Accumulation of procollagen in the degenerative articular cartilage of dogs with osteoarthritis

Collagen metabolism was studied in degenerative articular cartilage of dogs with spontaneous, early onset osteoarthritis. A fraction of collagen which represented about 1.5.% of the total was extracted from cartilage samples with dilute phosphate buffer (pH 7.4) containing 0.2% sodium dodecyl sulfate. Agarose gel filtration in the presence of sodium dodecul sulfate revealed that extracts of degenerative cartilage had about 24% procollagen whereas extracts of normal samples had only 3%. The isolated procollagen fraction was rechromatographed on agarose columns in the presence of mercaptoethanol. This resulted in the identification of a collagen species which migrated between marker β and α collagen chains. The molecular weight of this collagen was estimated to be 150000. Based on incorporation of [¹⁴C]proline, its ratio of hydroxy[¹⁴C]proline to total ¹⁴C was 0.32. Procollagen was not found after limited pepsin digestion (pH 3,4°C, 16 h) of degenerative cartilage samples.Since the total collagen content (μg hydroxyproline/mg cartilage), hydroxy[¹⁴C]proline/mg cartilage, specific radioactivity of hydroxy[¹⁴C]proline (cpm/μg), in the whole cartilage, and the specific radioactivity of hydroxyproline in the extractable collagen fraction were similar for normal and degenerative cartilage we propose that procollagen accumulated in the degenerative cartilage due to a partial defect in conversion of procollagen to collagen.     

Metabolism of tyramine and feruloyltyramine in TMV inoculated leaves of Nicotiana tabacum

TMV inoculation is known to stimulate tyramine N-feruloyl-CoA transferase activity in Nicotiana tabacum cv Xanthi n.c. leaves during the hypersensitive reaction. When [2-¹⁴C]-tyramine is fed for 2 hr to TMV inoculated leaf discs or detached leaves, ca 1 % of the supplied radioactivity is integrated into cinnamoyl-, p-coumaroyl- and feruloyltyramine and up to 14 % is integrated into the cell wall residue. [2-¹⁴C]-tyramine can only be partially released from this residue by acid hydrolysis. After nitrobenzene oxidation, 97 % of the radioactivity found in the cell walls is made soluble but only 13 % is recovered in p-hydroxybenzaldehyde. Feruloyltyramine is very rapidly metabolised, ca 20 % of the administrated radioactivity is found after 2 hr feeding in unindentified methanoi soluble metabolites. Acid hydrolysis of the cell wall fraction, which hydrolyses the amide bond of feruloyltyramine, releases labelled tyramine, while radioactivity is still detected in the acid insoluble residue. Label from [¹⁴C]-feruloyltyramine is integrated into this residue more quickly than from free [2-¹⁴C]-tyramine.     

Genetic alterations of ribonuclease-sensitive glycoprotein subunits of amino acid transport systems in Neurospora crassa conidia.

Neurospora crassa conidia have multiple and constitutive amino acid transport systems. Extraction by KCl releases amino acid-binding glycoproteins which have been purified by arginine affinity chromatography. Disappearance of certain fractions is coordinate with genetic lesions which reduce amino acid transport. Two such affinity fractions contain radioactivity when cells are grown on l-[¹⁴C]phenylalanine or on [¹⁴C]uridine, but not when cells are grown on [¹⁴C ]glucosamine. One purified arginine-binding fraction (B) contains 113 amino acid residues per minimum molecular weight. This glycoprotein also contains eight types of neutral sugar residues. No amino sugars were detected. Electrophoresis of crude extracts reveals five major Coomassie blue-staining species. The number of species is reduced, and the electrophoretic pattern is altered in extracts from transport-deficient strains. Tryptic “fingerprints” of these extracts indicate that mutations that reduce transport result in amino acid substitutions in the extractable glycoproteins. Nondialyzable material which absorbs light in the 260-nm region becomes dialyzable after digestion with RNase. Digestion of conidia with RNase reduces the amount of l-phenylalanine accumulated by the cells after 10 min of incubation with the amino acid.     

