Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence

Infectious pancreatic necrosis virus (IPNV) is the causative agent of infectious pancreatic necrosis (IPN) disease in salmonid fish. Recent studies have revealed variation in virulence between isolates of the Sp serotype, associated with certain residues of the structural protein VP2. The isolates are also highly heterogenic in the coding region of the nonstructural VP5 protein. To study the involvement of this protein in the pathogenesis of disease, we generated three recombinant VP5 mutant viruses using reverse genetics. The "wild-type" recombinant NVI15 (rNVI15) virus is virulent, having a premature stop codon at nucleotide position 427, putatively encoding a truncated 12-kDa VP5 protein, whereas rNVI15-15K virus encodes a 15-kDa protein. Recombinant rNVI15-deltaVP5 virus contains a mutation in the initiation codon of the VP5 gene that ablates the expression of VP5. Atlantic salmon postsmolts were challenged to study the virulence characteristics of the recovered viruses in vivo. The role of VP5 in persistent infection was investigated by challenging Atlantic salmon fry with the recovered viruses, as well as with the low-virulence field strain Sp103 and a naturally occurring VP5-deficient mutant of Sp103. The results show that VP5 is not required for viral replication in vivo, and its absence does not alter the virulence characteristics of the virus or the establishment of persistent IPNV infection.     

Tissue tracking imaging for identifying the origin of idiopathic ventricular arrhythmias: a new role of cardiac ultrasound in electrophysiology

Several strategies for mapping ventricular outflow tract tachycardia have been reported as useful indices for differentiating between those originating from the right and the left side. Recently, tissue tracking imaging (TTI) has been demonstrated as a novel non-invasive modality for identifying the origin of outflow tract tachycardias. Tissue tracking imaging is an ultrasonographic technique that measures the myocardial motion amplitude towards the transducer in each region during systole, identifying regional myocardial displacement on the basis of myocardial velocities using color Doppler myocardial imaging principles. In this technique, the origin of the arrhythmia could be recognized as the site where the earliest color-coded signal (ECCS) appeared on the myocardium at the onset of the systole. In preliminary studies this modality was found to be useful in differentiating outflow tract ventricular tachycardias. ECCS was always found below or at the level of the pulmonary valve in all arrhythmias which could be ablated from the right ventricular outflow tract, while in those where the ECCS was above and close to the pulmonary valve it could be ablated from the left sinus of valsalva. These results indicate that TTI can provide detailed and accurate information on the arrhythmia origin of outflow tract tachycardia and may be useful for differentiating between an outflow tract tachycardia originating from the LV epicardium remote from the LSV and that from the LSV. Newer advances in echocardiographic technologies like high resolution, high frame rate real time three dimensional echocardiography with speckle tracking may further improve the precise localization of arrhythmias in the future.     

Impaired gastric acid secretion in mice with a targeted disruption of the NHE4 Na+/H+ exchanger

The NHE4 Na+/H+ exchanger is abundantly expressed on the basolateral membrane of gastric parietal cells. To test the hypothesis that it is required for normal acid secretion, NHE4-null mutant (NHE4-/-) mice were prepared by targeted disruption of the NHE4 (Slc9a4) gene. NHE4-/- mice survived and appeared outwardly normal. Analysis of stomach contents revealed that NHE4-/- mice were hypochlorhydric. The reduction in acid secretion was similar in 18-day-old, 9-week-old, and 6-month-old mice, indicating that the hypochlorhydria phenotype did not progress over time, as was observed in mice lacking the NHE2 Na+/H+ exchanger. Histological abnormalities were observed in the gastric mucosa of 9-week-old NHE4-/- mice, including sharply reduced numbers of parietal cells, a loss of mature chief cells, increased numbers of mucous and undifferentiated cells, and an increase in the number of necrotic and apoptotic cells. NHE4-/- parietal cells exhibited limited development of canalicular membranes and a virtual absence of tubulovesicles, and some of the microvilli had centrally bundled actin. We conclude that NHE4, which may normally be coupled with the AE2 Cl-/HCO3- exchanger, is important for normal levels of gastric acid secretion, gastric epithelial cell differentiation, and development of secretory canalicular and tubulovesicular membranes.     

Host limits to accurate human growth hormone production in multiple plant systems

Human growth hormone (hGH) is not only a valuable recombinant therapeutic protein for hormone deficiency indications, but is also an extensively characterized molecule both from recombinant bacterial systems and as circulating in humans. We describe the characterization of hGH produced in three different plant systems: tobacco cell culture, soy seed, and maize seed. The data indicate highest production in the maize seed system, with continued productivity over multiple generations, and when bred to a new host genotype for improved productivity. Purification indicated significant material of the correct structure from both plant cell culture and maize seed, with maize seed also showing correct activity relative to that produced by Escherichia coli. However, all systems showed some proteolyzed hGH, with data from gel electrophoresis, mass spectrometry, and peptide mapping localizing to a region of the protein also prone to cleavage in some other systems. Together, the data indicate the dependence of recombinant protein accumulation on posttranslational processes in different host systems.     

