Collagen biosynthesis by human skin fibroblasts III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. The results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased tha amount of [³H]hydroxyproline syntehsized. Ascorbic acid, however, did not increase the synthesis of ³H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and selection of procollagen.     

Leukoregulin, a T-cell derived cytokine, upregulates stromelysin-1 gene expression in human dermal fibroblasts: evidence for the role of AP-1 in transcriptional activation.

Leukoregulin (LR), a product of activated T-cells, has been recently shown to modulate the metabolism of extracellular matrix components in human skin fibroblast cultures (Mauviel et al., J Cell Biol 113:1455-1462, 1991). In this study we focused our attention on the effects of LR on the expression of stromelysin-1 gene. This matrix metalloprotease has a broad spectrum of degradative activity and it is also required for maximal activation of interstitial collagenase. Incubation of skin fibroblast cultures with LR resulted in a dose- and time-dependent elevation of stromelysin-1 mRNA levels, the maximum enhancement being up to approximately sevenfold. This effect was abolished by cycloheximide, suggesting a requirement for ongoing protein synthesis. Transient cell transfections with a promoter/reporter gene construct containing 1.3 kb of 5' flanking DNA of the human stromelysin-1 gene linked to the chloramphenicol acetyl transferase (CAT) gene, indicated enhancement of promoter activity by LR. This enhancement was abolished by a single base substitution in the AP-1 binding site of the promoter. Furthermore, gel mobility shift assays demonstrated enhanced AP-1 binding activity in nuclear extracts from cells incubated with LR. However, LR did not alter the activity of a construct containing three AP-1 sequences in front of the thymidine kinase promoter linked to the CAT gene. These results collectively suggest that activation of stromelysin-1 gene expression by LR is mediated by AP-1 regulatory elements which are necessary, but not sufficient, for gene response.     

Human laminin a chain (LAMA) gene: Chromosomal mapping to locus 18p11.3

Laminin, an integral component of basement membranes, consists of three subunit polypeptides, A, B1, and B2 chains. We have recently isolated cDNAs corresponding to human laminin A chain. These cDNAs were utilized for chromosomal in situ hybridizations to establish the genomic location of the laminin A chain gene. Metaphase chromosomes of PHA-stimulated human peripheral blood leukocytes were examined by in situ hybridization with 3H-labeled cDNAs, and the chromosomes were identified by R-banding (fluorochrome-photolysis-Giemsa method). The results indicated that the human laminin A chain is at locus 18p11.3. Since human laminin B1 and B2 chain genes have been previously mapped to chromosomes 7 and 1, respectively, the results indicate that genes encoding human laminin chains reside in separate chromosomes.     

Laminin chains in the basement membranes of human thymus

Summary In recent studies, the α2 chain of laminin (Ln) has been suggested to be the only laminin α chain expressed in mouse and human thymus. We have now used chain-specific monoclonal antibodies and indirect immunofluorescence microscopy to study the expression of laminin chains in samples of foetal and 6-year-old human thymus. The subepithelial basement membrane of the capsule of foetal 16- to 18-week thymus presented a bright immunoreactivity for Ln α1, α3, β1, β3 and γ1 chains but not for α2 chain, suggesting the expression of laminins-1 and-5. Most cortical and medullary epithelial cells, including Hassall's corpuscles, however, lacked laminin immunoreactivity. Immunoreactivity for Ln β2 chain was only seen in basal laminae of larger blood vessels. In thymic specimens from 6-year-old children, immunoreactivity for the laminin α1, α3, β1, β3 and γ1 chains was invariably found in subepithelial basement membrane of the capsule and that for laminin α2 chain was now also distinct but more heterogeneous. Furthermore, the thymic subepithelial basement membrane of the capsule at all stages showed immunore-activity for collagen type VII, forming the anchoring fibres in epithelial basement membranes. The subcapsular thymic epithelium also showed immunoreactivity for the BP 230 antigen and β₄ integrin subunit, both components of hemidesmosomes. The present results show that the thymic subepithelial basement membrane of the capsule presents properties which are commonly seen in stratified and combined epithelia, and are compatible with suggestions of the antigenic similarity of thymic epithelial cells and keratinocytes.     

