Origin, structure, and biological activities of peroxidases in human saliva

Human whole saliva contains two peroxidases, salivary peroxidase (hSPO) and myeloperoxidase (hMPO), which are part of the innate host defence in oral cavity. Both hSPO as well as human milk lactoperoxidase (hLPO) are coded by the same gene, but to what extent the different producing glands, salivary and mammary glands, affect the final conformation of the enzymes is not known. In human saliva the major function of hSPO and hMPO is to catalyze the oxidation of thiocyanate (SCN(-)) in the presence of hydrogen peroxide (H(2)O(2)) resulting in end products of wide antimicrobial potential. In addition cytotoxic H(2)O(2) is degraded. Similar peroxidation reactions inactivate some mutagenic and carcinogenic compounds, which suggests another protective mechanism of peroxidases in human saliva. Although being target of an active antimicrobial research, the structure-function relationships of hSPO are poorly known. However, recently published method for recombinant hSPO production offers new tools for those investigations.     

Matrix association of latent TGF‐beta binding protein‐2 (LTBP‐2) is dependent on fibrillin‐1

The components of the extracellular matrix (ECM) and their differential expression patterns play important roles in tissue formation. The deposition of latent TGF‐β binding proteins (LTBPs) to the ECM exhibit distinct distribution profiles. We have analyzed here the temporal and spatial ECM association of latent TGF‐β binding protein LTBP‐2 in cultured human embryonic lung fibroblasts. We found that LTBP‐2 was not assembled to the ECM until by confluency of cultures following the deposition of fibronectin (FN) and fibrillin‐1. In 5‐day‐old cultures LTBP‐2 was rapidly secreted from cells and it subsequently associated with the ECM as shown by metabolic labeling and immunoprecipitation. LTBP‐2 colocalized transiently with fibronectin and failed to assemble to the ECM of FN deficient mouse fibroblasts. Analysis of different cultured human cell lines revealed partial colocalization of LTBP‐2 and fibrillin‐1 in the ECM of fibroblasts, MG‐63 osteosarcoma cells and human vascular endothelial cells. Silencing of fibrillin‐1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP‐2. Current results suggest that LTBP‐2 is not an element of the provisional ECM of fibroblasts but is more likely a component of more mature ECM and indicate that matrix association of LTBP‐2 depends on a pre‐formed fibrillin‐1 network. J. Cell. Physiol. 221: 586–593, 2009. © 2009 Wiley‐Liss, Inc.     

A comparison of growth patterns between a stunted and two large predatory Arctic charr populations under identical hatchery conditions

Arctic charr are characterized by an extensive variability in growth and body size in natural waters. Although growth traits may involve a significant heritable component, most of this intraspecific variation presumably is environmentally induced and thus attributable to phenotypic plasticity. In the present study, size-at-age and length–weight relationship (body condition) were assessed for three Finnish Arctic charr populations of different geographical origins and extreme size forms (a stunted vs. two large-growing, predatory charr) held under standardized rearing conditions for 3 years (up to 37 months after hatching). In particular, our interest was to investigate whether the differences in growth between the large and the stunted charr as observed in the wild populations would diminish when the fish are offered suitable food in abundance. Population-specific mean body size and condition differed significantly in 0+, 1+, 2+ and 3+ fish. However, the identical rearing conditions resulted in the originally stunted charr reaching a comparable final mean size (317 mm/427 g) as the large charr populations (343 mm/510 g and 359 mm/497 g). Some individuals were of the same size as their parents at spawning already at the age of 0+ years. Furthermore, length–weight regression residuals of the stunted charr developed to a notably high level, indicating the largest final condition mean. The increase of size variation (CV of weight) in stunted charr lasted for over two growth seasons, whereas in large charr it remained stable since the end of the first summer. Variations in mortality and sexual maturation at age 2 seemed to be less relevant factors affecting overall growth performance. The study demonstrates an example of the high plasticity involved in the growth of fish: the stunted charr possess a tremendous capacity for growth in a benign environment, virtually corresponding to that observed in the large predatory populations.     

