Properties of heart fibroblasts of adult rats in culture

Summary In the heart of the adult rat, fibroblasts are mainly responsible for the synthesis and deposition of the collagenous matrix. Because these cells in vitro may serve as an important model system for studies of collagen metabolism in heart tissue, we have cultured and characterized rat-heart fibroblasts from young adult and old animals. Conditions included use of media of different compositions with and without addition of ascorbate. Cell used were either cultured directly from fresh tissues or thawed previously frozen cells. Cultured cells were studied with respect to growth properties, morphology and ultrastructure and patterns of collagen. Heart fibroblasts generally resembled fibroblasts cultured from other tissues, but were more like skeletal muscle fibroblasts in that they deposited, in addition to type I collagen, type IV collagen and laminin. The fibroblasts showed a typical appearance in phase-contrast microscopy and electron microscopy. In the case of cells grown with added ascorbate, aligned collagen fibrils in the extracellular matrix showed a periodicity typical of type I collagen. The deposition of type I collagen occurred only in medium supplemented with ascorbate, and in that circumstance increased as a function of time past confluence; this was independent of the age of the animal from which the cells were obtained or of other changes of medium composition studied. Immunofluorescence studies with specific antibodies revealed that the cells deposited types I and IV collagens, laminin and fibronectin. In contrast to the case of type I collagen, the deposition of type IV collagen occurred in cells grown either with or without ascorbate. Direct observation of type IV collagen is consistent with the previous finding of type IV mRNA in cardiac fibroblasts in situ and in freshly isolated populations of these cells.     

Studies on well-coupled Photosystem I-enriched subchloroplast vesicles — energy-dependent switching between two different active states of the proton-translocation adenosine triphosphatase

Single-turnover flash-induced ATP synthesis coupled to natural cyclic electron flow in Photosystem I-enriched subchloroplast vesicles (from spinach) was continuously followed by the luciferin-luciferase luminescence. The ATP yield per flash was maximal (1 ATP per s per 1000 Chl) around a flash frequency of 0.5–2 Hz. It decreased both at lower and higher flash frequencies. The decrease at high flash frequency was due to limitation by the electron-transfer rate, while the decrease at low flash frequency was directly due to intrinsic properties of the ATPase itself. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) decreased the yield at low frequency more than at high frequency. The same behaviour was observed if electron transfer was artificially mediated by pyocyanin. If the ADP concentration was increased from 40 to at least 80 μM, or if the vesicles were preincubated with 5 mM dithiothreitol (DTT), the decrease of the yield at flash frequencies below 0.5 Hz was no longer observed. Incubation with DTT increased the rates of ATP hydrolysis and synthesis at any flash frequency. The decrease of the yield could be elicited again by addition of 50 nM FCCP. It is concluded that at low levels of the protonmotive force (Δ gm_H⁺), the ATPase is converted into an active ATP-hydrolyzing state in which ATP synthesis activity is decreased due to a decreased affinity towards ADP and/or to a decreased release of newly synthesized ATP, that can be cancelled by increasing the ADP concentration or by addition of DTT in the absence of uncoupler.     

Phylogenetic Survey of Proteins Related to Synapsin I and Biochemical Analysis of Four Such Proteins from Fish Brain

A phylogenetic survey of proteins immunologically related to Synapsin I, a major synaptic vesicle-associated phosphoprotein in mammals was carried out. Proteins antigenically related to Synapsin I were found by use of radioimmunoassay and other radioimmunochemical techniques in the nervous systems of several vertebrate and invertebrate species, which included birds, reptiles, amphibians, fish, echinoderms, arthropods, and mollusks. Four proteins present in fish brain, antigenically related to Synapsin I, were further studied and found to resemble mammalian Synapsin I in several respects. Like Synapsin I, the fish proteins were present in high amounts in nervous tissue, were enriched in synaptosomal fractions of brain where they were substrates for endogenous protein kinases, were acid extractable, and were sensitive to digestion by collagenase. In addition, two-dimensional peptide-mapping analysis revealed some homology between major phosphopeptide fragments of Synapsin I and the fish proteins. The results indicate that proteins related to Synapsin I are wide-spread in the animal kingdom.     

Characteristics of the relative maximum in the decay of delayed light emission from Pothos leaf following far-red excitation

Following illumination with wavelengths longer than 700 nm, the intensity of light emission from Pothos aurea leaf falls for 1 min and then increases to a maximum after 2 min in the dark. The spectrum of this minute-range liminescence matches that of prompt fluorescence excited at the same wavelength, but differs from that of prompt or minute-range delayed emission excited by wavelengths shorter than 700 nm. This emission is less sensitive to heat damage than millisecond delayed emission, and may originate from photosystem I.     

