Incorporation of proline and aromatic amino acids into cell walls of maize coleoptiles

Sections excised from maize coleoptiles incorporated radioactivity from proline, tyrosine, and phenylalanine into structural components of the cell wall. Only about 2% of radioactivity from proline taken up by sections was incorporated into cell wall; about 24% of that incorporated was in hydroxyproline and the rest remained in proline. In contrast, as much as 40% of the radioactivity from phenylalanine and 30% from tyrosine was incorporated into cell wall material. Most of this radioactivity was in saponifiable ferulic acid. Small amounts of p-coumaric and diferulic acid were found, but only a small fraction of the hemicellulose can possibly be immobilized directly through cross-linking of diferulic esters. Substantial amounts of radioactivity from aromatic amino acids remained insoluble after strong alkali extractions of wall material, and a large fraction of polysaccharide was solubilized by dilute alkali following oxidation of phenolics by acidic NaClO₂. Hence, hemicellulosic material in the cell walls of maize coleoptiles may be organized and cross-linked primarily through alkali-resistant etherified aromatics.     

Prostaglandin D2 metabolite stimulates collagen synthesis by human osteoblasts during calcification

We investigate the effect of the prostaglandin D₂ metabolite Δ¹²−PGD₂ (9−Deoxy−Δ⁹, Δ¹²−13,14-dihydroprostaglandin D₂) on collagen synthesis in human osteoblast. Δ¹²-PGJ₂ at 10⁻⁵M enhanced collagen synthesis in the presence of 2 mM α-glycerophosphate-2Na. The stimulative effect appeared as early as 3 days after addition and continued until 22 days. The enhancement of type I collagen synthesis was confirmed by polyacrylamide gel electrophoresis. The potency was the same as 10^1t-8M 1 α, 25 dihydroxy vitamine D₃ (1,25(OH)₂D₃). Northern blot analysis showed that 10⁻⁵M Δ ¹²-PGD₂ and 10⁻⁸M 1,25(OH)₂D₃ enhanced the transcribtion of type 1 procollagen (α₁) mRNA levels in osteoblasts.     

The regulatory influence of prostaglandins on protein synthesis in the human non pregnant cervix

Cervical connective tissue was obtained from non pregnant fertile women undergoing hysterectomy. Tissue specimens were mechanically chopped into 1 mm thick slices which were preincubated in Krebs-Ringer bicarbonate buffer containing PGE₂ or PGF_2α (300 ng/ml). After 60 min the slices were transferred into fresh buffer with the corresponding PG-concentration and ³H-labelled proline or hydroxyproline. Following incubation (60 min) the protein bound radioactivity was determined and related either to the dry weight or to the protein content of each slice. The two methods used did not show any qualitative differences. The experiments showed that PG:s had a marked effect on protein synthesis in the cervical tissue. During the follicular phase incubation with both types of PG:s was followed by a decreased incorporation indicating decreased net synthesis of collagen while there was an increased incorporation and hence increased synthesis in the luteal phase. There was no significant influence on the distribution of the model amino acid ¹⁴C-AIB in the presence of PG:s neither in the follicular nor in the luteal phase. The present data point to an acute effect of PGE₂ and PGF_2α on cervical collagen metabolism and indicate furthermore that the process is steroid hormone dependent. It is concluded that these substances may exert their effect by modulating the incorporation of amino acids into protein.     

Characterization of the stimulatory effects of PGF2α on the release of arachidonic acid

Fibroblasts derived from a rat carrageenin granuloma were cultured in the presence of radioactive arachidonic acid, palmitic acid and linoleic acid. More than 90% of each labeled fatty acid was incorporated into a phospholipid fraction by the cells in 18 hrs. Arachidonic acid was evenly incorporated into phosphatidylcholine and phosphatidylethanolamine, while both palmitic acid and linoleic acid were almost entirely incorporated into phosphatidylcholine. The position of phosphatidylcholine where the fatty acids were incorporated was different for each fatty acid. The ratio of the amount of fatty acid incorporated into the 2-position to the amount incorporated into the 1-position of phosphatidylcholine for each fatty acid was >90% for arachidonic acid, 2:1 for palmitic acid and 5:1 for linoleic acid. In the case of phosphatidylethanolamine, most arachidonic acid (>90%) was incorporated into the 2-position. PGF_2^α caused the stimulation of arachidonic acid release but not of palmitic acid and linoleic acid from pre-labeled fibroblasts.The serum in the medium was completely replaceable by bovine serum albumin. The effect of PGF_2^α increased with an increasing concentration of bovine serum albumin, suggesting that serum only acts as a ‘trap’ for released arachidonic acid. The effect of PGF_2^α was greater than bradykinin, and no synergistic effect was seen, although an additive effect was observed.The effect of PGF_2^α depended on the concentration of calcium ions under magnesium-supplemented conditions.     