The role of the cotyledons in the photocontrol of hypocotyl extension in Cucumis sativus L.

Summary When pea seedlings lose about 5% of their water content the abscisic acid ((+)-ABA) level of the shoots increases ca. 20 times and the level of bound ABA, in all probability ABA-glucose, ca.7-10 times. After watering both ABA and bound ABA contents decrease within 24–48 h to the level in the control plants.After application of (±)-[2-¹⁴C] ABA to wilted pea shoots at the time of watering radioactive substances appear in the water-soluble, ether-insoluble fraction of ethanolic extracts and increase with time whereas radioactivity in the acidic ether fraction decreases. The neutral ether fraction remains free of radioactivity. Three radioactive zones, A, B, and C, are seen on chromatograms of the water-soluble fraction. A increases considerably within the entire experimental time, whereas B increases in the first 4–8 h after application and subsequently decreases. The third substance, C, which releases free ABA after hydrolytic treatment, does not change during the experiment. Chromatograms of the acidic ether fraction yield ABA and a substance staying at the origin, possibly phaseic acid and/or dihydrophaseic acid. Only the activity of ABA decreases during the experiment.     

Presence of di- and polyamines covalently bound to protein in rat liver

Acid hydrolysis of trichloroacetic acid precipitate from rat tissue (liver, kidney and testis) homogenate released significant amounts of acid-insoluble putrescine, spermidine and spermine. Following incubation of liver homogenate with [1,4-¹⁴C]putrescine, 1.4% of total radioactivity and 1.0% of labelled diamine were recovered in the acid-insoluble fraction. Exhaustive digestion of acid-precipitable material with proteinases (Pronase, aminopeptidase M, carboxipeptidase A, B and Y) revealed the presence of di- and polyamines and of N¹-(γ-glutamyl)spermidine, N¹-(γ-glutamyl)sperminine and N¹, N¹²-bis(γ-glutamyl)spermine. These derivatives were identified both by chromatographic analysis and by enzymatic digestion with purified γ-glutamylamine cyclotransferase. The finding of di- and polyamine γ-glutamyl derivatives in the proteinase-digested acid-insoluble fraction of homogenate may be considered as a proof of the in vivo transglutaminase-catalyzed binding of polyamines to proteins. This evidence suggests that di- and polyamines might have an important role in mammalian tissues through covalent binding to proteins by either one or both the primary amino groups.     

Synthesis of lipids in isolated nuclei from rat thymus and liver cells

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-¹⁴C]acetate and [1-¹⁴C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-¹⁴C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.     

Involvement of dolicholmonophosphate in the formation of specific mannosyl-linkages in yeast glycoproteins

A membrane fraction from Saccharomyces cerevisiae catalyzes the transfer of mannosyl residues from GDP-Man partly via dolicholmonophosphate into a heterogenous glycoprotein fraction. The pattern of radioactive products obtained after mannosylation with GDP-[¹⁴C]Man is similar to that obtained with dolicholmonophosphate-[¹⁴C]mannose. In each case more than 70% of the radioactivity can be released by β-elimination. Evidence is presented, that only the mannosyl residue directly linked to protein is incorporated via dolicholmonophosphate.     

