Three β-tubulin cDNAs from the parasitic nematode Haemonchus contortus

Experimental evidence indicates that tubulin is the site of action of the anthelmintic benzimidazoles. Furthermore, certain residues of β-tubulin seem to be critical for this mechanism. Although the benzimidazoles selectively affect nematode vs. mammalian β-tubulin, the molecular basis for this differential action is not known. To enhance our understanding of this phenomenon, and to provide the basis for investigating benzimidazole resistance in parasitic nematodes, we undertook the cloning of β-tubulin cDNAs from the ruminant parasite, Haemonchus contortus. We have cloned and sequenced three β-tubulin cDNAs from this organism, β12–16, β12–164, and β8–9. The first 2 differ at only 23 nucleotides, which give rise to 4 amino acid changes, β8–9 represents a different isotype class from the other two, since it differs extensively in the carboxyterminus. By comparing the sequences of these and other nematode β-tubulins with mammalian β-tubulins, several regions of consistent difference can be recognized; the functional significance of these regional differences has not been defined. Sequences very similar or identical to β8–9 and β12–16 are present in both benzimidazole-sensitive and benzimidazole-resistant populations of H. contortus. However, it appears that drug-resistant organisms may differ in the presence of a gene product which is closely related to β8–9.     

TcR-α/β CD4CD8 T Cells in Humans with the Autoimmune Lymphoproliferative Syndrome Express a Novel CD45 Isoform That Is Analogous to Murine B220 and Represents a Marker of Altered O-Glycan Biosynthesis

Autoimmune lymphoproliferative syndrome (ALPS), caused by inherited defects in apoptosis secondary to mutations in genes encoding Fas/CD95/APO-1 and Fas ligand (Fasl)/CD95L, is characterized by nonmalignant lymphadenopathy and splenomegaly, increased T cell receptor α/β⁺ CD4⁻CD8⁻ T cells (α/β⁺ double-negative T cells [α/β⁺-DNT cells]), autoimmunity, hypergammaglobulinemia, and cytokine abnormalities. The α/β⁺-DNT cells are immunophenotypically and functionally similar to α/β⁺-DNT cells that accumulate in lpr and gld mice, which bear genetic mutations in Fas and FasL. In these mice, α/β⁺-DNT cells express the B-cell-specific CD45R isoform B220. We show that α/β⁺-DNT cells of ALPS patients, with either Fas or FasL mutations, also express B220. In addition, also similar to LPR/gLD mice, they have an unusual population of B220-positive CD4⁺ T cells. B220 expression, together with our finding of characteristic lectin binding profiles, demonstrates that cell surface O-linked glycoproteins have undergone specific modifications, which may have consequences for lymphocyte trafficking, cell–cell interactions, and access to alternative apoptosis pathways.     

High and low potency interferon-α subtypes induce (2′–5′) oligoadenylate synthetase with similar efficiency

Three major subtypes of human interferon-α (IFN-α), isolated from virus-induced leukocytes, were compared for their antiviral and anticellular activities on one hand, and for their ability to induce (2′–5′) oligoadenylate synthetase on the other hand. One subtype, IFN-α_l, was found to have low specific antiviral (6.10⁶-5.10⁷ units/mg) and anticellular activities when measured on a variety of human cells. A second subtype, exhibiting an unusually high molecular weight (26,000) by SDS-polyacrylamide gel electrophoresis (IFN-α 26K), was found to have the highest known specific antiviral (8.10⁸-2.10⁹ units/ mg) and anticellular activities. Thus, these two subtypes of IFN-α differ by a factor of 330 and represent the two extremes in the antiviral scale on human cells. A third subtype, IFN-α₂, was tested as well and was found to have intermediate antiviral and anticellular activities. The ability of these three subtypes to induce (2′–5′) oligoadenylate synthetase in human cells was then measured. It was found that on a weight basis, the three subtypes were equally effective in inducing the enzyme. Since the level of (2′–5′) adenylate oligomers is affected also by the interferon-induced (2′–5′) phosphodiesterase, the ability of these subtypes to induce this enzyme was compared as well and was found to be very similar. We therefore conclude that the differences in potency between these IFN-α subtypes are not related to their ability to induce (2′–5′) oligoadenylate synthetase.     

Molecular characterization of novel interferon gamma receptor 1 isoforms in zebrafish (Danio rerio) and goldfish (Carassius auratus L.)

