Sequence diversity of the nucleoprotein gene of iris yellow spot virus (genus Tospovirus, family Bunyaviridae) isolates from the western region of the United States

Summary. Iris yellow spot virus (IYSV), a tentative virus species in the genus Tospovirus and family Bunyaviridae, is considered a rapidly emerging threat to onion production in the western United States (US). The present study was undertaken to determine the sequence diversity of IYSV isolates from infected onion plants grown in California, Colorado, Idaho, Oregon, Utah and Washington. Using primers derived from the small RNA of IYSV, the complete sequence of the nucleoprotein (NP) gene of each isolate was determined and the sequences compared. In addition, a shallot isolate of IYSV from Washington was included in the study. The US isolates of IYSV shared a high degree of sequence identity (95 to 99%) with one another and to previously reported isolates. Phylogenetic analyses showed that with the exception of one isolate from central Oregon and one isolate from California, all the onion and shallot isolates from the western US clustered together. This cluster also included onion and lisianthus isolates from Japan. A second distinct cluster consisted of isolates from Australia (onion), Brazil (onion), Israel (lisianthus), Japan (alstroemeria), the Netherlands (iris) and Slovenia (leek). The IYSV isolates evaluated in this study appear to represent two distinct groups, one of which largely represents isolates from the western US. Understanding of the population structure of IYSV would potentially provide insights into the molecular epidemiology of this virus.     

A micro cell culture method utilizing modified microscope slides for use in fluorescent antibody and enzyme immunoassays

Summary A technique for culturing small quantities of mammalian cells on modified microscope slides is described. The modified microscope slides were Bellco Glass, Inc., toxoplasmosis slides and the cell cultures used were early passage bovine embryonic lung cells and continuous cell lines of porcine and canine origins. The slide cell cultures were either uninfected or infected with selected viruses or the obligate intracellular protozoanEncephalitozoon caniculi for utilization in direct and indirect fluorescent antibody testing or in peroxidase antiperoxidase immunosorbant assays.     

Lack of protection in mice and necrotizing bronchointerstitial pneumonia with bronchiolitis in guinea pigs immunized with vaccines directed against the hsp60 molecule of Mycobacterium tuberculosis

In this study, the hsp60 and hsp70 heat shock protein antigens of Mycobacterium tuberculosis were tested as potential vaccine candidates, using purified recombinant protein antigens or antigens encoded in the form of a DNA plasmid vaccine. Guinea pigs vaccinated with a mixture of the two proteins showed no evidence of resistance to low-dose aerosol challenge infection and quickly developed severe lung damage characterized by necrotizing bronchointerstitial pneumonia and bronchiolitis. As a result, we turned instead to a DNA vaccination approach using a plasmid encoding the hsp60 antigen of M. tuberculosis. Although immunogenic in mice, vaccination with plasmid DNA encoding hsp60 was not protective in that model or in the guinea pig model and again gave rise to similar severe lung damage. This study seriously questions the safety of vaccines against tuberculosis that target highly conserved heat shock proteins.     

Assessment of fibronectin conformation adsorbed to polytetrafluoroethylene surfaces from serum protein mixtures and correlation to support of cell attachment in culture

Surfaces of polytetrafluoroethylene (PTFE) were exposed to buffered aqueous solutions containing radio-labeled human fibronectin ([125I]Fn), Fn/bovine serum albumin (BSA) binary mixtures of various ratios or whole human plasma dilutions for 1 h. Total adsorbed Fn and albumin adsorption following rinsing was quantified on this surface. 125I-labeled monoclonal antibodies against either the tenth type-III Fn repeat unit (containing the cell-binding RGDS integrin recognition motif) or the Fn amino-terminal domain were used to probe the accessibility of each of these respective Fn regions post-adsorption. Human umbilical vein endothelial cells (HUVECs) were cultured on PTFE surfaces pre-exposed to each of these protein adsorption conditions and compared to identical conditions on tissue culture polystyrene (TCPS). Fn adsorption to PTFE is dependent upon the concentration of albumin co-adsorbing from solution: albumin out-competes Fn for PTFE surface sites even at non-physiological Fn/HSA ratios 10-100-fold biased in Fn. Antibodies against Fn do not readily recognize Fn adsorbed on PTFE as the HSA co-adsorption concentration in either binary mixtures or in plasma increases, indicating albumin masking of adsorbed Fn. At Fn/HSA ratios rich in Fn (1:1, 1:100), albumin co-adsorption actually improves anti-Fn antibody recognition of adsorbed Fn. HUVEC attachment efficiency to PTFE after protein adsorption correlates with amounts of Fn adsorbed and levels of anti-Fn antibody recognition of Fn on PTFE, linking cell attachment to integrin recognition of both adsorbed Fn density and Fn adsorbed conformation on PTFE surfaces.     

