A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

Drosophila basement membrane procollagen IV. I. Protein characterization and distribution

A collagen was isolated from Drosophila E85, Schneider line 2L and Kc cell cultures. The purified protein was characterized and antibodies were raised against it. Immunofluorescence microscopy locates this material to the regions of basement membranes of Drosophila embryos, larvae, and adults. The molecules are mostly, or entirely, homotrimers of one polypeptide chain linked by interchain disulfide bonds. The partial amino acid sequences of a cyanogen bromide cleavage product of this chain are identical with a part of the virtual translation product of the Drosophila pro alpha 1(IV) nucleotide sequence that is reported in the accompanying paper. This gene is at Drosophila chromosome location 25C and was identified by the high homology of one part of it with the noncollagenous carboxyl terminus (NC1) of vertebrate type IV basement membrane collagens (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). In the electron microscope each molecule appears as a thread with a knob at one end, which contains the carboxyl peptide domains. The variation of flexibility of the thread was mapped along its length. Pulse-chase labeling of cell cultures showed that these molecules associate into disulfide-linked dimers and higher oligomers that can be partly separated by velocity sedimentation and are resolved by sodium dodecyl sulfate-agarose gel electrophoresis. Dimers and higher oligomers formed by overlap of the amino ends of molecules were found. Mild pepsin digestion of Drosophila embryos and larvae solubilized the corresponding disulfide-linked collagen molecules, and Staphylococcus aureus V8 protease peptide maps showed the identity of the collagen derived from animals and from cell cultures. Individual, native molecules have a sedimentation coefficient s20,w = 4.1 S, the dichroic spectrum and amino acid composition of a collagen, and a Tm = 31 degrees C. Positive in situ hybridization with a specific probe for this collagen began 6-8 h after egg laying and showed message in the locations of embryos and larvae which reacted with the antibodies. This included some prominent individual cells in the hemolymph.     

Large complex globular domains of type VII procollagen contribute to the structure of anchoring fibrils

Type VII collagen, in the form of an antiparallel dimer, is a major protein component of anchoring fibrils. The ultrastructural appearance of these fibrils suggests that they may serve to anchor the basement membrane zone to the underlying connective tissue matrix. We report here the identification and initial characterization of Type VII procollagen, recovered from the media of epidermoid carcinoma cell cultures. Immunoblotting using monospecific antibodies to Type VII procollagen identifies a single, homogeneous band of at least Mr 320,000 following disulfide bond reduction. This chain contains 170 kDa of collagen triple helix and 150 kDa of non-helical domain at the carboxyl terminus. Pepsin digestion of this material yields Type VII collagen identical to that isolated from whole tissue and a series of quasi-stable peptides derived from the carboxyl-terminal region. Cell extracts contain procollagen chains identical in size to those secreted into the media. There is no evidence for processing of this material in cell culture. Partial purification by velocity sedimentation and transmission electron microscopic observation following rotary shadowing reveals both monomers (426 nm) and dimers (785 nm). Dimers are antiparallel and interact through 60-nm overlap, with amino-terminal globular domains present at the ends of the overlap. The multi-domain carboxyl-terminal region appears as three similar arms originating from a centralized globular region adjacent to the collagen helix. The carboxyl globular domain is present in whole tissue and may participate in the unique fibril form of this collagen. The amino-terminal globule may function in the antiparallel assembly of dimers.     

Production of DON-related trichothecenes byFusarium graminearum Schw from Krasnodarski krai of the USSR

34Fusarium graminearum Schw isolates produced 4-deoxynivalenol to form significant amounts of 4, 7 — dideoxynivalenol and lesser amounts of 4 — deoxynivalenol monoacetates on grain substratesin vitro. This is the first report on the capability a large group of naturally occurring isolates to produce 4,7-dideoxynivalenol. The average levels of 4,7-dideoxynivalenol on rice, corn, barley, and wheat as a substrate were respectively 26.8, 14.0, 12.8, and 10.5% of the level of 4-deoxynivalenol. 4, 7 — dideoxynivalenol was present in all examined naturally contaminated wheat kernel samples at levels of 1.7 to 7.9% of the level of 4-deoxynivalenol. These findings suggest that more attention should be given to the occurrence of 4,7-dideoxynivalenol in cereals.     

