The buffering power of plasma in brown bullhead (Ameiurus nebulosus)

Brown bullhead (Ameiurus nebulosus) blood plasma was found to exhibit an unusually high non-bicarbonate buffer capacity (beta) in relation to that of other teleost fish. In brown bullhead, the non-bicarbonate buffer capacity of plasma (beta(plasma)), at -5.72 +/- 0.34 mmol l(-1) pH unit(-1) (mean +/- S.E.M., N=30), constituted 37% of whole blood beta and was 2.5 times higher than beta(plasma) in rainbow trout (-2.33 +/- 0.42 mmol l(+/-1) pH unit(-1); N=7). The strong buffering power of bullhead plasma was not the result of unusually high plasma protein levels. Size separation chromatography in conjunction with a spectrophotometric assay for buffering capacity were used to isolate a plasma fraction of high buffering power. SDS-polyacrylamide gel electrophoresis revealed that this fraction contained four proteins, but was dominated by a protein of approximately 68-70 kDa molecular mass. On the basis of the amino acid composition of this fraction, the dominant protein was identified as albumin. In comparison to other fish albumins, bullhead albumin appears to be histidine-rich (6.7%). Thus, the unusually high non-bicarbonate buffer capacity of bullhead plasma appears to stem from the presence in the plasma of a histidine-rich albumin.     

Half pint regulates alternative splice site selection in Drosophila

Alternative splicing is used by metazoans to increase protein diversity and to alter gene expression during development. However, few factors that control splice site choice in vivo have been identified. Here we describe a factor, Half pint (Hfp), that regulates RNA splicing in Drosophila. Females harboring hypomorphic mutations in hfp lay short eggs and show defects in germline mitosis, nuclear morphology, and RNA localization during oogenesis. We find that hfp encodes the Drosophila ortholog of human PUF60 and functions in both constitutive and alternative splicing in vivo. In particular, hfp mutants display striking defects in the developmentally regulated splicing of ovarian tumor (otu). Furthermore, transgenic expression of the missing otu splice form can rescue the ovarian phenotypes of hfp.     

Changes in Nitrogen-Fixing and Ammonia-Oxidizing Bacterial Communities in Soil of a Mixed Conifer Forest after Wildfire

This study was undertaken to examine the effects of forest fire on two important groups of N-cycling bacteria in soil, the nitrogen-fixing and ammonia-oxidizing bacteria. Sequence and terminal restriction fragment length polymorphism (T-RFLP) analysis of nifH and amoA PCR amplicons was performed on DNA samples from unburned, moderately burned, and severely burned soils of a mixed conifer forest. PCR results indicated that the soil biomass and proportion of nitrogen-fixing and ammonia-oxidizing species was less in soil from the fire-impacted sites than from the unburned sites. The number of dominant nifH sequence types was greater in fire-impacted soils, and nifH sequences that were most closely related to those from the spore-forming taxa Clostridium and Paenibacillus were more abundant in the burned soils. In T-RFLP patterns of the ammonia-oxidizing community, terminal restriction fragments (TRFs) representing amoA cluster 1, 2, or 4 Nitrosospira spp. were dominant (80 to 90%) in unburned soils, while TRFs representing amoA cluster 3A Nitrosospira spp. dominated (65 to 95%) in fire-impacted soils. The dominance of amoA cluster 3A Nitrosospira spp. sequence types was positively correlated with soil pH (5.6 to 7.5) and NH₃-N levels (0.002 to 0.976 ppm), both of which were higher in burned soils. The decreased microbial biomass and shift in nitrogen-fixing and ammonia-oxidizing communities were still evident in fire-impacted soils collected 14 months after the fire.     

Gene expression analysis of host innate immune responses during Lethal H5N1 infection in ferrets

How viral and host factors contribute to the severe pathogenicity of the H5N1 subtype of avian influenza virus infection in humans is poorly understood. We identified three clusters of differentially expressed innate immune response genes in lungs from H5N1 (A/Vietnam/1203/04) influenza virus-infected ferrets by oligonucleotide microarray analysis. Interferon response genes were more strongly expressed in H5N1-infected ferret lungs than in lungs from ferrets infected with the less pathogenic H3N2 subtype. In particular, robust CXCL10 gene expression in H5N1-infected ferrets led us to test the pathogenic role of signaling via CXCL10's cognate receptor, CXCR3, during H5N1 influenza virus infection. Treatment of H5N1-infected ferrets with the drug AMG487, a CXCR3 antagonist, resulted in a reduction of symptom severity and delayed mortality compared to vehicle treatment. We contend that unregulated host interferon responses are at least partially responsible for the severity of H5N1 infection and provide evidence that attenuating the CXCR3 signaling pathway improves the clinical course of H5N1 infection in ferrets.     