Interferon gamma (IFNγ) is a highly pleotropic pro-inflammatory and anti-viral cytokine that mediates its effects by binding to a receptor complex composed of interferon gamma receptors 1 and 2 (IFNGR1 and IFNGR2). Using gene synteny analysis, we identified a distinct isoform of the zebrafish IFNGR1. The two zebrafish IFNGR1 called here IFNGR1-1 and IFNGR1-2 were used to identify the respective cDNA sequences of the goldfish IFNGR1-1 and IFNGR1-2. Analysis of protein sequences revealed that all fish IFNGR1 species have potential JAK1 and STAT1 docking sites. Phylogenetically, teleost IFNGR1 proteins grouped separately from those of higher vertebrates. Q-PCR analysis revealed that while the constitutive mRNA levels of the two zebrafish IFNGR1 isoforms were comparable in different tissues examined, the goldfish IFNGR1-1 tissue expression was substantially higher than that of IFNGR1-2. Q-PCR analysis of goldfish immune cell populations revealed highest expression of both receptor isoforms in monocytes. Incubation of goldfish macrophages with recombinant goldfish IFNγ2 (rgIFNγ2) up-regulated expression of both IFNGR1-1 and IFNGR1-2, while treatment of cells with rgTNFα2 only increased the expression of IFNGR1-1. Treatment with rgTGFβ resulted in more modest increases in expression of both receptor isoforms only after prolonged treatment. In vitro binding studies indicated that rgIFNGR1-1 bound to rgIFNγ1 but not rgIFNγ2, while the rgIFNGR1-2 bound to rgIFNγ2. Thus, unlike mammals that have a single IFNGR1, cyprinid fish have two distinct IFNGR1 isoforms that preferentially bind corresponding ligands, IFNγ1 and IFNγ2, respectively, suggesting that the type II interferon system of these fish species is distinct from that of higher vertebrates.     

Molecular Characterization of a Distinct Begomovirus Species and its Associated Satellite DNA Isolated from <Emphasis Type="Italic">Malvastrum Coromandelianum</Emphasis> in China

A virus isolate Y160 was obtained from Malvastrum coromandelianum showing yellow vein symptoms in Baoshan, Yunnan province of China. The complete nucleotide sequence of DNA-A was determined, it contains 2747 nucleotides and has typical genomic organization of a begomovirus. Comparisons show that the total DNA-A of Y160 has the highest sequence identity (82.2%) with that of Malvastrum yellow vein virus-[Y47] (AJ457824), while less than 77.6% identities are found when compared with the other begomoviruses. The molecular data show that Y160 is a distinct begomovirus species, for which the name Malvastrum yellow vein Yunnan virus (MYVYNV) is proposed. Satellite DNA molecule (Y160β) was found to be associated with Y160 using the abutting primers beta0l and beta02 specific for DNAβ. Y160β consists of 1350 nucleotides, with a functional ORF (βC1) in complementary-sense DNA. Y160β has the highest sequence identity (73.5%) with DNAβ molecule associated with Malvastrum yellow vein virus-[Y47] (AJ421482).     

Relevance of IL-1 and TNF gene polymorphisms on interleukin-1β and tumor necrosis factor-α gastric mucosal production

We aimed to evaluate the influence of Helicobacter pylori infection and IL-1/TNF gene polymorphisms on interleukin (IL)-1β and tumor necrosis factor (TNF)-α gastric mucosal production. IL-1β and TNF-α levels in homogenized biopsy specimens taken from the antrum and corpus of 81 patients were measured by enzyme-linked immunosorbent assay. Genomic DNA was typed for the IL1B-511, IL1B+3954, variable number of tandem repeat (VNTR) IL1RN, TNFA-308, TNFA-238, LTA NcoI, and LTA Bsi gene polymorphisms by polymerase chain reaction, restriction fragment length polymorphism, and TaqMan assays. H. pylori infection and CagA/VacA antibody status were determined by Western blot. IL-1β and TNF-α protein levels were significantly higher in the gastric antrum of patients infected with H. pylori compared with uninfected patients [9.54 (5.07–16.28) vs. 4.55 (3.69–8.28) pg IL-1β/mg protein, p = 0.004, and 1.5 (0.7–2.71) vs. 0.63 (0.3–1.26) pg TNF-α/mg protein, p = 0.001]. Among H. pylori-infected individuals, carriers of the IL1RN*2 allele had significantly higher antrum mucosal IL-1β levels than noncarriers [15.97 (9.59–26.6) vs. 10.08 (7.72–13.33), p = 0.008]. No association between gastric mucosal TNF-α levels and genotypes of the TNFA and LTA gene polymorphisms was reported. Our results indicate that the VNTR polymorphism of the IL1RN gene influences IL-1β gastric mucosal production in patients infected with H. pylori.     

