Transforming growth factor-beta and platelet-derived growth factor synergistically stimulate contraction by testicular peritubular cells in culture in serum-free medium

We report investigations on factors influencing contractility by testicular peritubular cells (PC) maintained in culture in a three-dimensional collagen gel system, and the behavior of PC in culture on a two-dimensional system. At low and moderate cell densities, PC embedded in collagen gels in serum-free Eagle's minimal essential medium (MEM) have a lesser degree of contractility than PC in culture in MEM containing calf serum. The contractility by PC, measured by determining changes in diameter of the collagen gel, was increased by addition of transforming growth factor-beta (TGF-beta) to serum-free MEM, and this was further enhanced by supplementing the medium with platelet-derived growth factor (PDGF). In the absence of TGF-beta, however, PDGF had no detectable effects on PC contractility. Other growth factors examined (epidermal growth factor, insulin, and fibroblast growth factor) did not influence the degree of contractility of PC in serum-free MEM in the presence or absence of TGF-beta. PC maintained in MEM supplemented with platelet-poor serum (PPS) have a lesser degree of contractility than their counterparts in MEM containing 2.5% calf serum. The addition of TGF-beta and PDGF to PPS-supplemented MEM restored contractility by PC to a level comparable to that observed by PC in MEM containing complete serum. The addition of nonpurified bovine serum albumin (BSA) to MEM greatly increased PC contractility. By contrast, highly purified BSA had no such effect, suggesting that one or more components adsorbed to the impure BSA was implicated. Polyclonal antibody against fibronectin did not influence the contractility of PC in collagen gels in the presence or absence of serum. Antiserum against TGF-beta partially blocked the enhancement of contractility of PC in MEM containing non-purified BSA. In PC plated on top of a collagen gel lattice, the attachment, spreading, and cell shape were greatly influenced by the presence of TGF-beta and PDGF, both singly and together. Data presented are interpreted to indicate that effects elicited by serum on the properties of PC in culture, and on the contractility of PC, can be attributed in part to the combined influences of TGF-beta and PDGF in serum.     

O-Methylated and sulfoconjugated catecholamines: differential activities at human platelet alpha 2-adrenoceptors.

The physiological effects of the sulfoconjugates of epinephrine, norepinephrine, and the 3-O-methylated catecholamines, metanephrine, normetanephrine, and methoxytyramine were examined with regard to their alpha 2-adrenoceptor binding properties and aggregation activity in human platelets. Sulfoconjugation of catecholamines resulted in the loss of both their competitive potency for [3H]yohimbine binding and their influence on platelet aggregation. O-Methyl substituted catecholamines showed attenuation of their alpha 2-adrenoceptor binding affinities when compared with those of the corresponding non-esterified amines. Unlike the free amine epinephrine, which stimulated platelet aggregation, the O-methylated catecholamine derivatives inhibited aggregation. Inhibition was dose-dependent and restricted to the alpha 2-adrenoceptor mediated aggregation response stimulated by epinephrine (1 microM) or potentiated by subthreshold concentrations of epinephrine (30-300 nM) in the presence of subaggregatory doses of vasopressin (10-30 nM). Collagen- and ADP-induced platelet aggregation was not affected. The hydrophilic beta-antagonist CGP 12177 displayed no effects. However, high concentrations (0.1 mM) of both isomers of the strongly lipophilic beta-adrenoceptor antagonist propranolol inhibited the actions of all aggregators by stabilizing the membrane. Such a nonspecific membrane interaction of the methylated catecholamines could be excluded because of their low lipid solubility calculated in a n-octanol-phosphate buffer system at pH 7.4. We suggest therefore that methylated catecholamines are biological alpha 2-adrenoceptor antagonists acting on alpha 2-adrenoceptor stimulated reactions of human platelets. Whether this receptor antagonism is relevant to other human tissues needs clarification. Sulfated catecholamines, however, are wholly ineffective at this receptor site and may constitute a pathway to control the concentration of the active free catecholamines.     

Biotechnological production of unnatural L-amino acids from D,L-5-monosubstituted hydantions. II. L-α- and L-β-naphthylalanine

Summary Resting cells ofArthrobacter sp. (DSM 3745) with the ability to form L-tryptophan from D,L-5-(3-indolylmethy)hydantoin were used for the bioconversion of D,L-5-- and D,L-5--naphthylmethylhydantoin (D,L-5-- and D,L-5--NMH) to the corresponding L-amino acids. Under the optimal reaction conditions of pH 9.7 and 40°C specific productivities of 0.2 (-naphtylalanine) and 0.6 (-naphtylalanine) mM amino acid x g cell dry mass^–1 x h^–1 were obtained in a 0.1 M Na₂CO₃/NaHCO₃-buffer in a strirred bioreactor.     