Innate immunity mediated by TLR5 as a novel antiinflammatory target for cystic fibrosis lung disease

Novel therapies to target lung inflammation are predicted to improve the lives of people with cystic fibrosis (CF) but specific antiinflammatory targets have not been identified. The goal of this study was to establish whether TLR5 signaling is the key molecular pathway mediating lung inflammation in CF, and to determine whether strategies to inhibit TLR5 can reduce the damaging inflammatory response. The innate immune responses were analyzed in both airway epithelial cells and primary PBMCs from CF patients and matched controls. Additionally, 151 clinical isolates of Pseudomonas aeruginosa from CF patients were assessed for motility and capacity to activate TLR5. Blood and airway cells from CF patients produced significantly more proinflammatory cytokine than did control cells following exposure to the CF pathogens P. aeruginosa and Burkholderia cepacia complex (p < 0.001). Stimulation with pure TLR ligands demonstrated that TLR signaling appears to mediate the excessive cytokine production occurring in CF. Using complementary approaches involving both neutralizing Ab targeting TLR5 and flagellin-deficient bacteria, we established that inhibition of TLR5 abolished the damaging inflammatory response generated by CF airway cells following exposure to P. aeruginosa (p < 0.01). The potential therapeutic value of TLR5 inhibition was further supported by our demonstration that 75% of clinical isolates of P. aeruginosa retained TLR5 activating capacity during chronic CF lung infection. These studies identify the innate immune receptor TLR5 as a novel antiinflammatory target for reducing damaging lung inflammation in CF.     

Activation of gamma delta T cells by Borrelia burgdorferi is indirect via a TLR- and caspase-dependent pathway

Activation of the innate immune system typically precedes engagement of adaptive immunity. Cells at the interface between these two arms of the immune response are thus critical to provide full engagement of host defense. Among the innate T cells at this interface are gammadelta T cells. gammadelta T cells contribute to the defense from a variety of infectious organisms, yet little is understood regarding how they are activated. We have previously observed that human gammadelta T cells of the Vdelta1 subset accumulate in inflamed joints in Lyme arthritis and proliferate in response to stimulation with the causative spirochete, Borrelia burgdorferi. We now observe that murine gammadelta T cells are also activated by B. burgdorferi and that in both cases the activation is indirect via TLR stimulation on dendritic cells or monocytes. Furthermore, B. burgdorferi stimulation of monocytes via TLR, and secondary activation of gammadelta T cells, are both caspase-dependent.     

T regulatory cells contribute to the attenuated primary CD8+ and CD4+ T cell responses to herpes simplex virus type 2 in neonatal mice

The first weeks of life are characterized by immune tolerance and increased susceptibility to intracellular pathogens. The neonatal adaptive response to HSV is attenuated compared with adult control models in humans and mice. T Regulatory cells (Tregs) control autoimmunity and excessive immune responses to infection. We therefore compared Treg responses in the draining lymph nodes (LN) of HSV-infected neonatal and adult C57BL/6 mice with the effect of Treg depletion/inactivation by anti-CD25 (PC61) treatment before infection on Ag-specific T cell effector responses at this site. There was a small, but significant increase in the frequency of CD4(+)Foxp3(+) Tregs at day 3 postinfection (p.i.) in the LN of neonatal and adult mice, compared with age-matched mock-infected controls. Depletion of Tregs before HSV infection significantly enhanced HSV-specific CD8(+) T cell cytotoxicity in vivo, cell number, activation, and granzyme B expression 4 days p.i. only in neonatal mice, and significantly enhanced CD8(+) and CD4(+) T cell IFN-gamma responses in both infected adults and neonates. Treg depletion also reduced the titer of infectious virus in the draining LN and nervous system of infected neonates on days 2 and 3 p.i. Treg suppression of the neonatal CTL response p.i. with HSV was associated with increased expression of TGF-beta in the draining LN at day 4 p.i. compared with uninfected neonates, but IL-10 was increased in infected adults alone. These experiments support the notion that the newborn primary T cell effector responses to HSV are suppressed by Tregs.     

Babesia divergens: identification and characterization of BdHSP-20, a small heat shock protein

This study describes the identification and characterization of the Babesia divergens α-crystallin/small heat shock protein 20 (BdHSP-20). BdHSP-20 was recognized by the DG7 monoclonal antibody (DG7 mAb) originally produced by Precigout et al. [Precigout, E., Valentin, A., Carcy, B., Gorenflot, A., Nakamura, K., Aikawa, M., Schrevel, J. 1993. Babesia divergens: characterization of a 17-kDa merozoite membrane protein. Experimental Parasitology 77, 425-434] against B. divergens merozoites. We used DG7 mAb to immunoscreen a B. divergens cDNA library to clone the gene encoding the small heat shock protein. Bdhsp-20 is a single copy gene interrupted by one intron. The deduced gene product (BdHSP-20) clearly belongs to the α-crystallin family and shows significant homology to Babesia bovis, Plasmodiumfalciparum and Toxoplasma gondii sHSPs, with the highest degree of sequence identity around the catalytic domain. Nutritient stress (serum depletion) treatment of the parasites induced the upregulation of BdHSP-20 gene expression observed by semi-quantitative PCR and immunoprecipitation. This regulation pattern suggests that BdHSP-20 could probably be of importance for parasite survival in the case of environmental stress. BdHSP-20 has previously been shown to be highly conserved among different strains and antibodies against the protein drastically reduce parasitemia in vitro.