Basement membrane and epithelial features of fetal-type Leydig cells in rat and human testis

The basement membranes of developing Leydig cells in fetal and newborn testis of rat were studied by ultrastructural and immunocytochemical methods. Fetal-type Leydig cells in prenatal rats were organized in irregularly outlined groups in the interstitium and were extensively surrounded by ultrastructurally identifiable basement membranes and immunocytochemically localized laminin and collagen type IV. Prenatal Leydig cell precursors had small patches of laminin and collagen type IV on their surfaces, which indicated that changes in extracellular matrix took place during their differentiation to mature fetal-type Leydig cells. Additionally, ultrastructural evidence was obtained for a basement membrane surrounding the fetal human Leydig cells similar to that in fetal rats. Soon after birth the rat fetal-type cells gathered into distinct clusters surrounded by delicate envelope cells and a discontinuous basement membrane. Basement-membrane structures, laminin, and collagen type IV were observed between the clustered cells as well. The basement membranes covering large cell surface areas of the fetal-type Leydig cells in fetal and newborn rats differed from those of the adult-type cells, which, according to our earlier study, are covered only by small patches of basement membrane. The difference between the basement membranes of the fetal- and adult-type rat Leydig cells further supports the concept of two different Leydig cell populations. The earlier findings of the epithelial nature of the Leydig cells agree with the observation of basement membranes in the Leydig cells.     

Field test of resource regulation by the bud-galling sawfly, Euura mucronata, on Salix cinerea

The attack by the bud-galling sawfly, Euura mucronata , on its willow host was simulated by removing every other living bud from previous year's shoots in a natural stand of Salix cinerea . The impact of simulated attack on the growth of the willow and the subsequent attack by E. mucronata was estimated after one growing season. Experimental bud-killing resulted in a growth reaction in S. cinerea characterized by significant increase in the length of new shoots produced by the remaining buds Also the number of buds and E. mucronata galls per shoot were significantly higher on bud-removal branches as compared with control branches. The overall survival of E. mucronata larvae was better on long shoots than on short shoots. The result shows that sawfly attack increases the amount of high quality resources for the subsequent generation of the same sawfly as predicted by "resource regulation hypothesis". We conclude that these highly specialized insect herbivores have adapted to utilize and to maintain the willow's juvenile traits. Rapid regrowth of willows after damage may have originally arisen as an adaptation in response to other, less specific pressures such as mammal browsing or snow and ice damage.     

Differentiation of smooth muscle cells in the fetal rat testis and ovary: localization of alkaline phosphatase,smooth muscle myosin,F-actin,and desmin

Summary The histochemical demonstration of alkaline phosphatase (AP) activity and localization of smooth muscle myosin (SMM), F-actin, and desmin were carried out on frozen sections of testes and ovaries from 15-day-old fetal to newborn rats. The presence of immunocytochemically localized SMM and desmin was confirmed by Western blot analysis of proteins from isolated gonads. The development of smooth muscle cells was predominant in the testis. The first SMM-positive cells with an increasing intensity for F-actin and desmin appeared in the testicular tunica albuginea and around the testicular cords by the age of 16 days. A continuous layer of SMM- and F-actin-positive (but not uniformly desmin-positive) myoid cells was detected in the newborn testis. In the early gonads and in the newborn ovary, a majority of the interstitial cells expressed desmin, indicating that, in undifferentiated tissues, non-myogenic cells may also express desmin. During fetal development, male and female gonocytes showed a decrease in F-actin content but retained their high AP activity. In the cortex of the newborn rat ovary, the observed high AP activity and the presence of desmin may be associated with the postnatal histogenesis of the follicles. The presence of SMM-containing cells in the hilus of the ovary may be required for the demarcation of the ovary from the mesonephros by the constriction of the mesovarium. The occurrence of SMM-positive cells predominantly in male fetuses suggests that the development of the contractile cells in the fetal testis may be induced by testicular androgens.     