Two type XII-like collagens localize to the surface of banded collagen fibrils

Two recently identified collagen molecules, termed twelve-like A and twelve-like B (TL-A and TL-B) have properties similar to type XII collagen. These molecules have been localized in human and calf tissues by immunoelectron microscopy. The observations strongly suggest that both molecules are located along the surface of banded collagen fibers. The epitopes recognized by the antibodies are contained in large, nontriple-helical domains at one end of the collagen helix. The epitopes are visualized at a distance from the surface of the banded fibers roughly equal to the length of the nonhelical domains, suggesting that the nonhelical domains extend from the fibril, while the triple-helical domains are likely to bind directly to the fibril surface. Occasionally, both TL-A and TL-B demonstrate periodic distribution along the fibril surface. The period corresponds to the primary interband distance of the banded fibrils. Not all fibrils in a fiber bundle are labeled, nor is the labeling continuous along the length of labeled fibrils. Simultaneous labeling of TL-A and type VI collagen only rarely shows colocalization, suggesting that TL-A and TL-B do not mediate interactions between the type VI collagen beaded filaments and banded collagen fibrils. Also, interfibrillar distances are approximately equivalent in the presence and absence of these type XII-like molecules. While the results do not directly indicate a specific function for these molecules, the localization at the fibril surface suggests that they mediate interactions between the fibrils and other matrix macromolecules or with cells.     

Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments

Basal keratinocytes attach to the underlying dermal stroma through an ultrastructurally unique and complex basement membrane zone. Electron-dense plaques along the basal surface plasma membrane, termed hemidesmosomes, appear to attach directly to the lamina densa of the basement membrane through fine strands, called anchoring filaments. The lamina densa is secured to the stroma through a complex of type VII collagen containing anchoring fibrils and anchoring plaques. We have identified what we believe is a novel antigen unique to this tissue region. The mAbs to this antigen localize to the anchoring filaments, just below the basal-dense plate of the hemidesmosomes. In cell culture, the antigen is deposited upon the culture substate by growing and migrating human keratinocytes. Addition of mAb to the cultures causes the cells to round and detach, but does not impair them metabolically. Skin fragments incubated with antibody extensively de-epithelialize. These findings strongly suggest that this antigen is intimately involved in attachment of keratinocytes to the basement membrane. This antigen was isolated from keratinocyte cultures by immunoaffinity chromatography. Two molecules are observed. The most intact species contains three nonidentical chains, 165, 155, and 140 kD linked by interchain disulfide bonds. The second and more abundant species contains the 165- and 140-kD chains, but the 155-kD chain has been proteolytically cleaved to 105 kD. Likewise, two rotary-shadowed images are observed. The larger of the two, presumably corresponding to the most intact form, appears as an asymmetric 107-nm-long rod, with a single globule at one end and two smaller globules at the other. The more abundant species, presumably the proteolytically cleaved form, lacks the distal small globule. We propose the name "kalinin" for this new molecule.     

The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor.

Kalinin, a recently characterized novel protein component of anchoring filaments, has been shown to be involved in keratinocyte attachment to culture substrates and to dermis in vivo, and to exist in keratinocyte-conditioned culture medium in two heterotrimeric forms of 440 and 400 kDa (Rousselle, P., Lunstrum, G.P., Keene, D.R., and Burgeson, R.E. (1991) J. Cell Biol. 114, 567-576). This study demonstrates that kalinin is initially synthesized in a cell-associated form estimated to be 460 kDa. By second dimension reduced electrophoresis, V8 protease digestion, and immunoblot analysis, we demonstrate that the cell form contains nonidentical subunits of 200, 155, and 140 kDa. The 440-kDa medium form is derived from the cell form by extracellular processing of the 200-kDa subunit to 165 kDa, a step which also occurs in skin organ culture. The 400-kDa form is derived from the 440-kDa form by extracellular processing of the 155 kDa-subunit to 105 kDa. The cell form is secreted by keratinocytes, deposited onto culture substratum, and is the form which facilitates attachment and adhesion of growing and spreading keratinocytes. It is also the form initially synthesized in skin organ culture. Kalinin purified from tissue, which appears to facilitate epithelial-mesenchymal cohesion in vivo, is closely related to the 400-kDa medium form purified from culture.     

