New haemoglobin genotypes in Atlantic cod, Gadus morhua: possible relation with growth

In a preliminary study, 121 individually tagged juvenile Atlantic cod (Gadus morhua) were classified according to their haemoglobin genotypes into four groups, i.e., two main haemoglobin genotypes [Hb-I(1/2), Hb-I(2/2)] and two sub-types [Hb-I(1/2b), Hb-I(2/2b)], and reared for 3 months at 10 degrees C, 13 degrees C and T-step (fish reared at 16 degrees C and then subsequently moved to 13 and later to 10 degrees C). Overall growth rates across temperatures were 10% and 19% higher in the Hb-I(2/2b), Hb-I(1/2b) sub-types compared to corresponding Hb-I(2/2) and Hb-I(1/2) main types, respectively. Individual growth rate trajectories varied between the genotypes at all temperatures studied. Our study indicates that under certain environmental conditions higher growth in the two sub-types compared to the main genotypes could be expected. This may indicate difference in other physiological characters not studied here, but seen in previous studies, i.e., oxygen affinity and competitive performance.     

Kallikrein-related peptidase 14 may be a major contributor to trypsin-like proteolytic activity in human stratum corneum

We have previously presented evidence that two human kallikrein-related peptidases, KLK5 (hK5, stratum corneum tryptic enzyme, SCTE) and KLK7 (hK7, stratum corneum chymotryptic enzyme, SCCE), which are abundant in the stratum corneum, may be involved in desquamation. Since we had noted that not all trypsin-like activity in the plantar stratum corneum could be ascribed to KLK5, we set out to identify other skin proteases with similar primary substrate specificity. Here we describe purification of a protease identified as KLK14 from plantar stratum corneum, and show that this enzyme may be responsible for as much as 50% of the total trypsin-like activity in this tissue, measured as activity towards a chromogenic substrate cleaved by a wide variety of enzymes with trypsin-like specificity. This was in spite of very low levels of KLK14 protein compared to KLK5 and KLK7. KLK14 could be detected by immunoblotting in normal superficial stratum corneum of all individuals examined. The majority of KLK14 in the plantar stratum corneum is present in its catalytically active form. KLK14 could be immunohistochemically detected in sweat ducts, preferentially in the intraepidermal parts (the acrosyringium), and in sweat glands. The role played by this very efficient protease under normal and disease conditions in the skin remains to be elucidated.     

The contributions of integrin affinity and integrin-cytoskeletal engagement in endothelial and smooth muscle cell adhesion to vitronectin

The serine proteinase inhibitor, plasminogen activator inhibitor type-1 (PAI-1), binds to the adhesion protein vitronectin with high affinity at a site that is located directly adjacent to the vitronectin RGD integrin binding sequence. The binding of PAI-1 to vitronectin sterically blocks integrin access to this site and completely inhibits the binding of purified integrins to vitronectin; however, its inhibition of endothelial and smooth muscle cell adhesion to vitronectin is at most 50-75%. Because PAI-1 binds vitronectin with approximately 10-100-fold higher affinity than purified integrins, we have analyzed the mechanism whereby these cells are able to overcome this obstacle. Our studies exclude proteolytic removal of PAI-1 from vitronectin as the mechanism, and show instead that cell adhesion in the presence of PAI-1 is dependent on integrin-cytoskeleton engagement. Disrupting endothelial or smooth muscle cell actin polymerization and/or focal adhesion assembly reduces cell adhesion to vitronectin in the presence of PAI-1 to levels similar to that observed for the binding of purified integrins to vitronectin. Furthermore, endothelial cell, but not smooth muscle cell adhesion to vitronectin in the presence of PAI-1 requires both polymerized microtubules and actin, further demonstrating the importance of the cytoskeleton for integrin-mediated adhesion. Finally, we show that cell adhesion in the presence of PAI-1 leads to colocalization of PAI-1 with the integrins alphavbeta3 and alphavbeta5 at the cell-matrix interface.     

The complete cDNA sequence of human hexabrachion (Tenascin). A multidomain protein containing unique epidermal growth factor repeats.

Hexabrachion (Tenascin) is a large glycoprotein that appears in extracellular matrices as a disulfide-linked multimer. It is synthesized in an ordered fashion at particular sites during development, is made in large amounts by certain tumors, and is found in restricted tissue locations in the adult. In this report, we describe the sequence of a full length cDNA of human hexabrachion. The encoded protein contains a total of 2203 amino acids and is a linear array of discrete reiterated domains. At the 5' end are encoded hydrophobic residues and 8 flanking cysteines predicted to be responsible for assembly of hexabrachion polypeptides into a radially arranged, six-armed complex. Following this region are 14 1/2 contiguous 31-amino acid epidermal growth factor-like repeats that have a unique structure with respect to the known examples of this type of domain. Immediately adjacent to these repeats lie 15 uninterrupted segments of approximately 90 amino acids which are similar to the Type III units found in fibronectin. At the carboxyl terminus of the protein is a 210-amino acid domain that is similar to fibrinogen. The domain structure of this protein is consistent with the potential for interaction with multiple ligands and for roles in cell adhesion and/or signaling.     