Hydrogen Sulfide Alleviates Myocardial Collagen Remodeling in Association with Inhibition of TGF-β/Smad Signaling Pathway in Spontaneously Hypertensive Rats

The study was designed to explore the role and possible mechanisms of hydrogen sulfide (H₂S) in the regulation of myocardial collagen remodeling in spontaneously hypertensive rats (SHRs). We treated nine-week-old male SHRs and age- and sex-matched Wistar–Kyoto rats (WKYs) with NaHS (90 μmol/kg⁻¹·day⁻¹) for 9 wks. At 18 wks, plasma H₂S, tail arterial pressure, morphology of the heart, myocardial ultrastructure and collagen volume fraction (CVF), myocardial expressions of collagen I and III protein and procollagen I and III mRNA, transforming growth factor-β1 (TGF-β1), TGF-β type I receptor (TβR-I), type II receptor (TβR-II), p-Smad2 and 3, matrix metalloproteinase (MMP)-13 and tissue inhibitors of MMP (TIMP)-1 proteins were determined. TGF-β1-stimulated cultured cardiac fibroblasts (CFs) were used to further study the mechanisms. The results showed that compared with WKYs, SHRs showed a reduced plasma H₂S, elevated tail artery pressure and increased myocardial collagen, TGF-β1, TβR-II, p-Smad2 and p-Smad3 expressions. However, NaHS markedly decreased tail artery pressure and inhibited myocardial collagen, TGF-β1, TβR-II, p-Smad2 and p-Smad3 protein expressions, but H₂S had no effect on the expressions of MMP-13 and TIMP-1. Hydralazine reduced blood pressure but had no effect on myocardial collagen, MMP-13 and TIMP-1 expressions and TGF-β1/Smad signaling pathway. H₂S prevented activation of the TGF-β1/Smad signaling pathway and abnormal collagen synthesis in CFs. In conclusion, the results suggested that H₂S could prevent myocardial collagen remodeling in SHR. The mechanism might be associated with inhibition of collagen synthesis via TGF-β1/Smad signaling pathway.     

Binding and receptor-mediated endocytosis of pregnancy zone protein-proteinase complex in rat macrophages

¹²⁵I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3–4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4°C was 2–3-times higher than binding of the homologous α₂-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (K_d approx. 60 pM). After binding to the receptor, this ligand was internalised within 2–3 min at 37°C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the α₂-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes.     

Synthesis of procollagen by odontogenic cells of rabbit tooth germ

Studies were performed to determine whether cultured odontogenic cells from rabbit tooth germ (RP cell) could synthesize dentine-like collagen. When cells were cultured with [¹⁴C]proline, 33% of the total incorporated proteins present were collagenous. Cultured RP cells were labelled with [¹⁴C]proline in the presence of β-aminopropionitrile. The resulting fractions, on analysis by CM-cellulose chromatography, contained three radioactive protein peaks, α1(I), [α1(III)]₃, α2. From the radioactive measurements, RP cells synthesized a significant amount of type III collagen, comparable to type I collagen.DEAE-cellulose chromatography was used to separate collagen molecules from collagen precursors. The results showed that 60% of total collagen precursor was type III precursor and the remainder was type I precursor.CM-cellulose chromatography of CNBr peptides of collagen from culture medium and cell extract revealed the presence of type I and type III collagen. Thus, the RP cell, which is a diploid cell, is unique in the predominance of type III collagen in culture, differing thereby from the character of collagen in vivo.     

