pH dependence was observed as judged from the formation of BSA-centered radicals, which were monitored at room temperature by electron spin resonance spectroscopy, and the formation of dityrosine. The reaction between ferrylmyoglobin, MbFe(IV)=O, and BSA was pH-dependent. BSA-centered radicals and dityrosine were formed in low levels at neutral pH and increased at low pH to the same levels as observed in the reaction of *MbFe(IV)=O with BSA. The present results demonstrate that protein-centered radicals can be formed from the non-radical MbFe(IV)=O under mildly acidic conditions, and this should be taken into account when considering oxidation in cellular compartments of low pH and in meat-related products.     

The crystal structure of the glutathione S-transferase-like domain of elongation factor 1Bgamma from Saccharomyces cerevisiae

The crystal structure of the N-terminal 219 residues (domain 1) of the conserved eukaryotic translation elongation factor 1Bgamma (eEF1Bgamma), encoded by the TEF3 gene in Saccharomyces cerevisiae, has been determined at 3.0 A resolution by the single wavelength anomalous dispersion technique. The structure is overall very similar to the glutathione S-transferase proteins and contains a pocket with architecture highly homologous to what is observed in glutathione S-transferase enzymes. The TEF3-encoded form of eEF1Bgamma has no obvious catalytic residue. However, the second form of eEF1Bgamma encoded by the TEF4 gene contains serine 11, which may act catalytically. Based on the x-ray structure and gel filtration studies, we suggest that the yeast eEF1 complex is organized as an [eEF1A.eEF1Balpha.eEF1Bgamma]2 complex. A 23-residue sequence in the middle of eEF1Bgamma is essential for the stable dimerization of eEF1Bgamma and the quaternary structure of the eEF1 complex.