Identification of virus found in mouse lymphomas induced by HIX murine oncornavirus.

Lymphomas induced by pure HIX murine leukemia virus in two mouse strains contained large amounts of virus with amphotropic properties. Analysis of tumor derived virus purified by limiting dilution techniques indicated that its interference and neutralization spectra were essentially identical to parental HIX virus and different from eco- and xenotropic viruses. Examination of virus progeny from many individual foci induced by virus derived directly from lymphomas indicated that each infectious unit contained only HIX virus. No evidence of ecotropic virus presence was observed.     

Evolution of arthropod hemocyanins and insect storage proteins (hexamerins)

Crustacean and cheliceratan hemocyanins (oxygen-transport proteins) and insect hexamerins (storage proteins) are homologous gene products, although the latter do not bind oxygen and do not possess the copper- binding histidines present in the hemocyanins. An alignment of 19 amino acid sequences of hemocyanin subunits and insect hexamerins was made, based on the conservation of elements of secondary structure observed in X-ray structures of two hemocyanin subunits. The alignment was analyzed using parsimony and neighbor-joining methods. Results provide strong indications for grouping together the sequences of the 2 crustacean hemocyanin subunits, the 5 cheliceratan hemocyanin subunits, and the 12 insect hexamerins. Within the insect clade, four methionine- rich proteins, four arylphorins, and two juvenile hormone-suppressible proteins from Lepidoptera, as well as two dipteran proteins, form four separate groups. In the absence of an outgroup sequence, it is not possible to present information about the ancestral state from which these proteins are derived. Although this family of proteins clearly consists of homologous gene products, there remain striking differences in gene organization and site of biosynthesis of the proteins within the cell. Because studies on 18S and 12S rRNA sequences indicate a rather close relationship between insects and crustaceans, we propose that hemocyanin is the ancestral arthropod protein and that insect hexamerins lost their copper-binding capability after divergence of the insects from the crustaceans.      

Adaptation of a luciferase gene reporter and lac expression system to Borrelia burgdorferi

The development of new genetic systems for studying the complex regulatory events that occur within Borrelia burgdorferi is an important goal of contemporary Lyme disease research. Although recent advancements have been made in the genetic manipulation of B. burgdorferi, there still remains a paucity of basic molecular systems for assessing differential gene expression in this pathogen. Herein, we describe the adaptation of two powerful genetic tools for use in B. burgdorferi. The first is a Photinus pyralis firefly luciferase gene reporter that was codon optimized to enhance translation in B. burgdorferi. Using this modified reporter, we demonstrated an increase in luciferase expression when B. burgdorferi transformed with a shuttle vector encoding the outer surface protein C (OspC) promoter fused to the luciferase reporter was cultivated in the presence of fresh rabbit blood. The second is a lac operator/repressor system that was optimized to achieve the tightest degree of regulation. Using the aforementioned luciferase reporter, we assessed the kinetics and maximal level of isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent gene expression. This lac-inducible expression system also was used to express the gene carried on lp25 required for borrelial persistence in ticks (bptA). These advancements should be generally applicable for assessing further the regulation of other genes potentially involved in virulence expression by B. burgdorferi.     

Elevated ATP via enhanced miRNA-30b, 30c,and 30e downregulates the expression of CD73 in CD8+ T cells of HIV-infected individuals

CD8⁺ T cells play a crucial role against chronic viral infections, however, their effector functions are influenced by the expression of co-stimulatory/inhibitory receptors. For example, CD73 works with CD39 to convert highly inflammatory ATP to adenosine. However, its expression on T cells in the context of viral infections has not been well defined. Here, we analyzed the expression of CD73 on human T cells in a cohort of 102 HIV-infected individuals including those on antiretroviral therapy (ART), ART-naïve, and long-term non-progressors who were not on ART. We found that the frequency of CD73⁺ T cells was markedly lower among T cell subsets (e.g. naïve, effector or memory) in the peripheral blood of all HIV-infected individuals. Notably, CD73 was decreased at the cell surface, intracellular and gene levels. Functionally, CD8⁺CD73⁺ T cells exhibited decreased cytokine expression (TNF-α, IFN-γ and IL-2) upon global or antigen-specific stimulation and impaired expression of cytolytic molecules at the gene and protein levels. In contrast, CD8⁺CD73⁺ T cells expressed elevated levels of homing receptors such as CCR7, α4β7 integrin, which suggests a migratory advantage for these cells as observed in vitro. We also observed significant migration of CD73⁺CD8⁺ T cells into the cerebrospinal fluids of multiple sclerosis (MS) patients at the time of disease relapse. Moreover, we found that elevated levels of ATP in the plasma of HIV-infected individuals upregulates the expression of miRNA30b-e in T cells in vitro. In turn, inhibition of miRNAs (30b, 30c and 30e) resulted in significant upregulation of CD73 mRNA in CD8⁺ T cells. Therefore, we provide a novel mechanism for the downregulation of CD73 via ATP-induced upregulation of miRNA30b, 30c and 30e in HIV infection. Finally, these observations imply that ATP-mediated downregulation of CD73 mainly occurs via its receptor, P2X1/P2RX1. Our results may in part explain why HIV-infected individuals have reduced risk of developing MS considering the role of CD73 for efficient T cell entry into the central nervous system.     