Interaction of abscisic acid and indole-3-acetic acid-producing fungi with Salix leaves

The molds Botrytis cinerea, Cladosporium cladosporioides, and the yeast Aureobasidium pullulans, isolated from the leaves of three short-rotation Salix clones, were found to produce indole-3-acetic acid (IAA). Abscisic acid (ABA) production was detected in B. cinerea. The contents of IAA and ABA in the leaves of the Salix clones and the amounts of fungal propagules in these leaves were also measured, in order to evaluate whether the amounts of plant growth regulators produced by the fungi would make a significant contribution to the hormonal quantities of the leaves. The content of ABA, and to a lesser degree that of IAA, showed a positive correlation with the frequency of infection by the hormone-producing organisms. The amounts of hormone-producing fungi on leaves that bore visible colonies were, however, not sufficiently high to support the claim that either the fungal production of ABA or IAA would significantly contribute to the hormonal contents of the leaves of the Salix clones. It is therefore suggested that the effect of fungal IAA production on plants is limited to the rhizosphere and that B. cinerea, which is a known pathogen, induces ABA production by the mother plant as a response to physiological stress.Abbreviations ABA abscisic acid - ABA-Me abscisic acid methyl ester - GC-MS-SIM gas chromatography-selected ion monitoring-mass spectrometry - IAA indole-3-acetic acid - IAA-Me indole-3-acetic acid methyl ester Author for correspondence.     

Spatial variation in infection by digenetic trematodes in a population of freshwater snails (Potamopyrgus antipodarum)

Larval digenetic trematodes commonly castrate their first intermediate hosts, and should therefore impose strong selection on the timing and mode of host reproduction. Here we examine spatial variation in infection by trematodes in the freshwater snail Potamopyrgus antipodarum. Snails were collected at 11 different sites at Lake Alexandrina on the South Island of New Zealand from transects that ran perpendicular to the shore and across several different habitat types (from 0 to 8 m deep). Logistic regression was used to analyze the relationships between the frequency of trematode infection and snail size, habitat type, and transect location. On average, the probability of infection increased 3.3 times with each 1 mm increase in shell length. Prevalence of infection by the most common species of trematode, Microphallus sp., was highest in the shallow-water habitats where its final hosts (waterflow) spend most of their time. Prevalence of infection by another parasite, Telogaster ophistorchis (final host: eels) increased with depth, but because Microphallus was much more common, total infection by all trematodes decreased with depth. The effects of transect location were minor for Telogaster, but there was significant variation in Microphallus prevalence among transects, especially in the shore-bank habitat. Taken together, these results suggest that the risk of infection is spatially variable, but generally higher in shallow-water habitats, which may explain the greater frequency of sexual individuals as well as earlier reproduction among individuals near shore.     

Phenotypic plasticity and priority rules for energy allocation in a freshwater clam: a field experiment

We studied resource allocation among maintenance, reproduction and growth in the freshwater clam Anodonta piscinalis. Recent theoretical and empirical studies imply that organisms with indeterminate growth may have priority rules for energy allocation. That being so, the traits involved should potentially be capable of considerable phenotypic modulation, as a mechanism to adjust allocation. We tested this hypothesis using a 1-year reciprocal transplant experiment at six sites. Experimental clams were caged at higher than natural densities in order to detect any phenotypic modulation of the traits and discover the putative priority rules in energy allocation. We recorded the survival and shell growth of clams during the experiment, and the reproductive output, somatic mass and fat content of clams at the end of the experiment. Shell growth, somatic mass, and the reproductive output of females varied more among transplant sites than among the populations of origin, suggesting a high capacity for phenotypic modulation. However, the reproductive investment, somatic mass and shell growth were also affected by origin; clams from productive habitats invested more in reproduction and were heavier. In comparison to undisturbed clams, the reproductive output of the experimental clams was similar and their fat content was higher, whereas their shell growth was considerably slower and their somatic mass lower. These results suggests that when resources are limiting (due to high density) reproductive allocation overrides allocation to somatic growth. The highest mortality during the experiment coincided with the period of reproductive stress in the spring. Additionally, the proportion of reproducing females was lower in those transplant groups where the survival rate was lowest, suggesting that maintenance allocation overrides allocation to reproduction when available resources are scarce. The results of this field experiment support theoretical predictions and results of previous laboratory experiments that suggest that there are priority rules for energy allocation in organisms with indeterminate growth.     

