Characterisation of a type-I collagen trimeric cross-linked peptide from calf aorta and its cross-linked structure. Detection of pyridinoline by time-of-flight secondary ion-mass spectroscopy and evidence for a new cross-link

A collagenous trimeric cross-linked peptide has been isolated from the insoluble matrix of calf aorta, using trypsin solubilisation, and purified by gel filtration, cation-exchange chromatography and reversed-phase HPLC. Molecular mass and amino acid composition indicated that the C-terminal, non-helical region of type I collagen in its dimer form, designated as [ColC(I)]2, is cross-linked to a tryptic peptide TN(I) from the N-terminal helical cross-link region of an adjacent type I molecule, forming the cross-linked peptide [ColC(I)]2 X TN(I). Amino acid sequence analysis of the peptide yielded a series of sequences corresponding to the cross-linking domains ColC(I) and TN(I) and furnished the first direct chemical evidence for the 4D staggered arrangement of type I molecules within native fibers. The trifunctional cross-linking amino acid pyridinoline was shown to occur in the peptide, confirming the peptides three-chain structure. Pyridinoline was isolated from the cross-linked peptide by preparative amino acid analysis and reversed-phase HPLC and identified by its ultraviolet absorption spectra, its fluorescence excitation and emission spectra and, for the first time, its time-of-flight secondary ion-mass spectrum. The high sensitivity of the latter method, exceeding that of fast-atom-bombardment mass spectroscopy by three orders of magnitude, allowed detection of pyridinoline in the picomole range. The occurrence of pyridinoline in non-stoichiometric amounts, the presence of hydroxylysine in hydrolysates of all cross-linked peptides and the finding that hydrolysates also contained an unidentified component indicated that there is at least one cross-link form that is different from pyridinoline and is hydrolysable.     

Combining Affymetrix microarray results

Background  

As the use of microarray technology becomes more prevalent it is not unusual to find several laboratories employing the same microarray technology to identify genes related to the same condition in the same species. Although the experimental specifics are similar, typically a different list of statistically significant genes result from each data analysis.     

The Sero-epidemiology of Coxiella burnetii in Humans and Cattle,Western Kenya: Evidence from a Cross-Sectional Study

Evidence suggests that the intracellular bacterial pathogen Coxiella burnetii (which causes Q fever) is widespread, with a near global distribution. While there has been increasing attention to Q fever epidemiology in high-income settings, a recent systematic review highlighted significant gaps in our understanding of the prevalence, spatial distribution and risk factors for Q fever infection across Africa. This research aimed to provide a One Health assessment of Q fever epidemiology in parts of Western and Nyanza Provinces, Western Kenya, in cattle and humans. A cross-sectional survey was conducted: serum samples from 2049 humans and 955 cattle in 416 homesteads were analysed for C. burnetii antibodies. Questionnaires covering demographic, socio-economic and husbandry information were also administered. These data were linked to environmental datasets based on geographical locations (e.g., land cover). Correlation and spatial-cross correlation analyses were applied to assess the potential link between cattle and human seroprevalence. Multilevel regression analysis was used to assess the relationships between a range of socio-economic, demographic and environmental factors and sero-positivity in both humans and animals. The overall sero-prevalence of C. burnetii was 2.5% in humans and 10.5% in cattle, but we found no evidence of correlation between cattle and human seroprevalence either within households, or when incorporating spatial proximity to other households in the survey. Multilevel modelling indicated the importance of several factors for exposure to the organism. Cattle obtained from market (as opposed to those bred in their homestead) and those residing in areas with lower precipitation levels had the highest sero-prevalence. For humans, the youngest age group had the highest odds of seropositivity, variations were observed between ethnic groups, and frequent livestock contact (specifically grazing and dealing with abortion material) was also a risk factor. These results illustrate endemicity of C. burnetii in western Kenya, although prevalence is relatively low. The analysis indicates that while environmental factors may play a role in cattle exposure patterns, human exposure patterns are likely to be driven more strongly by livestock contacts. The implication of livestock markets in cattle exposure risks suggests these may be a suitable target for interventions.     