Conversion of atriopeptin II to atriopeptin I by atrial dipeptidyl carboxy hydrolase

We are examining the substrate specificity of atrial dipeptidyl carboxyhydrolase, a membrane-bound metallo enzyme that we isolated from bovine atrial tissue homogenates. This enzyme readily removes the dipeptide, Phe-Arg, from Bz-Gly-Ser-Phe-Arg, a stand-in substrate for atriopeptin II, one of several atrial natriuretic factors. We now report that the atrial enzyme cleaves the C-terminal dipeptide, Phe-Arg, from atriopeptin II to form atriopeptin I. The km (pH 7.5) is 25 microM and the ratio of relative Vmax/km as a measure of substrate specificity indicates that atriopeptin II is a 240-fold better substrate than Bz-Gly-His-Leu. Only Phe-Arg was detected as a hydrolysis product, indicating that sequential cleavage of Asn-Ser from atriopeptin II does not occur, and that atriopeptin I is not a substrate. Bz-Gly-Asn-Ser was as good a substrate for the atrial enzyme as Bz-Gly-His-Leu, but Bz-Cys(bzl)-Asn-Ser was not hydrolyzed. This result suggests that the presence of an intact disulfide bond or an S-alkylated residue in the P1 position of a substrate (as in atriopeptin I) prevents hydrolysis by the atrial enzyme. Comparative studies were made with the angiotensin I converting enzyme. Atriopeptin II was not a substrate. The stand-in substrates for atriopeptin I, Bz-Cys(bzl)-Asn-Ser and Bz-Gly-Asn-Ser were barely hydrolyzed, which by itself suggests that atriopeptin I is not a substrate of the angiotensin converting enzyme. Our results strongly suggest that atriopeptin II is converted to atriopeptin I and that hydrolysis is mediated by the atrial enzyme. The angiotensin I converting enzyme plays no role in processing these peptides. We suggest that the atrial enzyme be named atrial peptide convertase.     

Diversity in the immune system: “Preconceived ideas” or ideas preconceived?

Two concepts of the evolution and regulation of expression of the combining site repertoire of the immune system, are compared. One view is based on the Associative Recognition Theory as formulated by the author and the other is based on the Idiotype Network Idea as conceived by Jerne. The two concepts are analyzed from the point of view of their logic, internal consistency and factual support.     

Phosphoenolpyruvate-dependent phosphorylation of a 55-kDa protein of Streptococcus faecalis catalyzed by the phosphotransferase system

Abstract A protein with an M _r of 55000 was isolated from glucose-grown Streptococcus faecalis cells. The protein becomes phosphorylated in a phosphoenolpyruvate-dependent reaction catalyzed by enzyme I and HPr of the bacterial phosphotransferase system. It did not stimulate phosphoenolpyruvate-dependent glucose phosphorylation. Several sugars were tested for their ability to dephosphorylate the phosphorylated protein in the presence of membrane fragments. Even though some of the sugars were able to dephosphorylate phospho-HPr quickly, the factor III-like 55-kDa protein remained phosphorylated. We therefore assumed that this protein is not involved in any sugar uptake reaction but that it exerts a regulatory function in Gram-positive bacteria comparable to the function of factor III specific for glucose in Escherichia coli .     

Alpha 1- and beta 2-adrenergic receptors co-expressed on cloned MDCK cells are distinct glycoproteins

We have explored the molecular differences between alpha 1- and beta 2-adrenergic receptors that are co-expressed by a clonally-derived cell line, Madin-Darby canine kidney clone D (MDCK-D). MDCK-D membranes were pre-labeled with selective alpha 1- and beta-adrenergic radioligands and were then solubilized with the non-ionic detergent digitonin. Solubilized alpha 1- and beta 2-adrenergic receptors were retained by immobilized wheat germ agglutinin and were eluted following addition of N-acetyl-D-glucosamine or sialic acid. Both receptors were also retained by immobilized Limax flavus lectin, a sialic acid-binding lectin. Lectins that were specific for N-acetyl-D-glucosamine residues did not bind to these receptors. These results indicate that both alpha 1 and beta 2 receptors are sialylated glycoproteins. The solubilized alpha 1- and beta 2-adrenergic receptors migrated with different elution profiles from an Ultragel AcA 34 column. The apparent molecular sizes of the digitonin-receptor complexes were 68A for the alpha 1 receptor and 55A for the beta 2 receptor. These results show that alpha 1- and beta 2-adrenergic receptors can be present on the same cell as distinct sialic acid-containing glycoproteins.     

Triterpenes from Periandra dulcis roots

Three oleanane triterpenes were isolated from the roots of Periandra dulcis,and identified as 3β-hydroxy-25-al-olean-18-en-30-oic acid (periandric acid I), 3β-hydroxy-25-al-olean-12-en-30-oic acid (periandric acid II) and 3-oxo-25-hydroxy-olean-12-en-30-oic acid. The former two compounds (periandric acids I and II) were identical with the aglycones obtained by hydrolysis of periandrin I and II, respectively and the latter one was a new triterpene.