Proteolytic cleavage and phosphorylation of a tumor-associated ErbB4 isoform promote ligand-independent survival and cancer cell growth

The ErbB1 and ErbB2 receptors are oncogenes with therapeutic significance in human cancer, whereas the transforming potential of the related ErbB4 receptor has remained controversial. Here, we have addressed whether four alternatively spliced ErbB4 isoforms differ in regulating cellular responses relevant for tumor growth. We show that the two tumor necrosis factor-α converting enzyme (TACE)-cleavable ErbB4 isoforms (the juxtamembrane [JM]-a isoforms) were overexpressed in a subset of primary human breast cancers together with TACE. The overexpression of the JM-a cytoplasmic (CYT)-2 ErbB4 isoform promoted ErbB4 phosphorylation, survival of interleukin-3-dependent cells, and proliferation of breast cancer cells even in the absence of ligand stimulation, whereas activation of the other three ErbB4 isoforms required ligand stimulation. Ligand-independent cellular responses to ErbB4 JM-a CYT-2 overexpression were regulated by both tyrosine kinase activity and a two-step proteolytic generation of an intracellular receptor fragment involving first a TACE-like proteinase, followed by γ-secretase activity. These data suggest a novel transforming mechanism for the ErbB4 receptor in human breast cancer that is 1) specific for a single receptor isoform and 2) depends on proteinase cleavage and kinase activity but not ligand activation of the receptor.     

Interrelationships between low density lipoprotein receptor defect, serum fatty acid composition, and serum cholesterol concentration

It is known that, in the general human population, serum fatty acid composition is correlated with serum triacylglycerol and cholesterol concentrations. The goal of the present study was to analyze whether the same is true of individuals who have a low density lipoprotein receptor (LDL-R) defect. Concentrations of 16 different fatty acids, cholesterol, triacylglycerol, and major lipoproteins in serum were determined in eight individuals who had (FH-North Karelia), the most common LDL-R defect in Finland, which causes familial hypercholesterolemia, and in their 30 relatives belonging to a single large pedigree as controls. The average number of double bonds (i.e., degree of desaturation) in serum fatty acids correlated negatively with the concentrations of serum total cholesterol (r = 0.27, P < 0.05) and total triacylglycerol (r = -0.71, P < 0.001) and positively with the number of fish meals per week (r = 0.50, P < 0.01), which was analyzed in all pedigree members jointly. These effects were similar in individuals having LDL-R defect, in which group the correlation coefficients were -0.31 (P = NS), -0.99 (P < 0.001), and 0.79 (P = NS) for serum total cholesterol, triacylglycerol, and weekly fish meals, respectively. Thus, LDL-R defect does not impair the correlation between serum fatty acid composition and serum triacylglycerol concentration. This result is in agreement with dietary studies that have shown that familial hypercholesterolemia patients respond very favorably to dietary therapy.     

Co-recycling of MT1-MMP and MT3-MMP through the trans-Golgi network. Identification of DKV582 as a recycling signal

Members of the membrane-type matrix metalloproteinases (MT-MMPs) have been implicated in a wide range of physiological and pathological processes from normal development to tumor growth. Tethered on plasma membrane, these enzymes are potentially regulated by the trafficking machinery of the cells. Here we demonstrate that both MT1-MMP and MT3-MMP are internalized, transported to the trans-Golgi network through early endosomes, and recycled back to cell surface in 60 min in a manner distinct from the one employed by transferrin receptor. Interestingly, co-expressed MT1-MMP and MT3-MMP are localized and routed in the same vesicles throughout the trafficking process. We further demonstrated that the carboxyl-terminal sequence DKV(582) of MT1-MMP is required for its recycling, thus defining a novel recycling motif. These results suggest that MT-MMPs may coordinate their proteolytic activities through the cellular trafficking machinery.     

Partitioning of pyrene-labeled phospho- and sphingolipids between ordered and disordered bilayer domains

Here we have studied how the length of the pyrene-labeled acyl chain (n) of a phosphatidylcholine, sphingomyelin, or galactosylceramide affects the partitioning of these lipids between 1), gel and fluid domains coexisting in bovine brain sphingomyelin (BB-SM) or BB-SM/spin-labeled phosphatidylcholine (PC) bilayers or 2), between liquid-disordered and liquid-ordered domains in BB-SM/spin-labeled PC/cholesterol bilayers. The partitioning behavior was deduced either from modeling of pyrene excimer/monomer ratio versus temperature plots, or from quenching of the pyrene monomer fluorescence by spin-labeled PC. New methods were developed to model excimer formation and pyrene lipid quenching in segregated bilayers. The main result is that partition to either gel or liquid-ordered domains increased significantly with increasing length of the labeled acyl chain, probably because the pyrene moiety attached to a long chain perturbs these ordered domains less. Differences in partitioning were also observed between phosphatidylcholine, sphingomyelin, and galactosylceramide, thus indicating that the lipid backbone and headgroup-specific properties are not severely masked by the pyrene moiety. We conclude that pyrene-labeled lipids could be valuable tools when monitoring domain formation in model and biological membranes as well as when assessing the role of membrane domains in lipid trafficking and sorting.     