The Swelling-Activated Anion Conductance in the Mouse Renal Inner Medullary Collecting Duct Cell Line mIMCD-K2

Swelling-activated Cl⁻ currents (I _Cl,swell ) have been characterized in a mouse renal inner medullary collecting duct cell line (mIMCD-K2). Currents activated by exposing the cells to hypotonicity exhibited characteristic outward rectification and time- and voltage-dependent inactivation at positive potentials and showed an anion selectivity of I⁻ > Br⁻ > Cl⁻ > Asp⁻. NPPB (100 μm) inhibited the current in a voltage independent manner, as did exposure to 10 μm tamoxifen and 500 μm niflumic acid (NFA). In contrast, DIDS (100 μm) blocked the current with a characteristic voltage dependency. These characteristics of I _Cl,swell in mIMCD-K2 cells are essentially identical to those of heterologously expressed cardiac CLC-3. A defining feature of CLC-3 is that activation of PKC by PDBu inhibits the conductance. In mIMCD-K2 cells preincubation with PDBu (100 nm) prevented the activation of I _Cl,swell by hypotonicity. However, PDBu inhibition of I _Cl,swell was reversed after PDBu withdrawal, but this was refractory to subsequent PDBu inhibition. Activation of either the cystic fibrosis transmembrane conductance regulator (CFTR) or Ca²⁺ activated Cl⁻ conductance (CaCC), which are coexpressed in mIMCD-K2 cells prior to PDBu treatment, abolished the PDBu inhibition of I _Cl,swell . Control of I _Cl,swell by PKC therefore depends on the physiological status of the cell. In intact mIMCD-K2 layers in Ussing chambers, forskolin stimulation of an inward short-circuit current (due to transepithelial Cl⁻ secretion via apical CFTR) was inhibited by cell swelling upon hypotonic exposure at the basolateral surface. Activation of I _Cl,swell is therefore capable of regulating transepithelial Cl⁻ secretion and suggests that I _Cl,swell is located at the basolateral membrane. PDBu exposure prior to or during hypotonic challenge was ineffective in reversing the swelling-activated inhibition of Cl⁻ secretion, but tamoxifen (100 μm) abolished the hypotonic inhibition of forskolin-stimulated short-circuit current (I _sc ). RT-PCR analysis confirmed expression of mRNA for members of the CLC family, including both CLC-2 and 3, in the mIMCD-K2 cell line. Received: 24 February 2000/Revised: 26 May 2000     

Analysis of cyanogen bromide peptides of type I collagen from a patient with lethal osteogenesis imperfecta.

The CNBr peptides of type I collagen from bone of a patient with lethal osteogenesis imperfecta and age-matched controls were isolated by molecular-sieve chromatography and their amino acid compositions were determined. No differences were found between the compositions of the peptides from the patient and those from the controls, except for an increase in the degree of hydroxylation of lysine in all peptides from the patient. Type I collagen CNBr peptides from chick-embryo skin [Barnes, Constable Morton & Kodicek (1971) Biochem. J. 125, 925--928] and guinea-pig scar tissue [Shuttleworth, Forrest & Jackson (1975) Biochim. Biophys. Acta 379, 207--216] also have an increased degree of hydroxylation of lysine with an otherwise normal amino acid composition, and it was believed that this could be an embryonic form of collagen. As a similar collagen was present in the bones of the patient studied, it seems possible that the same 'embryonic' collagen is synthesized during development, in repair process and also in genetic disorders of collagen metabolism.     

Blood pressure of Amerindians from Surinam

Blood pressures of members of the Trio and Wajana tribes from Surinam have been analysed in relation to age, stature and weight. The sample is representative of a group living in a hunting and gathering economy but beginning to come into jarring contact with an alien culture. Diastolic pressure does not alter significantly with age in adults of either sex. There is a small but significant increase in systolic pressure with age in adult females but in males there is an apparent decrease. This decrease is probably an artifact of the cross-sectional survey approach. The high pressure in some young adults is discussed with reference to psychosocial stress. Mean blood pressures are given for children from five years of age onwards.     

Action of rheumatoid synovial collagenase on cartilage collagen. Different susceptibilities of cartilage and tendon collagen to collagenase attack.