Glucolipid Synthesis in Acanthamoeba castellanii*

SYNOPSIS. Cell-free preparations of Acanthamoeba castellanii trophozoites transfer glucose from UDP-[U-¹⁴C]glucose to a chloroform-soluble form. This radioactive material has been isolated by thin-layer chromatography; it contains an alkali-labile and an alkali-stable (unsaponifiable) component. Treatment of the enzymic product with 0.1 N KOH for 15 min at 0 C or 20 C releases radioactivity into the aqueous phase as glucose. During this treatment, 30–60% of the original glycolipid remains chloroform-soluble. It is considered to be an alkali-stable glycolipid because no further loss of radioactivity occurs during an additional 45-min of treatment with 0.1 N KOH. During incubation with 0.1 N HCI at 100 C glucose is released quantitatively from both the untreated glycolipid and the alkali-stable glycolipid with a half-time of 6 min. Glycolipid formation is inhibited by UDP and is reversible; extracts catalyze the formation of UDP-glucose from the alkali-stable glucolipid and UDP. The chemical and physical properties of the alkali-stable glycolipid are consistent with a glucosyl phosphoryl polyprenol structure. Extracts prepared from cysts catalyze the formation of glycolipids aiso, but the glucosyltransferase activity/cell decreases during the course of encystment. Radioactivity is incorporated into the fraction insoluble in chloroform-methanol-water (1:1:1:) during these incubations when UDP-[U-¹⁴C]glucose or [¹⁴C]glycolipid is the substrate.     

Comparison of in vivo and in vitro binding of polycyclic hydrocarbons to DNA

The in vivo binding of [³H]benzo(a)pyrene (BP) and 3-[³H]methylcholanthrene (3MC) to liver and lung DNA was studied in A/J mice. Only in liver was there any reduction in total DNA-bound radioactivity between 4 h and 24 h after administration of the hydrocarbon. DNA was fractionated on Sephadex LH-20 after enzymatic digestion. A single deoxyribonucleoside-BP adduct was detected whereas two major 3MC-adducts were observed. With both BP and 3MC, three additional peaks of radioactivity eluted rapidly in the lung DNA experiments while a fourth was noted with liver DNA. The nucleoside-bound adducts from lung represented a much larger proportion of the total radioactivity than with liver. In vitro analysis of 3MC binding to DNA showed the nucleoside-bound adducts to be predominantly deoxyguanosine-dependent but that the early peaks were independent of base suggesting binding to another part of the DNA molecule, perhaps phosphate, i.e., phosphotriesters.     

Metrizamide density gradients of sea urchin chromatin: fractions rich and poor in nascent DNA.

A crude, lightly sheared chromatin preparation obtained from a mixture of [methyl-3H] thymidine pulse and [2-14C] thymidine long-labeled sea urchin embryos (swimming blastulae), was centrifuged in metrizamide to form an isopycnic gradient. The buoyant density of the 3H pulse labeled chromatin was slightly higher than that of the 14C labeled bulk chromatin. The 3H/14C ratios in the higher and lower density regions of the overlapping radioactivity peaks, indicated the presence of fractions rich and poor in nascent DNA in these two density regions. After 15 min chase, the difference disappeared, indicating that the chromatin fractions with nascent DNA have a half-life shorter than 15 min.     

Tumor uptake studies of d,l-[5-14C]ornithine and d,l-2-difluoromethyl[5-14C]ornithine

The feasibility of d,l-[5-¹⁴C]ornithine ([¹⁴C]ornithine), a precursor for polyamine synthesis, and d,l-2-difluoromethyl[5-¹⁴C]ornithine ([¹⁴C]DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) were investigated for tumor localization. As an animal model, mice bearing mammary carcinoma, FM3A, were used. After i.v. injection of [¹⁴C]ornithine accumulation of radioactivity was observed in the FM3A, in which 43% of the ¹⁴C radioactivity was measured in the polyamine pool and 41% in the amino acid pool at 60 min after injection. Tumor uptake of [¹⁴C]DFMO was relatively low but constant during 60 min after injection. At 60 min after injection, 11% of the ¹⁴C was present in the acid-precipitable fraction of the FM3A, which suggests the formation of an irreversible complex of [¹⁴C]DFMO with ODC. For both compounds rapid blood clearance and high tumor-to-organ ratios were observed. Our results indicate that in connection with an enhanced polyamine synthesis in the tumors, the compounds investigated have potential as tracers for tumor detection.     