Interrelationships among human aldo-keto reductase: immunochemical, kinetic and structural properties

We have propsed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α, β subunits, and aldehyde reductase II is a monomer of δ subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (α and β) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (α subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li₂SO₄(0.4 M) for the expression of maximum enzyme activity, and its K_m for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li₂SO₄ and its K_m for glucose is more than 200 mM. The β subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The β subunits hybridized with the α subunits of placenta aldehyde I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristics properties of placenta aldehyde reductase I.     

Thermal denaturation of soluble calf-skin collagen

1. Soluble calf-skin collagen has been denatured thermally between 37° and 60° and the component proteins have been separated on carboxymethylcellulose. 2. Four main fractions have been separated; α and β (in the nomenclature in common usage) and two other fractions. (The α and β components are complex owing to the presence of α₁, α₂, β₁ and β₂ parts). 3. Fractions 3 and 4 undergo rapid denaturation between 39° and 40° whereafter fraction 4 remains virtually unchanged even at 60°. 4. That portion of fraction 4 which remains at 60° is thought to be identical with the fraction designated γ by other workers, this fraction being composed of three α-chains in covalent linkage (such bonds are alkali-labile). 5. The equilibrium between α, β and fractions 3 and 4 is apparently reversible since acid-soluble collagen after denaturation at 45° or 60° followed by cooling to 0° for 30min. was found to contain only fraction 4 when chromatographed at 37°.     

Soluble Integrin Ligands and Growth Factors Independently Rescue Neuroblastoma Cells from Apoptosis under Nonadherent Conditions

We have investigated the role of extracellular matrix (ECM) and growth factors in the survival of nonadherent human neuroblastoma cells (line SK-N-BE). Cells cultured in serum-free medium under nonadherent conditions died with apoptotic-like features (cromatin condensation and nuclear fragmentation). SK-N-BE cells underwent neuronal differentiation in response to retinoic acid (RA). While RA itself did not induce apoptosis, differentiation increased the susceptibility of SK-N-BE cells to detachment-induced apoptosis. The appearance of the apoptotic-like phenotype required the maintenance in suspension of SK-N-BE cells for at least 16 h (12.43 ± 1.40% of cells undergoing apoptosis) and the percentage increased up to 46.84 ± 3.15% after 24 h. Suspension-induced apoptosis did not depend on increased intracellular Ca²⁺levels nor onde novoprotein synthesis and was not associated with extensive DNA degradation. Stimulation by soluble collagen I rescued suspended cells from apoptosis, even in the absence of cell adhesion and spreading. The survival promoting effect of ECM was mediated by the integrin receptors, since ([1]) the protective effect of soluble collagen I was blocked by anti-integrin antibodies to β₁and α₁subunits and ([2]) the antibody-induced clustering of α₁, α₃, α_v, β₁, and β₃integrins rescued SK-N-BE cells cultured in suspension from apoptosis. As expected, adhesion on immobilized ECM proteins, collagen I, or laminin (0.1 to 10 μg/ml) also rescued SK-N-BE cells from apoptosis in a dose-dependent manner. Thede novoprotein synthesis was required to promote the survival effect of ECM, since cycloheximide completely abolished the protective effect of collagen I and protection from apoptosis by ECM or by anti-β₁antibody was associated with the increased expression ofbcl-2.In addition to integrin stimulation, serum, insulin, and nerve growth factor inhibited suspension-induced apoptosis of SK-N-BE cells. The survival effect of serum and growth factors did not require the synthesis of new proteins, unlike the ECM effect. These data show that matrix proteins can promote cell survival in neuronal cells via integrin receptors. This effect does not require cell adhesion and the subsequent changes in cell shape as it can be mediated by soluble integrin ligands in suspended cells and involves a signaling pathway different from that triggered by growth factors.     