Proinflammatory cytokine–induced and chemical mediator–induced IL-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase–dependent pathway

The p38 mitogen-activated protein (MAP) kinase is activated in various cells by proinflammatory cytokines and environmental stresses. However, little is known about the role of p38 MAP kinase in proinflammatory cytokine– and chemical mediator–induced cytokine expression in human bronchial epithelial cells (BECs). In this study we examined the role of p38 MAP kinase in IL-8 expression in BECs to clarify the signal transduction pathway regulating IL-8 expression in BECs stimulated with tumor necrosis factor-α (TNF-α), IL-1α, and platelet-activating factor (PAF). We used TNF-α, IL-1α, and PAF as inducers for the analysis of the signal transduction pathway and determined IL-8 expression in BECs because TNF-α, IL-1α, and PAF are known to induce cytokine expression in BECs, and these proinflammatory cytokines and PAF are described to have a role in the production of allergic inflammation. The results showed that TNF-α, IL-1α, and PAF induced tyrosine phosphorylation of p38 MAP kinase in a dose- and time-dependent manner. The specific p38 MAP kinase inhibitor, SB 203580, completely inhibited TNF-α–, IL-1α–, or PAF-induced IL-8 protein and mRNA expression in BECs. These results indicated that p38 MAP kinase plays an important role in TNF-α–, IL-1α–, or PAF-activated signaling pathway, which regulates IL-8 expression in BECs. In addition, these results provide new evidence on a strategy for treatment of airway inflammation with the specific p38 MAP kinase inhibitor. (J Allergy Clin Immunol 1998;101:825-31.)     

IL-1 receptor–type expression in relation to atopy

Background: IL-1 has 2 receptors, type I (IL-1RI) and type II (IL-1RII), which have 2 forms each, membrane (m) and soluble (s). When IL-1 binds to mIL-1RI, the active receptor, an inflammatory response is initiated, which does not occur when IL-1 binds to mIL-1RII, the decoy receptor. Both sIL-1RI and sIL-1RII function as IL-1–mopping mechanisms. We hypothesized that the ratio of active (mIL-1RI) to inactive (mIL-1RII, sIL-1RI, and sIL-1RII) receptors is important in determining the amount of inflammation produced in allergic reactions. Objective: Our aim was to compare the concentrations of mIL-1RI and mIL-1RII on cultured PBLs and sIL-1RI, sIL-1RII, and IL-1β in sera and supernatants of cultured PBMCs from atopic and nonatopic subjects. Methods: The membrane receptors, soluble receptors, and IL-1β concentrations were measured by ELISA with specific mAbs. Results: Although there was no difference in the level of serum IL-1β between the 2 groups, PBMCs from atopic persons spontaneously secreted higher levels of IL-1β than those from nonatopic donors (P < .05). PBLs from atopic subjects compared with those from nonatopic individuals expressed higher mIL-1RI (P < .0001) and mIL-1RII (P < .05). Levels of both the soluble receptors from both serum (P < .0001) and PBMCs (P < .05) of nonatopic donors were higher than those found in atopic donors. Conclusion: This augmentation of mIL-1RI concomitant with a reduction in soluble receptors may be an important contributory factor to the inflammation that occurs with allergen exposure. (J Allergy Clin Immunol 1999;103:1100-7.)     

Effect of deoxymannojirimycin and Brefeldin A onYersinia pseudotuberculosisinvasin–eukaryotic cell interaction

The cell surface receptors of theYersinia pseudotuberculosisinvasin protein are theα_3–6β₁integrins. Invasin and the extracellular matrix protein fibronectin bind to the same or closely located sites onα₅β₁integrin, although invasin bind with a much greater affinity. Invasin-integrin interaction promotes bacterial penetration into eukaryotic cells. Binding of fibronectin to its integrin receptor seems to be dependent on terminal oligosaccharides processing. In this paper, we have examined the effect of 1-deoxymannojirimycin (dMNJ), an inhibitor of Golgiα-mannosidases involved in processing of N-glycan precursors, and of Brefeldin A (BFA), a natural product of fungi which has profound effects on the structure and function of the Golgi apparatus, on invasin–cell interaction. We found that unlike fibronectin, the interaction of invasin with cells was resistance to dMNJ. However, preincubating cells with BFA caused a dose dependent inhibition of invasin-mediated cell entry, while cell invasion bySalmonella typhimuriumwas not affected.     