Somatoform disorders among patients attending walk-in clinics in Trinidad: prevalence and association with depression and anxiety

Objectives Somatoform disorders are common in international primary care settings, but have been little studied in the developing world. The objective of this study was to determine the prevalence of severe undifferentiated somatoform disorder, and its relationship to depression and anxiety, among patients attending walk-in clinics in Trinidad.Methods The study participants, who were all aged 18 years or older and attending walk-in clinics at 16 randomly selected health centres, were surveyed between May and August 2007 using the PRIME-MD questionnaire.Results There were 594 participants (the response rate was 92%), of whom 72.7% were female. Their ages ranged from 18 to 93 years, and 54.5% were over 50 years of age. In total, 37.2% were married and 25.9% were single. Indo-Trinidadians represented 43.1% and Afro-Trinidadians represented 36% of the study sample; 56.5% of the participants reported that their income was less than US$ 400 per month, and 65.7% were unemployed. At walk-in clinics in Trinidad, the estimated prevalence of severe undifferentiated somatoform disorder was 10.3% (95% CI: 7.86–12.74), that of hypochondriasis was 28.5% (95% CI: 24.9–32.1), and that of body dysmorphic disorder was 15.8% (95% CI: 11.9–18.7). Severe undifferentiated somatoform disorder was statistically significantly associated with gender and ethnicity but not with age, level of education, employment status or income. Chi-square testing found significant associations between the presence of severe undifferentiated somatoform disorder and both depression and anxiety (P < 0.05), between hypochondriasis and both anxiety and depression (P < 0.05), and between body dysmorphic disorder and depression (P < 0.05) but not anxiety. Regression analysis suggested that the demographic features that predicted severe undifferentiated somatoform disorder were being female or Indo-Trinidadian.Conclusions Walk-in clinics in Trinidad that serve older patients on a lower income have a high proportion of patients with somatoform disorders as measured by the PRIME-MD scale. These patients exhibit many features of anxiety and depression. These findings have implications for medical training and service delivery.     

Inter-familial relationships of the shorebirds (Aves: Charadriiformes) based on nuclear DNA sequence data

Background  

Phylogenetic hypotheses of higher-level relationships in the order Charadriiformes based on morphological data, partly disagree with those based on DNA-DNA hybridisation data. So far, these relationships have not been tested by analysis of DNA sequence data. Herein we utilize 1692 bp of aligned, nuclear DNA sequences obtained from 23 charadriiform species, representing 15 families. We also test earlier suggestions that bustards and sandgrouses may be nested with the charadriiforms. The data is analysed with methods based on the parsimony and maximum-likelihood criteria.     

Secretion of cartilage oligomeric matrix protein is affected by the signal peptide

Cartilage oligomeric matrix protein (COMP) is a secreted glycoprotein found in the extracellular matrices of skeletal tissues. Mutations associated with two human skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia, disturb COMP secretion leading to intracellular accumulation of mutant COMP, especially in chondrocytes. Here we show that the manifestation of this secretory defect is dramatically influenced by the signal peptide that targets COMP for secretion. The comparison of wild type and mutant COMP secretion directed by the COMP or BM40 signal peptide in HEK-293 cells and rat chondrosarcoma cells revealed that the BM40 signal peptide substantially enhances secretion of mutant COMP that accumulates in endoplasmic reticulum-like structures when targeted by its own signal peptide. Additionally, we demonstrate that mutant COMP forms mixed pentamers with wild type COMP. Our findings suggest that the secretory defect in pseudoachondroplasia and multiple epiphyseal dysplasia is not specific for chondrocytes, nor does it require interaction of mutant COMP with other matrix proteins prior to transport from the cell. They also imply a previously unappreciated role for the signal peptide in the regulation of protein secretion beyond targeting to the endoplasmic reticulum.