An abundant chick gizzard integrin is the avian α1β1 integrin heterodimer and functions as a divalent cation-dependent collagen IV receptor

The α-subunit of an abundant chick gizzard integrin was isolated ([12.], J. Biol. Chem. 262, 17,189–17,199) and fragmented by proteolytic digestion. The N-terminal sequences of the intact polypeptide and of several internal peptides were determined and were found to be highly homologous to the mammalian integrin α₁-subunit. Monoclonal antibodies to the chick integrin β₁-chain react on immunoblots with the gizzard integrin β-subunit ([28.], J. Biol. Chem. 265, 14,561–14,565). The chain composition of the abundant chick gizzard integrin is therefore α₁β₁. Polyclonal antibodies to the avian integrin α₁-subunit block attachment of embryonic gizzard cells to human and chick collagen IV completely and inhibit attachment to mouse Engelbreth-Holm-Swarm (EHS) tumor laminin partially. In ELISA-style receptor assays, the isolated α₁β₁ integrin bound to human and chick collagen IV and to mouse EHS tumor and chick heart laminin. While the binding to collagen IV was abolished by removal of divalent cations, the binding to laminin was not sensitive to EDTA under the conditions used. Collagen I bound the isolated avian α₁β₁ integrin only weakly. As collagen IV was the only extracellular matrix protein for which a consistent, divalent cation-dependent, binding to the avian α₁β₁ integrin could be demonstrated in both cellular and molecular assays we suggest that it is a preferred ligand for this integrin.     

Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K_a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABA_A receptors in neuronal populations within the olfactory bulb.     

Granulocyte-macrophage colony stimulating factor is anabolic and interleukin-1β is catabolic for rat articular chondrocytes

Objective: To study the effects of GM-CSF and IL-1β, both implicated in tissue damage in arthritis, on articular chondrocyte proliferation and metabolism, and to explore their agonist/antagonist effects. Methods: Chondrocytes were obtained from 1-month-old rats. First-passage monolayers were incubated for 24 h with or without GM-CSF and/or IL-1β, and labeled with ³H-thymidine, ³⁵S–SO₄ and ¹⁴C-proline. Proteoglycan and collagen synthesis were analyzed by liquid chromatography and SDS–PAGE. Gene expression was measured by RT-PCR. Results: IL-1β exerts potent, and GM-CSF weak, inhibitory effects on DNA synthesis. GM-CSF strongly stimulates, and IL-1β inhibits, proteoglycan and collagen synthesis. IL-1β suppresses the effect of GM-CSF, and increases the release of radioactive molecules from pre-labeled cartilage fragments; GM-CSF decreases the IL-1β-induced effect. Interestingly, both cytokines induce the expression of each other’s gene. Conclusions: IL-1β appears to be a catabolic and anti-anabolic agent for chondrocytes, whereas GM-CSF is mainly anabolic, and blocks the IL-1β-induced catabolic effect. It is postulated that both agents are implicated in inflammation: IL-1β promotes tissue catabolism and destruction, whereas GM-CSF enhances tissue reconstruction.     

Influence of 6-keto-PGF1α on collagen synthesis in the human cervix during various phases of the menstrual cycle

Uterine cervix tissue, obtained from nonpregnant fertile women undergoing hysterectomy, was mechanically chopped into 1 mm thick slices and incubated in Krebs-Ringer bicarbonate buffer containing 6-keto-PGF_1α (0.03–10 μg/ml) and ³H-proline. After incubation of 30–120 min the incorporated radioactivity was determined and related to the protein content of each slice. 6-keto-PGF_1α had specific and significant effects on the incorporation of ³H-proline into cervical tissue. In the follicular phase of the cycle a decreased incorporation was registered, indicating a reduced net synthesis of protein. However, increased radiolabelling was observed in the luteal phase, reflecting an augmented protein synthesis. It is suggested that 6-keto-PGF_1α, the stable metabolite of prostacyclin (PGI₂), has the ability to influence cervical protein metabolism and that this effect is steroid hormone dependent.     

β3-Adrenoceptor agonists for the treatment of frequent urination and urinary incontinence: 2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)phenoxy]-2-methylpropionic acid

In a search for novel analogues of β₃-adrenoceptor (AR) agonists relaxing the bladder for treatment of urinary dysfunction, 2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)phenoxy]-2-methylpropionic acids (1a–e), into which a fibrate-like structure had been incorporated, were synthesised. Compound 1a was found to be a selective β₃-AR agonist in functional assays using the ferret detrusor (β₃-AR), rat uterus (β₂-AR), and rat atrium (β₁-AR); β₃: EC₅₀=7.8 nM, β₂: IC₅₀=7,300 nM, β₁: EC₂₀=23,000 nM. The introduction of a chlorine atom or methyl substituent at the ortho-position on the phenyl ring of 1a further improved β₃-AR selectivity. In an in vivo study, 1a lowered intrabladder pressure (ED₅₀=31 μg/kg) in rats, without increasing heart rate, in keeping with the in vitro results. Consequently, it is proposed that 1a and its analogues (1b–e), possess β₃-AR agonistic activity in the absence of undesirable β₁- or β₂-AR mediated actions, and may be useful for clinical treatment and pharmacological studies.     