Spatial and temporal patterns in the calling behavior of beluga whales,Delphinapterus leucas,in Cook Inlet,Alaska

Cook Inlet beluga whales (CIBs) are an endangered population residing in Cook Inlet, Alaska. We characterized the calling behavior of CIBs to improve our understanding of sounds produced by this population. Bottom‐moored hydrophones were deployed at Eagle Bay in summer 2009 and at Trading Bay in summer and winter 2009. CIB sounds were qualitatively analyzed and categorized as a whistle, pulsed call, or click train. A total of 4,097 calls were analyzed, and 66 unique whistle contours were identified. Whistles were quantitatively analyzed using a custom Matlab program. A chi‐square test showed the call category usage at Eagle Bay during summer 2009 and those at Trading Bay during summer 2009 and winter 2009–2010 differed significantly (P < 0.001). Pulsed calls were more common during summer months, and click trains within the frequency band (12.5 kHz) were more common in Eagle Bay. The variation in calling behavior suggests differences in habitat usage or in the surrounding environment, including background noise. With the proposed development projects in Cook Inlet and the potential increase in ambient noise level due to ocean acidification, it is important to understand how this endangered population uses sound, and what anthropogenic factors may influence that use.     

Ethylene production,cluster root formation,and localization of iron(III) reducing capacity in Fe deficient squash roots

Dicots and non-graminaceous monocots have the ability to increase root iron(III) reducing capacity in response to iron (Fe) deficiency stress. In squash (Cucurbita pepo L.) seedlings, Fe(III) reducing capacity was quantified during early vegetative growth. When plants were grown in Fe-free solution, the Fe(III) reducing capacity was greatly elevated, reached peak activity on day 4, then declined through day 6. Root ethylene production exhibited a temporal pattern that closely matched that of Fe(III) reducing capacity through day 6. On the 7th day of Fe deficiency, cluster root morphology developed, which coincided with a sharp increase in the root Fe(III) reducing capacity, although ethylene production decreased. Localization of Fe(III) reducing capacity activity was observed during the onset of Fe deficiency and through the development of the root clusters. It was noted that localization shifted from an initial pattern which occurred along the main and primary lateral root axes, excluding the apex, to a final localization pattern in which the reductase appeared only on secondary laterals and cluster rootlets. This revised version was published online in June 2006 with corrections to the Cover Date.     

Abrogation of ospAB constitutively activates the Rrp2‐RpoN‐RpoS pathway (sigmaN‐sigmaS cascade) in Borrelia burgdorferi

Molecular mechanisms underlying the reciprocal regulation of the two major surface lipoproteins and virulence factors of Borrelia burgdorferi, OspA and OspC, are not fully understood. Herein, we report that inactivation of the ospAB operon resulted in overproduction of OspC and many other lipoproteins via the constitutive activation of the Rrp2‐RpoN‐RpoS pathway. Complementing the ospAB mutant with a wild‐type copy of ospA, but not an ospA variant that lacks the lipoprotein signal sequence, restored normal regulation of the Rrp2‐RpoN‐RpoS pathway; these results indicate that the phenotype was not caused by spurious mutations. Interestingly, while most of the ospAB mutant clones displayed a constitutive ospC expression phenotype, some ospAB mutant clones showed little or no ospC expression. Further analyses revealed that this OspC‐negative phenotype was independent of abrogation of ospAB. While activation of the Rrp2‐RpoN‐RpoS pathway was recently shown to downregulate ospA, our findings suggest that reduction of OspA can also activate this pathway. We postulate that the activation of the Rrp2‐RpoN‐RpoS pathway and downregulation of OspA form a positive feedback loop that allows spirochaetes to produce and maintain a constant high level of OspC and other lipoproteins during tick feeding, a strategy that is critical for spirochaetal transmission and mammalian infection.     

Chemical characterization of lipopolysaccharide from Edwardsiella ictaluri, a fish pathogen

The chemical components of lipopolysaccharide (LPS) from the fish pathogen Edwardsiella ictaluri (Ed. ictaluri) were analyzed by SDS-PAGE, gas chromatography, and spectrophotometry, and compared with those of Salmonella typhimurium and Escherichia coli 0111:B4. Only four to five low molecular weight species of LPS from Ed. ictaluri were detected by silver staining after separation by polyacrylamide gel electrophoresis. The low molecular weight species, as well as a low sugar content, indicate that the LPS from Ed. ictaluri was of the rough type, compared with that of S. typhimurium and E. coli which were both of the smooth type LPS. Quantitatively, mannose was not a major sugar component in Ed. ictaluri, unlike S. typhimurium. Palmitic, palmitoleic, and cis-9,10-methylene-hexadecanoic acids were predominant fatty acids among the total cellular lipids of Ed. ictaluri. C14 fatty acids comprised 78% of the total in the LPS of this bacterium, with beta-hydroxy-myristate representing 55%. The results of this study suggest that the lipid A segment of the LPS molecule of Ed. ictaluri is similar to S. typhimurium and E. coli, at least with respect to fatty acid content; however, the core polysaccharide of E. ictaluri differs in that it has twice the heptose content.