Huntington disease in Finland: a molecular and genealogical study

Summary Huntington disease (HD) is found at exceptionally low frequency in the Finnish population. In this population, linkage disequilibrium was earlier established with markers from the D4S10 and D4S43 loci. We now report a continuation to the restriction fragment length polymorphism haplotype analysis, in combination with a genealogical study of all the Finnish HD families. When the HD pedigrees were systematically traced to the 18th century, only one consanguinity was found, and a high percentage (28%) of the families had foreign ancestors. The majority of the Finnish ancestors were localized to border regions or trade centers of the country following the old postal routes. The observed high risk haplotypes formed with markers from the D4S10 and D4S43 loci were evenly distributed among the HD families in different geographical locations. Consequently, the HD gene(s) has most probably arrived in Finland on several occasions via foreign immigrants during the last few centuries.     

Pericellular substrates of human mast cell tryptase: 72,000 dalton gelatinase and fibronectin.

Migrating cells degrade pericellular matrices and basement membranes. For these purposes cells produce a number of proteolytic enzymes. Mast cells produce two major proteinases, chymase and tryptase, whose physiological functions are poorly known. In the present study we have analyzed the ability of purified human mast cell tryptase to digest pericellular matrices of human fibroblasts. Isolated matrices of human fibroblasts and fibroblast conditioned medium were treated with tryptase, and alterations in the radiolabeled polypeptides were observed in autoradiograms of sodium dodecyl sulphate polyacrylamide gels. It was found that an M(r) 72,000 protein was digested to an M(r) 62,000 form by human mast cell tryptase while the plasminogen activator inhibitor, PAI-1, was not affected. Cleavage of the M(r) 72,000 protein could be partially inhibited by known inhibitors of tryptase but not by aprotinin, soybean trypsin inhibitor, or EDTA. Fibroblastic cells secreted the M(r) 72,000 protein into their medium and it bound to gelatin as shown by analysis of the medium by affinity chromatography over gelatin-Sepharose. The soluble form of the M(r) 72,000 protein was also susceptible to cleavage by tryptase. Analysis using gelatin containing polyacrylamide gels showed that both the intact M(r) 72,000 and the M(r) 62,000 degraded form of the protein possess gelatinolytic activity after activation by sodium dodecyl sulphate. Immunoblotting analysis of the matrices revealed the cleavage of an immunoreactive protein of M(r) 72,000 indicating that the protein is related to type IV collagenase. Further analysis of the pericellular matrices indicated that the protease sensitive extracellular matrix protein fibronectin was removed from the matrix by tryptase in a dose-dependent manner. Fibronectin was also susceptible to proteolytic degradation by tryptase. The data suggest a role for mast cell tryptase in the degradation of pericellular matrices.     

Transforming growth factor beta alters plasminogen activator activity in human skin fibroblasts

Adult human skin fibroblasts were used as a model to study the effects of transforming growth factor beta (TGF beta) on the secreted plasminogen activator (PA) activity of cultured cells. TGF beta, at nanogram concentrations, enhanced the secretion of pro-PA from two fibroblast strains in a time- and dose-dependent manner. The induced enzymatic activity was inhibited by anti-urokinase antibodies and it co-migrated with purified urokinase in polyacrylamide gels. The secretion of PA activity was abolished when cycloheximide (0.1 microgram/ml) was added to the cultures. The activity was thus dependent on protein synthesis rather than just on direct activation of a plasminogen proactivator. TGF beta had only a slight mitogenic effect on the test cells. Epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin were ineffective alone in inducing PA. Insulin, on the contrary, had an inhibitory effect on the TGF beta-induced PA activity. In addition to its effects on the secretion of PA, TGF beta enhanced the production of a proteinase inhibitor by these cells. The results suggest a role for TGF beta in the regulation of PA activity and pericellular proteolysis in fibroblastic cells.     

Polyamine-phospholipid interaction probed by the accessibility of the phospholipidsn-2 ester bond to the action of phospholipase A2

Summary Conditions were used where the action of porcine pancreatic phospholipase A2 on phospholipids can be followed in the absence of added calcium and the catalytic activity is supported by the calcium brought with the nanomolar enzyme. Therefore, alterations in the enzyme velocity resulting from the presence of spermine or spermidine could be specifically studied using 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) and 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphoglycerol (PPHPG) as substrates. Both spermine and spermidine activated the hydrolysis of PPHPG fourfold at polyamine/phospholipid molar ratios of approximately 11 and 121, respectively. Double-reciprocal plots of enzyme activityvs. PPHPG concentration revealed the enhancement to be due to increased apparentV _max while the apparentK _m was slightly increased. In the presence of 4mm CaCl₂ inhibition by polyamines of PPHPG hydrolysis by phospholipase A2 was observed. Using synthetic diamines we could further demonstrate that two primary amino groups are required for the activation. In the absence of exogenous CaCl₂ polyamines inhibited the hydrolysis of PPHPC by phospholipase A2. The presence of 4mm CaCl₂ reversed this inhibition and a twofold activation was observed at 10 m spermine. The results obtained indicate that the activation of PLA2 by spermine and spermidine is produced at the level of the substrate, PPHPG. This implies the formation of complexes of phosphatidylglycerol and polyamines with defined stoichiometries.     