Modulation of HeLa cell phospholipid metabolism and ornithine decarboxylase activity by tumour promoters: regulation of response to phorbol-12,13-dibutyrate by receptor occupancy time

Experiments were carried out to determine the effect of receptor occupancy time on the response of HeLa cells to the tumor promoter phorbol-12,13-dibutyrate. Cells were exposed to the promoter for varying times, and the promoter then removed by washing. The biological responses that were measured were the release of radioactivity from cells pre-loaded with [³H]choline or [¹⁴C]arachidonic acid, the incorporation of [³H]choline into phospholipids and the induction of ornithine decarboxylase activity. Removal of phorbol-12,13-dibutyrate from the cells resulted in a rapid cessation of radioactivity release and choline incorporation. It was therefore concluded that continued receptor occupation is required to maintain the promoter induced changes in phospholipid metabolism. In addition, induction of ornithine decarboxylase activity did not occur unless the cells were exposed to phorbol-12,13-dibutyrate for at least 4 h.     

Incorporation of L-azetidine-2-carboxylic acid into hemoglobin in rabbit reticulocytes

L-Azetidine-2-car?ylic acid (AZA), the lower homologue of proline, was incorporated into hemoglobin in rabbit reticulocytes in vitro. Hydrolysis of [¹⁴C] AZA-hemolobin with 3 N p-toluenesulfonic acid and subsequent amino acid analysis resulted in recovery of 90% of the initial radioactivity in fractions containing added free AZA standard. An additional 6% of the radioactivity eluted with homoserine, a known degradation product of AZA. After tryptic digestion of [¹⁴C] AZA-hemoglobin, less than 9% of radioactivity eluted near added free AZA standard, while 91% of radioactivity was located in tryptic peptides elsewhere on the chromatogram. These data provide the first demonstration of AZA incorporation into a mammalian protein.     

Transport and distribution of homocarnosine after intracerebroventricular and intravenous injection in the rat

Rats were injected intracerebroventricularly (i.c.v.) or i.v. with [¹⁴C]homocarnosine (250 nmol). Distribution of the dipeptide in brain structures, transport from the brain to the blood, distribution in peripheral organs, and excretion in the urine were studied by measuring radioactivity in tissue, plasma, and urine samples by liquid scintillation counting 15–120 min after injection. After i.c.v. injection, [¹⁴C]homocarnosine was taken up into all parts of the brain investigated (highest uptake in structures close to the site of injection), it was transported to the blood, and radioactive substances were found in low concentration in muscle, spleen, and liver, in high concentration in the kidneys, and very high concentration in the urine. Investigations using high pressure liquid chromatography (HPLC) showed that no degradation took place in the brain, all radioactivity was found in the homocarnosine fraction. In the plasma 86% of the radioactivity was found in the GABA fraction presumed to be formed by cleavage of the peptide, while in the kidneys 35% and in the urine 40% was found in the GABA fraction. After i.v. injection of [¹⁴C]homocarnosine, no radioactivity was measured in hippocampus, striatum, cerebellum and cerebral cortex 15 min after injection, however, 60 min after injection a very low activity was detected in these structures (estimated intravascular radioactivity subtracted). A low activity was also measured in the spinal cord both 15 and 60 min after injection. When homocarnosine and GABA were separated on HPLC, all radioactivity in brain tissue was found in the GABA fraction, indicating either that [¹⁴C]homocarnosine did not cross the blood-brain barrier in amounts that could be measured with the method used, or that peptide entering the brain was rapidly transported back to the blood. [¹⁴C]Homocarnosine was not taken up either into crude synaptosomal preparations from hippocampus, striatum, cerebellum, cortex and spinal cord, or into slices prepared from the hippocampus and striatum. Transport from the brain to the kidneys and excretion in the urine seems to be a major route for disposal of this peptide in the rat.     