Pentoxifylline treatment of mice with chronic pulmonary tuberculosis accelerates the development of destructive pathology

It is well established in animal models that production of the cytokine tumour necrosis factor-alpha (TNF-alpha) is essential to the proper expression of acquired specific resistance following infection with Mycobacterium tuberculosis. This gives rise to an apparent state of chronic disease which over the next 100-200 days is characterized by slowly worsening pathological changes in the lung. To determine whether continued TNF-alpha production was harmful during this phase mice were treated with a TNF-alpha inhibitor, pentoxifylline. It was observed that although this therapy did not alter the numbers of bacteria recovered from the lungs of the infected mice, tissue damage within the lung was accelerated. These data thus demonstrate that production of TNF-alpha, already known to be important during the early expression of resistance to tuberculosis, remains important and beneficial during the chronic stage of the disease.     

Inhibition of cellular responses to insulin in a rat liver cell line. A role for PKC in insulin resistance

The initial response of liver cells to insulin is mediated through exocytosis of Cl⁻ channel-containing vesicles and a subsequent opening of plasma membrane Cl⁻ channels. Intracellular accumulation of fatty acids leads to profound defects in metabolism, and is closely associated with insulin resistance. It is not known whether the activity of Cl⁻ channels is altered in insulin resistance and by which mechanisms. We studied the effects of fatty acid accumulation on Cl⁻ channel opening in a model liver cell line. Overnight treatment with amiodarone increased the fat content by ∼2-fold, and the rates of gluconeogenesis by ∼5-fold. The ability of insulin to suppress gluconeogenesis was markedly reduced indicating that amiodarone treatment induces insulin resistance. Western blot analysis showed that these cells express the same number of insulin receptors as control cells. However, insulin failed to activate exocytosis and Cl⁻ channel opening. These inhibitory effects were mimicked in control cells by exposures to arachidonic acid (15 μ m ). Further studies demonstrated that fatty acids stimulate the PKC activity, and inhibition of PKC partially restored exocytosis and Cl⁻ channel opening in insulin-resistant cells. Accordingly, activation of PKC with PMA in control cells potently inhibited the insulin responses. These results suggest that stimulation of PKC activity in insulin resistance contributes to the inhibition of cellular responses to insulin in liver cells.     

An Evaluation of Finger Pulse Volume as a Psychophysiological Measure of Anxiety

The present experiment explored the utility of finger pulse volume (FPV) as a measure of anxiety. Subjects were exposed to either a threatening or nonthreatening situation, and indices of physiological arousal (pulse rate (PR) and FPV) and self-report of anxiety (Affect Adjective Checklist (AACL)) were collected. Results indicated that FPV was responsive to changes in experimentally induced anxiety and significantly correlated with PR and AACL, although the strength of these relationships was not substantial. Relevance for psychophysiological theory and the clinical observation of anxiety is discussed.     

Separated fraternal twins: Resemblance for cognitive abilities

Thirty-four pairs of Swedish fraternal twins separated in the first 10 years of life were administered 12 tests of cognitive abilities. The average age of the twins was 59 years. Nineteen pairs were separeted before the first year of life and 26 pairs (76%) were separated by the age of 5 years. Moderate positive twin correlations were found for all tests, reaching significance for Raven's Progressive Matrices and Word Beginnings and Endings. A twin correlation of 0.52 emerged for the first principal component (a measure of general ability). Moderate correlations were found for Spatial Ability and for Verbal Ability/Perceptual Speed factors; for the Memory factor the correlation was lower. The effect of early environment on cognitive ability was assessed by analyzing the association between a degree of separation index and twin resemblance. The importance of the separation measures taken individually was also examined. Some significant effects were found, but these were consistently in a counterintuitive direction: Twins separated earlier weremore similar than those separated later.This research was supported in part by National Institute on Aging Grant AG-01592.