Growth control in chick embryo fibroblasts; no evidence for a specific role for cyclic purine nucleotides

Cyclic AMP and cyclic GMP concentrations were measured in cultures of normal chick embryo fibroblasts and those transformed by Rous sarcoma virus under different growth conditions. No significant and reproducible correlation between the nucleotide levels and the rate of proliferation was observed. Neither release of normal cells from density dependent inhibition of growth nor transformation of the cultures by different strains of Rous sarcoma virus affected the concentrations of cyclic AMP or cyclic GMP. Activities of cellular cyclic nucleotide phosphodiesterases, enzymes involved in regulating the level of the nucleotides, were not directly affected by growth-stimulating concentrations of insulin or neuraminidase. Growth stimulation by insulin did not alter the activities of cellular cAMP-dependent protein kinase. These results do not support the hypothesis that cyclic AMP or cyclic GMP has a specific role in the growth control of chick embryo fibroblasts.     

Transforming growth factor-beta does not alter interleukin-1 expression in cultured human macrophages

Transforming growth factor-beta (TGF beta) is a growth modulator that stimulates the growth of fibroblastic cells but inhibits the growth of cells of epithelial origin. TGF beta also influences the production of extracellular matrix proteins, and of proteases and the type 1 plasminogen activator inhibitor (PAI-1) by cultured cells. TGF beta appears also to have various immunoregulatory effects, suppressing both T- and B-cell activities. It has been proposed that it might increase the expression of interleukin-1 (IL-1) mRNA in cultured human monocytes, thus potentiating immune functions. To analyze the role of TGF beta in IL-1 production we have now quantitated the effect of this factor on the production of biologically active IL-1 as well as IL-1 beta mRNA expression. The effect of TGF beta on IL-1 production optimally activated with bacterial lipopolysaccharide (LPS) was also studied. It was found that IL-1 activity and mRNA levels were rapidly elevated by LPS but not by TGF beta. Culture fluids from monocytes treated with TGF beta alone or with TGF beta plus LPS inhibited the proliferation of the test thymocytes. After gel filtration, the media from TGF beta-treated cultures showed no activity in the molecular weight area of IL-1 (approx. 15 kD), while the supernatants from TGF beta plus LPS-induced cells contained IL-1 activity in these fractions, the magnitude of which was, however, at the same level as in the culture fluids derived from cells stimulated with LPS alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Does nitrogen fertilization have an impact on the trade-off between willow growth and defensive secondary metabolism?

The effect of nitrogen fertilization on the phytomass production, shoot length and leaf secondary phenolics in nine Salix myrsinifolia clones was investigated. Cuttings taken from 1-year-old and 2-year-old shoot parts of field cultivated clones were grown at three concentrations of nitrogen (7, 150 and 300 ppm) in a greenhouse for one growing season. The willow clones differed significantly in phytomass yield and secondary phenolics content. Nitrogen fertilization affected significantly the growth and secondary metabolism of willow clones. In most clones, the addition of nitrogen from a sub-optimum concentration (7 ppm) to an optimum concentration (150 ppm) appeared to reduce the amounts of salicortin, chlorogenic acid and unknown salicylate and increased shoot phytomass, but a supraoptimum nitrogen concentration (300 ppm) resulted in highly variable growth and secondary phenolic responses. A significantly negative correlation between leaf phytomass and amount of total phenolics at sub-optimum and optimum N-treatments indicates trade-off between growth and secondary metabolism in willow clones at these treatments. However, the leaf phytomass:total amount of phenolics ratio varied significantly among clones, and in all clones it was not significantly lower at sub-optimum N-treatment than at optimum N-treatment.