Protein Synthesis in the Isolated Forespores from Sporulating Cells of Bacillus subtilis

Developing forespores were isolated from Bacillus subtilis at different stages of sporulation and protein synthesis in the forespore compartment was examined. Pulse-labeling experiments indicated that [¹⁴C]phenylalanine was continuously incorporated into the sporangium throughout sporulation, and at t₅ (early stage V of sporulation) 58% of the radioactivity was located in the forespore compartment. Significantly high incorporation of [¹⁴C]phenylalanine was observed when the isolated forespores at t₅ were incubated with the corresponding mother-cell cytoplasmic fraction or an amino acid mixture. About 73% of the radioactivity incorporated into the isolated forespore at t₅ was found in the cytoplasmic fraction and 26% in the membranous fraction. Analysis by sodium dodecyl sulfate-gel electrophoresis showed that the ¹⁴C-labeled cytoplasmic protein had a molecular weight of about 20,000, and that a protein having the same molecular weight was present in the t₅ forespore as a slight protein band and also in the mature spore as a clear protein band. Gel electrophoresis also revealed that the ¹⁴C-labeled membranous-soluble protein (prepared by solubilization with detergents) had broad peaks with molecular weights of about 74,000, 33,000, 20,000, and 12,000.     

Differences between the biliary excretion of tri[14C]methyl-1-(3-hydroxyphenyl)ammonium iodide in Wistar and Gunn rats

1. The biliary excretion of [¹⁴C]trimophonium iodide [tri[¹⁴C]methyl(3-hydroxyphenyl)ammonium iodide] was studied in normal Wistar animals and in jaundiced homozygous Gunn rats. 2. In normal Wistar rats small amounts of radioactivity (approx. 3% of the dose in 4h) were excreted in bile as two glucuronide conjugates, i.e. [¹⁴C]trimophonium glucuronide [tri[¹⁴C]methyl-(3-oxyphenyl)ammonium glucuronide] (85%) and 3-di[¹⁴C]methylaminophenyl glucuronide (10–15%). Only minor amounts of the unchanged drug were detected in bile. 3. In the homozygous jaundiced Gunn rat large amounts of radioactivity (26% of the dose in 4h) were eliminated in bile as [¹⁴C]trimophonium glucuronide alone. The quantitative excretion of this metabolite in Gunn rat bile was about ten times that in normal animals. 4. It is proposed that the biochemical lesion in the homozygous Gunn rat may indirectly affect the biliary transport of exogenous glucuronides across the canalicular membrane.     

Incorporation of [14C]glucosamine into synaptosomes in vitro

Abstract— Synaptosomes isolated from rat cerebral cortex by zonal centrifugation in-corporated radioactive glucosamine into macromolecules in vitro as glucosamine, galactosamine, N-acetylneuraminic acid, and glucuronic acid. The largest percentage of incorporated radioactivity was recovered in the particulate fraction in which radioactive carbohydrates were bound in covalent linkage requiring acid hydrolysis or enzymatic digestion for release. Less than 20 per cent of the particulate radioactivity represented incorporation into gangliosides. Some 20 per cent of the radioactivity was incorporated into proteins as glucosamine, identified in hydrolysates by paper chromatography and by the amino acid analyser. After incubation, radioactivity was demonstrable in the proteins as sialic acid by paper chromatography and specific enzymic digestion; and as glucuronic acid by chromatography, electrophoresis, and digestion with hyaluronidase. Incorporation of carbohydrate was stimulated by sodium and potassium at concentrations demonstrated to enhance incorporation of amino acids, and involved the macro-molecules of all subsynaptosomal fractions. Significant incorporation of radioactivity was found in the synaptic plasma membrane. The synthesis of glycoproteins was suggested by simultaneous incorporation of [¹⁴C]glucosamine and [³H]leucine into glycopeptides subsequently hydrolysed and subjected to polyacrylamide gel electrophoresis and two-dimensional paper chromatography and electrophoresis. Such studies demonstrated that amino acids and carbohydrates may be incorporated into glycoproteins of the synaptic membrane and suggest the possibility of local synthesis as well as modification of material brought to the nerve ending by axoplasmic flow.     