Reversal of elution order for profen acid enantiomers in packed-column SFC on Chiralpak AD

Enantiomeric separations of four 2-substituted propionic acid drugs have been studied using packed-column supercritical fluid chromatography (SFC) with amylose tris(3,5-dimethylphenylcarbamate) coated on silica as support (Chiralpak AD). Under standard conditions (i.e., flow rate, 1.5 ml/min; column temperature, 30 degrees C; back-pressure, 150 bar), the order of elution could be reversed when the polar alcohol modifier methanol in carbon dioxide was replaced by 2-propanol for ibuprofen, ketoprofen, and naproxen. For flurbiprofen, with the highest selectivity factor, no reversal was observed, although selectivity was reduced significantly with higher alcohols. Naproxen and flurbiprofen were also investigated with 2-butanol and 2-pentanol. The former showed reversal of elution order but not the latter. For higher alcohol modifiers, including 2-propanol, the peak symmetry was poor but could be improved by addition of citric acid in the alcohol modifier. These results stress the importance to investigate enantiomer elution order during the development of enantioselective methods and when chromatographic conditions are optimized. Preliminary experiments with column temperatures over the range of -15 to 45 degrees C revealed that, in a few cases, reversal took place with a change in temperature only.     

Two dimensional electrophoresis of thylakoid membrane proteins and its application to microsequencing

A procedure of two-dimensional gel electrophoresis adapted for application on membrane proteins from the thylakoids is described. It involves isoelectric focusing in the first dimension and size dependent electrophoresis in the second dimension. About 100 polypeptides are clearly separated with relatively little streaking. About 20 polypeptides are identified by immunoblotting or location in the gel. They are the polypeptides of the PS I core, the 64 kDa protein, the and subunits of CF1 ATPase, cytochrome f, Rieske iron-sulfur protein, the 23 kDa and 33 kDa polypeptides of the oxygen evolving complexes, CP29, CP24, CP27 and CP25 (last two proteins belong to LHCII). Some proteins give rise to two or more separate spots indicating a separation of different isoforms of these proteins. Among them, the LHCII polypeptides (27 kDa and 25 kDa) were each resolved into at least three spots in the pH range 4.75–5.90; the Rieske FeS protein, as published elsewhere (Yu et al. 1994), was separated into two forms having different isoelectric points (pI 5.1 and 5.4), each of them was also microsequenced; the 64 kDa protein claimed to be a LHCII-kinase was found to be multiple forms appearing in at least two isoforms with pI 6.2 (K1) and 6.0 (K2) respectively, furthermore, K1 can be resolved into two subpopulations.The lateral distribution of these proteins in the thylakoid membrane was determined by analysing the vesicles originating from different parts of the thylakoids. The data obtained from this analysis can be partially used as markers for different thylakoid domains.This procedure for sample solubilization and 2-D electrophoresis is useful for the analysis of the polypeptide composition of vesicles originating from the thylakoid membrane and for microsequences of individual polypeptides isolated from the 2-D gel.     

Native TIMP-free 70 kDa progelatinase (MMP-2) secreted at elevated levels by RSV transformed fibroblasts

Rous sarcoma virus-transformed cultures of chicken embryo fibroblasts (RSVCEF) secrete elevated levels of a 70 kDa progelatinase, an avian form of the 72 kDa matrix metalloproteinase-2 (MMP-2). Affinity-purified preparations of secreted 70 kDa progelatinase are composed of two distinct populations of zymogen: a 70 kDa progelatinase tightly complexed with an avian form of TIMP-2 and a native 70 kDa progelatinase free of any detectable TIMP-2. These two forms of the progelatinase can be separated by Mono Q FPLC in the absence of denaturing agents. The homogeneity of the two separated forms is demonstrated by both SDS-PAGE and nondenaturing, native gel electrophoresis. The purified TIMP-free 70 kDa progelatinase is stable in aqueous conditions and does not spontaneously autoactivate. Treatment of the TIMP-free progelatinase with the organomercurial, p-aminophenylmercuric acetate (APMA), results in rapid (5-60 minutes) autolytic conversion of the 70 kDa progelatinase to 67 kDa, 62 kDa and lower molecular weight forms of the enzyme. APMA treatment of the TIMP-free progelatinase yields a preparation that is enzymatically active with a high specific activity towards a peptide substrate. Identical treatment of TIMP-complexed progelatinase with APMA results in a significantly slower conversion process in which the 70 kDa progelatinase is only 50% converted after 6-24 hours and the specific enzyme activity of the preparation is 8 to 18-fold lower. Purified avian TIMP-2 added to the TIMP-free progelatinase forms a complex with the progelatinase and prevents the rapid autolytic conversion induced by APMA. Comparative analysis of parallel cultures of transformed RSVCEF and normal CEF demonstrates that the transformed cultures contain threefold higher levels of the TIMP-free progelatinase than the normal CEF cultures which produce predominantly TIMP-complexed progelatinase. The presence in transformed cultures of elevated levels of a more readily activated TIMP-free progelatinase, the suppression of its rapid activation by TIMP-2, and the potential effect of the altered balance between TIMP-free and TIMP-complexed 70 kDa progelatinase on the invasive, malignant phenotype, are discussed. © 1994 Wiley-Liss, Inc.     

Localization of a gene for peripheral arterial occlusive disease to chromosome 1p31

Peripheral arterial occlusive disease (PAOD) results from atherosclerosis of large and medium peripheral arteries, as well as the aorta, and has many risk factors, including smoking, diabetes, hypertension, and hyperlipidemia. PAOD often coexists with coronary artery disease and cerebrovascular disease. Cross-matching a population-based list of Icelandic patients with PAOD who had undergone angiography and/or revascularization procedures with a genealogy database of the entire Icelandic nation defined 116 extended families containing 272 patients. A genomewide scan with microsatellite markers revealed significant linkage to chromosome 1p31 with an allele-sharing LOD score of 3.93 (P=1.04 x 10(-5)). We designate this locus as "PAOD1." Subtracting 35 patients with a history of stroke increased the LOD score to 4.93. This suggests that, although PAOD and other vascular diseases share risk factors, genetic factors specific to subtypes of vascular disease may exist.