NAK is recruited to the TNFR1 complex in a TNFalpha-dependent manner and mediates the production of RANTES: identification of endogenous TNFR-interacting proteins by a proteomic approach

Tumor necrosis factor alpha (TNFalpha) is a proinflammatory cytokine with pleiotropic immunological and biological activities. TNFalpha signaling is triggered by the engagement of soluble TNFalpha to two types of cell surface receptors, TNFR1 and TNFR2. This recruits cytosolic proteins to the intracellular domains of the receptors and initiates signaling to downstream effectors. In this study, we used a proteomic approach to identify these cytosolic proteins from affinity-purified, endogenous TNFalpha.TNFR complexes in human myelomonocytic U937 cells. Seven proteins were identified, including TRADD, TRAP2, and TRAF2, which are three proteins known to be recruited to TNFalpha receptors. NAK, RasGAP3, TRCP1, and TRCP2 were also identified. We further showed that NAK is recruited to TNFR1 in a temporally regulated and TNFalpha-dependent manner and that it mediates the TNFalpha-induced production of the chemokine RANTES (regulated on activation normal T cell expressed and secreted). These data demonstrate that NAK is a component of the TNFalpha.TNFR1 signaling complex and confirm the physiological role of NAK in the TNFalpha-mediated response.     

Membrane Curvature Induction and Tubulation Are Common Features of Synucleins and Apolipoproteins

Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly. Here, we find that α-synuclein, β-synuclein, and apolipoprotein A-1 have the conserved functional ability to induce membrane curvature and to convert large vesicles into highly curved membrane tubules and vesicles. The resulting structures are morphologically similar to those generated by amphiphysin, a curvature-inducing protein involved in endocytosis. Unlike amphiphysin, however, synucleins and apolipoproteins do not require any scaffolding domains and curvature induction is mediated by the membrane insertion and wedging of amphipathic helices alone. Moreover, we frequently observed that α-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that α-synuclein plays a role in vesicle trafficking and enhances endocytosis. Induction of membrane curvature must be under strict regulation in vivo; however, as we find it can also cause disruption of membrane integrity. Because the degree of membrane curvature induction depends on the concerted action of multiple proteins, controlling the local protein density of tubulating proteins may be important. How cellular safeguarding mechanisms prevent such potentially toxic events and whether they go awry in disease remains to be determined.     

The effect of osteoporosis on residual ridge resorption and masticatory performance in denture wearers

doi: 10.1111/j.1741‐2358.2011.00610.x The effect of osteoporosis on residual ridge resorption and masticatory performance in denture wearers Aim: To compare masticatory performance, masticatory efficiency and residual ridge resorption (RRR) in osteoporotic and non‐osteoporotic edentulous subjects after rehabilitation with complete dentures. Method: Thirty subjects fulfilling the inclusion criteria were enrolled from the patients visiting the Department of Prosthodontics for complete denture fabrication. Two groups consisting of control subjects (group I; N = 15) and osteoporotic subjects (group II; N = 15) were formed. Complete dentures satisfying certain criteria were fabricated for both groups. Masticatory performance and efficiency were measured 6 months after denture insertion. Areal measurements were taken on lateral cephalograms before and 6 months after denture fabrication. The data were then computed to analyse differences between groups I and II using SPSS statistical software version 15.0. Results: Six months after denture fabrication, the masticatory performance and efficiency were significantly higher (p < 0.001) for group I, with a significant decrease in maxillary and mandibular sagittal area seen in both groups. The rate of bone loss was more in group II compared with group I. Conclusion: Greater masticatory function was demonstrated by the non‐osteoporotic group, and the rate of RRR was more in the osteoporotic group compared with the normal group. In this pilot study, osteoporosis leads to greater RRR, decreased masticatory performance and efficiency in edentulous subjects 6 months after denture insertion. Screening for osteoporosis is suggested as a routine procedure for all edentulous subjects undergoing rehabilitation. Recall check‐ups for osteoporotic patients should be more frequent, and these patients may require more frequent denture remakes.     

Ammonia reduction with ornithine phenylacetate restores brain eNOS activity via the DDAH-ADMA pathway in bile duct-ligated cirrhotic rats

Ammonia is central in the pathogenesis of hepatic encephalopathy, which is associated with dysfunction of the nitric oxide (NO) signaling pathway. Ornithine phenylacetate (OP) reduces hyperammonemia and brain water in cirrhotic animals. This study aimed to determine whether endothelial NO synthase activity is altered in the brain of cirrhotic animals, whether this is associated with changes in the endogenous inhibitor, asymmetric-dimethylarginine (ADMA) and its regulating enzyme, dimethylarginine-dimethylaminohydrolase (DDAH-1), and whether these abnormalities are restored by ammonia reduction using OP. Sprague-Dawley rats were studied 4-wk after bile duct ligation (BDL) (n = 16) or sham operation (n = 8) and treated with placebo or OP (0.6 g/kg). Arterial ammonia, brain water, TNF-α, plasma, and brain ADMA were measured using standard techniques. NOS activity was measured radiometrically, and protein expression for NOS enzymes, ADMA, DDAH-1, 4-hydroxynonenol ((4)HNE), and NADPH oxidase (NOX)-1 were measured by Western blotting. BDL significantly increased arterial ammonia (P < 0.0001), brain water (P < 0.05), and brain TNF-α (P < 0.01). These were reduced significantly by OP treatment. The estimated eNOS component of constitutive NOS activity was significantly lower (P < 0.05) in BDL rat, and this was significantly attenuated in OP-treated animals. Brain ADMA levels were significantly higher and brain DDAH-1 significantly lower in BDL compared with sham (P < 0.01) and restored toward normal following treatment with OP. Brain (4)HNE and NOX-1 protein expression were significantly increased in BDL rat brain, which were significantly decreased following OP administration. We show a marked abnormality of NO regulation in cirrhotic rat brains, which can be restored by reduction in ammonia concentration using OP.