The production of recombinant human laminin-332 in a Leishmania tarentolae expression system

Laminin (LM)-332 (α3β3γ2), a large heterotrimeric glycoprotein, is an essential component of epithelial basement membranes that promotes cell adhesion and migration. Here, we expressed human LM-332 using a novel protein expression system based on the trypanosomatid protozoan host Leishmania tarentolae. Plasmids containing cDNA encoding full-length β3 and γ2 subunits and truncated α3 subunit were sequentially introduced into L. tarentolae. A recombinant strain harboring the three subunits of human LM-332 efficiently formed heterotrimer and secreted it into the culture medium. Heterotrimeric recombinant LM-332 (rLM-332) could be purified from culture medium with one-step immuno-affinity chromatography. The eluted fraction contained all three subunits, as confirmed by immunoprecipitation and immunoblotting. The purified rLM-332 showed similar cell adhesion activity to rLM-332 purified from mammalian cells, indicating its proper folding and assembly. The obtained expression level was not high; however, we suggest that this expression system has the potential for mass production of LMs for tissue engineering.     

Effects of products from macrophages, blood mononuclear cells and of retinol on collagenase secretion and collagen synthesis in chondrocyte culture

Collagenase secretion was studied in cultures of rabbit articular chondrocytes. Differentiation of the cells was assessed by characterizing the type of ³H-labelled collagen produced during treatment with (1) conditioned media from rabbit peritoneal macrophages and human blood mononuclear cells, and (2) with retinol, a potent cartilage resorbing agent in tissue culture. Conditioned media stimulated collagenase secretion. Total collagen synthesis was reduced due to a decrease of synthesis of α₁ chains; the amount of α₂ chains synthesized was unchanged. This is thought to be due to a reduction in type II synthesis. Retinol did not stimulate collagenase secretion. Total collagen synthesis was reduced by retinol. α₂ chain synthesis, however, was significantly increased, suggesting a switch of collagen synthesis in favor of type I collagen and, therefore, dedifferentiation. These results demonstrate that dedifferentiation of chondrocytes with respect to collagen synthesis is not necessarily associated with a stimulation of collagenase secretion.     

Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries

1. The molecular weights of the subunits of purified pig heart pyruvate dehydrogenase complex were determined by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis and were: pyruvate decarboxylase, α-subunit 40600, β-subunit 35100; dihydrolipoyl acetyltransferase 76100; dihydrolipoyl dehydrogenase 58200. 2. Inactivation of the pyruvate dehydrogenase complex by its integral kinase corresponded to the incorporation of 0.46nmol of P/unit of complex activity inactivated. 3. Further incorporation of phosphate into the complex occurred to a limit of 1.27nmol of P/unit of complex inactivated (approx. 3 times that required for inactivation). 4. Phosphate was incorporated only into the α-subunit of the decarboxylase. 5. The molar ratio of phosphate to α-subunits of the decarboxylase was estimated by radioamidination of amino groups of pyruvate dehydrogenase [³²P]phosphate complex by using methyl [1-¹⁴C]acetimidate, followed by separation of α-subunits by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis. Inactivation of the complex (0.46nmol of P/unit of complex inactivated) corresponded to a molar ratio of one phosphate group per two α-chains (i.e. one phosphate group/α₂β₂ tetramer). Complete phosphorylation corresponded to three phosphate groups per α₂β₂ tetramer. 6. Subunit molar ratios in the complex were also estimated by the radioamidination technique. Results corresponded most closely to molar ratios of 4 α-subunits:4 β-subunits:2 dihydrolipoyl acetyltransferase subunits:1 dihydrolipoyl dehydrogenase subunit.