Time-resolved fluorescence and fourier transform infrared spectroscopic investigations of lateral packing defects and superlattice domains in compositionally uniform cholesterol/phosphatidylcholine bilayers

Time-resolved fluorescence and Fourier transform infrared spectroscopies were used to investigate the lateral organization of lipids in compositionally uniform and fully equilibrated 1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPC/CHOL) liposomes prepared by a recently devised low-temperature trapping method. Independent fluorescence decay lifetime and rotational dynamics parameters of diphenylhexatriene (DPH) chain-labeled phosphatidylcholine (DPH-PC) in these liposomes were recovered from the time-resolved fluorescence measurements as a function of cholesterol molar fraction (X(CHOL)) at 23 degrees C. The results indicate significantly greater lifetime heterogeneity, shorter average lifetime, rotational correlation time, and lower order parameter of the DPH moiety at X(CHOL) approximately 0.40 and 0.50 as compared to the adjacent cholesterol concentrations. Less prominent changes were also detected at, for example, X(CHOL) approximately 0.20 and 0.33. These X(CHOL)'s coincide with the "critical" X(CHOL)'s predicted by the previously proposed superlattice (SL) model, thus indicating that POPC and cholesterol molecules tend to form SL domains where the components tend to be regularly distributed. The data also support another prediction of the SL model, namely that lateral packing defects coexist with the ordered SL domains. It appears that unfavorable interaction of the DPH-moiety of DPH-PC with cholesterol results in a preferential partition of DPH-PC to the defect regions. Fourier transform infrared analysis of the native lipid O=P=O, C=O, and C-H vibrational bands of POPC/CHOL liposomes in the absence of DPH-PC revealed an increase in the conformational order of the acyl chains and a decrease in the conformational order (or increased hydration) of the interfacial and headgroup regions at or close to the predicted critical X(CHOL)'s. This provides additional but probe-independent evidence for SL domain formations in the POPC/CHOL bilayers. We propose that the defect regions surrounding the putative SL domains could play an important role in modulating the activity of various membrane-associated enzymes, e.g., those regulating the lipid compositions of cell membranes.     

Ethylene insensitivity modulates ozone-induced cell death in birch

We have used genotypic variation in birch (Betula pendula Roth) to investigate the roles of ozone (O(3))-induced ethylene (ET), jasmonic acid, and salicylic acid in the regulation of tissue tolerance to O(3). Of these hormones, ET evolution correlated best with O(3)-induced cell death. Disruption of ET perception by transformation of birch with the dominant negative mutant allele etr1-1 of the Arabidopsis ET receptor gene ETR1 or blocking of ET perception with 1-methylcyclopropene reduced but did not completely prevent the O(3)-induced cell death, when inhibition of ET biosynthesis with aminooxyacetic acid completely abolished O(3) lesion formation. This suggests the presence of an ET-signaling-independent but ET biosynthesis-dependent component in the ET-mediated stimulation of cell death in O(3)-exposed birch. Functional ET signaling was required for the O(3) induction of the gene encoding beta-cyanoalanine synthase, which catalyzes detoxification of the cyanide formed during ET biosynthesis. The results suggest that functional ET signaling is required to protect birch from the O(3)-induced cell death and that a decrease in ET sensitivity together with a simultaneous, high ET biosynthesis can potentially cause cell death through a deficient detoxification of cyanide.     

Aggressiveness is associated with genetic diversity in landlocked salmon (Salmo salar)

The amount of intraindividual genetic variation has often been found to have profound effects on life history traits. However, studies concerning the relationship between behaviour and genetic diversity are scarce. Aggressiveness is an important component of competitive ability in juvenile salmonids affecting their later performance and survival. In this study, we used an experimental approach to test the prediction that juveniles with low estimated genetic diversity should be less aggressive than juveniles with high estimated genetic diversity in fry from a highly endangered population of land-locked salmon (Salmo salar). This was achieved by using a method enabling the accurate estimation of offspring genetic diversity based on parental microsatellite genotype data. This allowed us to create two groups of offspring expected to have high or low genetic diversity in which aggressive behaviour could be compared. Salmon fry with low estimated genetic diversity were significantly less aggressive than fry with high estimated genetic diversity. Closer analysis of the data suggested that this difference was due to differences in more costly acts of aggression. Our result may reflect a direct effect of genetic variation on a fitness-related trait; however, we cannot rule out an alternative explanation of allele-specific phenotype matching, where lowered aggression is expressed towards genetically more similar individuals.