Marine biosurfactants. IV. Production,characterization and biosynthesis of an anionic glucose lipid from the marine bacterial strain MM1

Summary During screening for biosurfactants among marine, n-alkane-utilizing bacteria, low- and high-molecular surface-active substances were detected. The marine bacterial strain MM1 was found to synthesize a novel glycolipid that has not so far been cited in the literature. Both ¹H, ¹³C-nuclear magnetic resonance spectroscopic and positive ion fast atom bombardment mass spectrometer studies led to the identification of a glucose lipid consisting of four 3-OH-decanoic acids, which are linked together by ester bonds. The lipophilic moiety is coupled glycosidically with C-1 of glucose. The glucolipid reduced the surface tension from 72 mN/m to 30 mN/m while the minimum interfacial tension towards n-hexadecane was lowered to values smaller than 5 mN/m. Correspondence to: S. Lang     

Directed mutagenesis of DNA cloned in filamentous phage: influence of hemimethylated GATC sites on marker recovery from restriction fragments.

Gapped duplex DNA molecules of recombinant genomes of filamentous phage are constructed in vitro. Denatured restriction fragments covering (part of) the precisely constructed gap are hybridized to the gapped duplex DNA molecules to form ternary duplices. The two strands of the ternary duplex molecules carry different genetic markers within the region spanned by the restriction fragment leading to a one base pair mismatch or to an insertion loop of 93 nucleotides, respectively. The two strands also vary with respect to A-methylation in GATC sites. In cases of asymmetrical methylation, transfection of E. coli with these heteroduplex molecules leads to marker recoveries with a pronounced bias in favour of the marker encoded by the methylated strand. This effect at least partly explains the comparably low marker yields achieved in previous directed mutagenesis experiments using filamentous phage as the vector. The results suggest how these procedures can be optimized. Precise construction of a 93 bp insertion of 9.5% marker yield is described.     

Spectroscopic and kinetic evidence for a cyclic geminal diamine intermediate in the reaction of O-aminoserine with pyridoxal 5'-phosphate in alkali

HT-29, a cell line derived from a human colon carcinoma, exhibits very low alkaline phosphatase activity. The enzyme is thermolabile and is of the intestinal type. Hyperosmolality and/or sodium butyrate induce increased levels of activity. The increase is most pronounced with HT-29 cells growing in hyperosmolar medium containing sodium butyrate. Under these conditions specific activity rises over 1000-fold. The effect of hyperosmolality is blocked by cycloheximide and that of sodium butyrate by thymidine, cordycepin, and cycloheximide. By contrast to other human cancer cell lines, the enzyme of HT-29 is not influenced by cell density and glucocorticoid hormones. 5-Bromo-2′-deoxyuridine and inhibitors of DNA synthesis cause a slight increase in specific activity.     

The tetracycline resistance transposons Tn1721 and Tn1771 have three 38-base-pair repeats and generate five-base-pair direct repeats

Summary The 10.7 kilobase (kb) tetracycline resistance transposons Tn1721 and Tn1771, isolated from disparate sources, are completely homologous on the basis of heteroduplex analyses. Both transposable elements are capable of forming multiple duplications of a 5.3 kb portion encompassing the resistance genes (tet region). A model accounting for both, recA-independent translocation and recA-dependent amplification, postulates two direct and one inverted repeat as essential constituents of the transposons. DNA sequence analyses of Tn1721 and Tn1771 have substantiated this model. They demonstrated three identical 38 base pair repeats identically in both transposons dividing them into a minor transposon and a tet region. Identical sequences of at least 87 base pairs providing recombination hot spots for gene duplication have been found at the ends of the repetitious tet region. Translocation of Tn1721 and Tn1771 generates five base pair direct repeats at the respective sites of insertion. On the basis of the heteroduplex molecules and sequences analyzed the two transposons are identical.To Professor Wolfram Heumann on the occasion of his 65th birthday     

Nectar parasitism of Asclepias syriaca by ants: Effect on nectar levels,pollinia insertion,pollinaria removal and pod production

Summary Milkweed (Asclepias syriaca L.) umbels and stems attended by ants (Lasius neoniger Emery and Tapinoma sessile (Say)) initiated significantly fewer pods and showed a trend to produce fewer mature pods than did umbels and stems not attended by ants. Since similar numbers of pollinia were inserted in ant-attended flowers, we hypothesize that ant-excluded flowers obtain more pollinia from other clones than do ant-attended flowers of this normally non-selfing species. If pollinators from other clones first visited flowers not depleted of nectar by ants, they could provide this source of foreign pollinia. Our experiments suggest that nocturnal noctuid and geometrid moths can provide these pollinia.     

An Analysis of γ-Aminobutyrate Receptors and Uptake by Isolated Purkinje Cells

Abstract: An isolated fraction of Purkinje perikarya was prepared. This fraction had [³H]GABA receptor binding and [³H]muscimol binding analogous to that reported for heterogeneous cerebellar membranes. The finding of two binding components with each ligand suggests that these components do not represent two binding sites, one presynaptic and the other postsynaptic, since both are clearly seen on purified Purkinje cell bodies. Subcellular fractionation indicates that synaptic endings and plasma membranes are enriched in GABA receptors compared with intracellular organelles. Purkinje cell bodies were also found to possess a high-affinity transport system for GABA, which was sensitive to inhibition by diaminobutyric acid (DABA) but not by β-alanine. They showed no evidence of homoexchange (the movement of label without net transport). This supports our suggestion that homoexchange is an artifact of synaptic particle formation.