Arginine vasopressin prevents amyloid β protein-induced impairment of long-term potentiation in rat hippocampus in vivo

Amyloid β protein (Aβ) is thought to be responsible for the loss of memory in Alzheimer's disease (AD). A significant decrease in [Arg⁸]-vasopressin (AVP) in the AD brain has been found. However, it is unclear whether the decrease in AVP is involved in Aβ-induced impairment of memory and whether AVP can protect against Aβ-induced neurotoxicity. The present study examines the effects of intracerebroventricular (i.c.v.) injection of AVP on hippocampal long-term potentiation (LTP), a synaptic model of memory, and investigates the potential protective function of AVP in Aβ-induced LTP impairment. The results showed that (1) i.c.v. injection of different concentrations of AVP or Aβ_25–35 did not affect the baseline field excitatory postsynaptic potentials (fEPSPs); (2) AVP administration alone induced a significant increase in HFS-induced LTP, while Aβ_25–35 significantly suppressed HFS-induced LTP; (3) Aβ_25–35-induced LTP suppression was significantly prevented by the pretreatment with AVP; (4) paired-pulse facilitation did not change after separate application or co-application of AVP and Aβ_25–35. These results indicate that AVP can potentiate hippocampal synaptic plasticity and dose-dependently prevent Aβ_25–35-induced LTP impairment. Thus, the present study provides further insight into the mechanisms by which Aβ impairs synaptic plasticity and suggests an important approach in the treatment of AD.     

Characterization of three novel β-defensin antimicrobial peptides in rainbow trout (Oncorhynchus mykiss)

An initial bioinformatics investigation followed by cloning and sequencing analysis, has led to the identification of three novel members (omDB-2, omDB-3, omBD-4) of the β-defensin family in rainbow trout (Oncorhynchus mykiss). The contiguous sequences could be translated to give predicted peptides of 62 (omDB-2), 63 (omDB-3) and 68 (omDB-4) amino acids (aa) in length, with mature peptides of 43 (omDB-2), 39 (omDB-3) and 42 (omDB-4) aa, with no obvious proregion present. Analysis of the gene organization found that all three new genes contained three exons divided by two introns, as seen in defensin genes of other fish species. Constitutive expression of all the trout defensins was detected by RT-PCR in a wide range of mucosal and systemic tissues from healthy fish, with omDB-3 and omDB-4 showing the highest expression levels. Following bacterial challenge in vivo, the defensin genes were induced at the three mucosal sites examined (skin, gill, gut), with levels of omDB-2 and omDB-3 increased some 16-fold in gut and gill respectively. Using polyinosinic polycytosinic RNA (polyI:C) as a viral mimic, all of the four trout β-defensin genes were induced in head kidney primary leucocyte cultures at 4 h post-stimulation, with omDB-1 and omDB-3 particularly highly expressed. These data suggest that β-defensins are likely an important component of the innate defences of fish, and reveal an added level of antimicrobial peptide complexity in fish to that known previously.     

The detection by immunofluorescence of distinct cell populations producing interleukin-1α and interleukin-1β in activated human peripheral blood

An immunofluorescent staining method using specific monoclonal antibodies was used to detect IL-1α and IL-1β in individuals cells in stimulated human peripheral blood. No staining was seen in unstimulated cells but intense, maximal staining of approximately 5% of the cells was seen 20–24 h after activation with PHA/PMA. The large irregularly shaped stained cells were surrounded by smaller unstained cells with lymphocyte-like morphology. By 44 h post activation a few cells only showed weak staining. The staining pattern was different for the two molecules studied, with a granular pattern for IL-1α staining and diffuse cytoplasmic staining for IL-1β. Staining post-activation could be abolished by preincubation of the monoclonal antibody with the appropriate recombinant IL-1, but not by pre-incubation with the other IL-1 type.When both anti-IL-1α and anti-IL-1β were used together two populations of cells were identified; one had the granular staining as seen with anti-IL-1α alone and the other had the diffuse staining pattern as seen with anti-IL-1β. The percentage of cells showing bright staining with anti-IL-α and anti-IL-1β together was approximately equal to the sum of the percentage of cell staining for IL-1α or IL-1β alone. This study demonstrates a method for the detection of individual IL-1α- and IL-1β- producing cells and suggests that in activated human peripheral blood IL-1α and IL-1β are produced by separate populations of cells.     