Changes in the distribution of integrins and their basement membrane ligands during development of human thyroid follicular epithelium

Interactions between cells and basement membrane components are crucial for the regulation of epithelial cell differentiation and polarization. We have studied by immunohistochemical methods the distribution of integrin adhesion proteins and some of their basement membrane ligands in foetal (16--19 weeks) and adult thyroid follicular epithelia. A diffuse immunoreactivity for only 3, v and 1 integrins was found in foetal follicular epithelium, whereas in adult follicular epithelium these integrins were expressed basally in a polarized manner. Additionally, 3 integrin was seen in a more basolaterally confined manner in adult follicular epithelium. Among basement membrane components, laminin 1, 1, 1 and 2 chains were found in epithelial basement membranes of the foetal thyroid gland, suggestive of the presence of laminins-1 and -3. In contrast, the basement membranes of adult follicular epithelium presented a much weaker immunoreactivity for the laminin 2 chain. Furthermore, immunoreactivity for the laminin 2 chain was occasionally seen in adult thyroid glands, apparently confined to myofibroblasts. Immunoreactivity for type IV collagen 1 and 2 (IV) chains was found in follicular basement membranes of foetal as well as adult thyroid gland. The results suggest that during maturation of foetal thyroid follicular epithelium a distinct polarization of integrins takes place. In mature thyroid follicular epithelium, the presumable adhesion-mediating integrin complexes are 31, v1 and/or v3 mediating adhesion to laminin-1 (1-1- 1) and type IV collagen trimer 12 (IV)     

Effects of bovine colostrum supplementation on serum IGF-I, IgG, hormone, and saliva IgA during training

Mero, Antti, Heidi Miikkulainen, Jarmo Riski, RaimoPakkanen, Jouni Aalto, and Timo Takala. Effects of bovinecolostrum supplementation on serum IGF-I, IgG, hormone, and saliva IgAduring training. J. Appl. Physiol.83(4): 1144-1151, 1997.The purpose of this study was to examinethe effects of bovine colostrum supplementation (Bioenervi) on seruminsulin-like growth factor I (IGF-I), immunoglobulin G, hormone, andamino acid and saliva immunoglobulin A concentrations during a strengthand speed training period. Nine male sprinters and jumpersunderwent three randomized experimental training treatments of 8 daysseparated by 13 days. The only difference in the treatments was thedrink of 125 ml consumed per day. Posttraining increases were noticedfor serum IGF-I in the 25-ml Bioenervi treatment (125 ml contained 25 ml Bioenervi) and especially in the 125-ml Bioenervi treatment (125 mlcontained 125 ml Bioenervi) compared with the placebo (normal milkwhey) treatment (P < 0.05). The change in IGF-I concentration during the 8-day periods correlated positively with the change in insulin concentration during the sameperiods with 25-ml Bioenervi treatment(r = 0.68;P = 0.045) and with 125-ml Bioenervitreatment (r = 0.69;P = 0.038). Serum immunoglobulin G,hormone, and amino acid and saliva immunoglobulin A responses weresimilar during the three treatments. It appears that a bovine colostrumsupplement (Bioenervi) may increase serum IGF-I concentration inathletes during strength and speed training.

     

Conversion of type II procollagen to collagen in Vitro: Removal of the carboxy-terminal extension is inhibited by several naturally occurring amino acids,polyamines, and structurally related compounds

Chick embryo sterna, which actively synthesize type II procollagen, were pulse-labeled with radioactive proline; protein synthesis was then inhibited by unlabeled proline and cycloheximide. After the inhibition of protein synthesis, several amino acids, polyamines, or structurally related compounds were added to the incubation medium. The conversion of procollagen, first to two intermediates, pC-collagen and pN-collagen, and then to collagen, was monitored by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The addition of 50 mm β-alanine, arginine, asparagine, glutamine, hydroxylysine, lysine, or ornithine, as well as agmatine, ?-aminocaproic acid, S-2-aminoethylcysteine, cadaverine, canavanine, putrescine, or spermine clearly inhibited the removal of the carboxy-terminal extension and pC-collagen accumulated; the removal of the amino-terminal extension was not affected. The inhibition of the conversion was reversible and unaffected by fetal calf serum. The results suggest that the conversion of type II procollagen to collagen requires at least two separate proteinases for the removal of amino-terminal and carboxy-terminal extensions. The results further suggest that naturally occurring molecules may be used to modulate the rate of conversion of procollagen to collagen, and development of analogs of these compounds may provide the means to interfere with excessive deposition of collagen in diseases with tissue fibrosis.