The labelling of nucleic acids by radioactive precursors in the blue-green algae Anacystis nidulans and Synechocystis aquatilis Sanv.

Summary The labelling of nucleic acids of growing cells of the blue-green algae Anacystis nidulans and Synechocystis aquatilis by radioactive precursors has been studies. A. nidulans cells most actively incorporate radioactivity from [2-¹⁴C]uracil into both RNA and DNA, while S. aquatilis cells incorporate most effectively [2-¹⁴C]uracil and [2-¹⁴C]thymine.Deoxyadenosine does not affect incorporation of label from [2-¹⁴C]thymidine into DNA, but weakly inhibits [2-¹⁴C]thymine incorporation into both nucleic acids and significantly suppresses the incorporation of [2-¹⁴C]uracil.The radioactivity from [2-¹⁴C]uracil and [2-¹⁴C]thymine is found in RNA uracil and cytosine and DNA thymine and cytosine. The radioactivity of [2-¹⁴C]thymidine is incorporated into DNA thymine and cytosine. These results and data of comparative studies of nucleic acid labelling by [2-¹⁴C]thymine and [5-methyl-¹⁴C]thymine suggest that the incorporation of thymine and thymidine into nucleic acids of A. nidulans and S. aquatilis is accompanied by demethylation of these precursors. In this respect blue-green algae resemble fungi and certain green algae.     

Sorption and Metabolism of Metolachlor by a Bacterial Community

A stable bacterial community absorbed and transformed the herbicide metolachlor [2-chloro-N-(2-ethyl- 6-methylphenyl)-N-(2-methoxy-1-methylethyl)-acetamide] from a liquid medium. About 80% of the added ring-[U-¹⁴C]metolachlor (50 μg/ml) disappeared from the medium and accumulated inside the cells. The ratio of cellular ¹⁴C to ¹⁴C in 1 mg of supernatant reached a value of 1.1 × 10⁴ in a 10-day-old culture. ¹⁴C remaining in the medium consisted primarily of two dechlorinated products of metolachlor with m/z 233 and 263 as determined by mass spectrometry. The ¹⁴C-labeled material absorbed by the cells was strongly bound; only 2% of the ¹⁴C was released into deionized water after shaking for 3 h. Approximately 96% of the ¹⁴C associated with the biomass was extracted with acetone, and high-performance liquid chromatographic analysis of this fraction showed six peaks containing radioactivity. Since no metolachlor was detected by chromatographic analysis, it was concluded that the radioactivity recovered from the cells represented transformed products of metolachlor. Pure cultures isolated from the bacterial mixed culture were less effective in transforming and accumulating metolachlor. These results suggest that it may be advantageous to seed an aquatic environment with a mixture of microorganisms, rather than individual microbial species, as a method for removal or detoxification of metolachlor.     

Enzymatic transfer of mannose from guanosine diphosphate mannose to yeast mannan-protein complexes

The radioactive products derived from transfer of [¹⁴C]mannose residues from GDP-[¹⁴C]mannose to endogenous acceptors of a Hansenula holstii particulate enzyme preparation have been solubilized by Pronase digestion. From this soluble mixture, glycopeptides containing [¹⁴C]mannose have been purified and have been shown by β-elimination-reduction experiments to contain radioactive mannose and oligosaccharides of mannose linked to serine and threonine residues. Radioactive macromolecular complexes of mannan-protein were extracted from the particulate enzyme fraction with hot, neutral citrate buffer. These components contained variable quantities of protein, mannose, and phosphate. The more neutral components were reduced in size by Pronase digestion and yielded glycopeptides similar to those obtained by direct Pronase digestion of the particulate fraction.