The action of purified rheumatoid synovial collagenase on purified cartilage collagen, alpha-1(II)-3, in solution at 25 degrees C has been characterised. The enzyme attacked cartilage collagen in solution producing a 58% reduction in specific viscosity and resulting in the appearance of two reaction products which represented approximately three-quarter and one-quarter fragments of the intact molecule as shown by disc electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. The alpha-chain fragments which comprised each of these components corresponded to molecular weights of approximately 74000 and 21000. Electron microscopy of segment-long-spacing crystallites of the reaction products revealed three-quarter (TC-a) and one-quarter (TC-b) length fragments, and permitted accurate localization of the cleavage locus between bands 41 and 42 (I-41). This cleavage site and the formation of TC-a and TC-b reaction products are very similar to those found for type-I collagen substrates. Cartilage collagen in solution was found to be more resistant to collagenase attack than tendon collagen, the rate of cartilage collagen degradation being six times slower than that for tendon collagen, as judged by viscometry. The mid-point melting temperatures (T-m) for lathyritic cartilage and tendon collagen were 40.5 and 41.5 degrees C, and for the collagenase-produced reaction products 38.5 and 37.5 degrees C, respectively. The significance of these findings is discussed in relation to the structure of type I and II collagens.     

An Anti-Human ICAM-1 Antibody Inhibits Rhinovirus-Induced Exacerbations of Lung Inflammation

Human rhinoviruses (HRV) cause the majority of common colds and acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Effective therapies are urgently needed, but no licensed treatments or vaccines currently exist. Of the 100 identified serotypes, ∼90% bind domain 1 of human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, making this an attractive target for development of therapies; however, ICAM-1 domain 1 is also required for host defence and regulation of cell trafficking, principally via its major ligand LFA-1. Using a mouse anti-human ICAM-1 antibody (14C11) that specifically binds domain 1 of human ICAM-1, we show that 14C11 administered topically or systemically prevented entry of two major groups of rhinoviruses, HRV16 and HRV14, and reduced cellular inflammation, pro-inflammatory cytokine induction and virus load in vivo. 14C11 also reduced cellular inflammation and Th2 cytokine/chemokine production in a model of major group HRV-induced asthma exacerbation. Interestingly, 14C11 did not prevent cell adhesion via human ICAM-1/LFA-1 interactions in vitro, suggesting the epitope targeted by 14C11 was specific for viral entry. Thus a human ICAM-1 domain-1-specific antibody can prevent major group HRV entry and induction of airway inflammation in vivo.     

Ehlers-Danlos syndrome type VIIB. Deletion of 18 amino acids comprising the N-telopeptide region of a pro-alpha 2(I) chain

A patient with Ehlers-Danlos syndrome Type VIIB was found to have an interstitial deletion of 18 amino acids in approximately half of the pro-alpha 2(I) chains of Type I procollagen. Analysis of pepsin-solubilized tissue and fibroblast collagen revealed an abnormal additional chain, alpha 2(I)', which migrated in sodium dodecyl sulfate-5% polyacrylamide gel electrophoresis between the normal alpha 1(I) and alpha 2(I) chains. The apparent ratio of normal alpha 1(I):mutant alpha 2(I)':normal alpha 2(I) was 4:1:1. Procollagen studies and enzyme digestion studies of native mutant collagen suggested defective removal of the amino propeptide. Sieve chromatography of CNBr peptides from purified alpha 2(I)' chains revealed the absence of the normal amino telopeptide fragment CB 1 and the appearance of a larger new peptide of approximately 60 residues (CB X). Compositional and sequencing studies of this peptide identified normal amino propeptide sequences. However, the most carboxyl-terminal tryptic peptide of CB X differed substantially in composition and sequence from the expected and was found to have an interstitial deletion of 18 amino acids corresponding to the N-telopeptide of the pro-alpha 2(I) chain. This deletion removes the normal sites of cleavage of the N-proteinase and also removes a critical cross-linking lysine residue. The 18 amino acids deleted correspond exactly to the residues encoded by exon 6 of the pro-alpha 2(I) collagen gene (COL 1 A2), and, therefore, the protein defect may be due to a genomic deletion, or alternatively, an RNA splicing defect.