Patterns of Chromosomal Imbalances in Advanced Urinary Bladder Cancer Detected by Comparative Genomic Hybridization

To identify genetic changes linked to bladder cancer progression we analyzed 90 invasive transitional cell carcinomas (37 pT1 and 53 pT2–4) by comparative genomic hybridization. The most frequent alterations included 1q+ (37%), 5p+ (24%), 6q− (19%), 8p− (29%), 8q+ (37%), 9p− (31%), 9q− (23%), 11p− (24%), 11q− (22%), 17q+ (29%), and 20q+ (28%). Interestingly, there were three groups of alterations that frequently occurred together (9p− and 11q13+/20q+ and 11q13+ or 17q+/1q+ and 3p+ or 11q−). These loci might carry genes that interact with each other in specific molecular pathways. There were remarkable genetic similarities between minimally and deeply invasive tumors of different histological grades, including a similar number of aberrations per tumor and an equal frequency of most individual alterations. However, deletions of 5q, 6q, and 15q and gains of 5p, 7p, and Xq were significantly more frequent in pT2–4 than in pT1 carcinomas. These loci may harbor genes that are important for bladder cancer progression.     

TLR2 and the NLRP3 inflammasome mediate IL-1β production in Prevotella nigrescens-infected dendritic cells

Prevotella nigrescens is an oral pathogen that is frequently observed in the subgingival plaque of periodontitis patients. Interleukin-1β (IL-1β) is known to be involved in the immunopathology of periodontal diseases and has been implicated in the destruction of bone. In this study, we investigated the mechanism of IL-1β production by P. nigrescens in murine bone marrow-derived dendritic cells (BMDCs). Our results showed that a host receptor, Toll-like receptor 2 (TLR2), but not TLR4 is required for pro-IL-1β induction and nucleotide-binding oligomerization domain like receptor pyrin domain containing 3 (NLRP3) priming in BMDCs in response to P. nigrescens and activation of the NLRP3 inflammasome is necessary for processing of pro-IL-1β into mature IL-1β. In addition, an inhibitor assay revealed that production of reactive oxygen species, P2X₇R activity, and release of cathepsin B are involved in IL-1β production in BMDCs in response to P. nigrescens.     

Higher Risk of Mismatch Repair-Deficient Colorectal Cancer in α1-Antitrypsin Deficiency Carriers and Cigarette Smokers

Microsatellite instability (MSI) is a genomic alteration observed in 15–30% of colorectal cancer (CRC). Two MSI phenotypes have been defined for CRC: MSI-H is characterized by MSI at ≥30% of the examined loci and MSI-L by MSI at 1–30% of the loci. An absence of MSI at any examined loci has been defined as a microsatellite stable (MSS) phenotype. Current data suggest the majority of MSI tumors are the result of defective DNA mismatch repair (MMR). In this study, we have determined the α₁-antitrypsin deficiency carrier (α₁ATD-ht) status of 161 CRC patients whose MSI phenotype and protein expression states had previously been determined. Cases were selected to enrich a larger number of MSI-H cases. Among 51 CRC patients with MSI-H tumors, the α₁ATD-ht rate was 21.6%; among 110 patients with MSI-L/MSS tumors, the rate was 9.1% (MSI-H vs MSI-L/MSS, P = 0.02); and among the 191 population-based controls the α₁ATD-ht rate was 9.4% (MSI-H vs controls, P = 0.02). The estimated relative risk of having MSI-H CRC among α₁ATD-ht was 3.1 after adjusting for age, gender, and smoking history. The risk of having MSI-H CRC among current and past smokers was 6.6 and 2.7, respectively. Patients who were α₁ATD-ht and smoked had a 20-fold increased risk of developing an MSI-H CRC compared to nonsmokers who were homozygous normal at the α₁ATD locus. Our findings suggest an etiologic link between α₁ATD alleles and development of CRC with defective MMR, and a synergistic effect between smoking and α₁ATD allele in the development of MSI-H CRC.