Radiolabeling of and macromolecular syntheses in Neisseria gonorrhoeae types 1 and 4.

Type 1 and type 4 cells of Neisseria gonorrhoeae were grown in a liquid medium that maintained colony type for several hours. Organisms from both types of colonies incorporated labeled glucose, as well as nucleic acid and protein precursors, into specific cellular fractions during growth in the medium. Ribonucleic acid and protein were synthesized actively by the bacteria, with incorporation of label chased by the addition of unlabeled precursors. The method can be used to label type 1 and type 4 cells of gonococci with specific isotopes for studies of metabolic and biochemical changes.     

Metabolism of rabbit skin collagen. Differences in the apparent turnover rates of type-I- and type-III-collagen precursors determined by constant intravenous infusion of labelled amino acids.

Growing rabbits were infused for up to 10 h with labelled proline, tyrosine and leucine to achieve plateau conditions within body free pools, for [3H]proline infusion, blood free-proline specific radioactivity remained constant after about 1 h. For individual animals, type-I- and type-III-collagen precursors were isolated by precipitation with (NH4)2SO4 and DEAE-cellulose chromatography. Experiments where 3H- and 14C-labelled proline and tyrosine were infused concurrently for different periods of time showed that type I procollagen reached plateau specific radioactivity within 3 h and 90% of the plateau value after 2 h infusion, corresponding to a calculated apparent t 1/2 of less than 26 min. Plateau values for type I procollagen were taken as precursor amino acid pool specific radioactivities. The type-III-collagen-precursor fractions consistently showed lower rates of label incorporation and, by assuming that both type I and type III collagens are synthesized from the same amino acid pools, kinetic analysis revealed an apparent t 1/2 for the isolated type-III-collagen precursors of 3.9 h. For proline, there were large variations between animals in the ratio between the precursor pool for collagen synthesis and the skin homogenate free pool (0.31 +/- 0.13, mean +/- S.D.), so that collagen-synthesis rates based solely on total tissue free-pool values for proline are subject to large and inconsistent errors.     

Lipid synthesis in vitro by rabbit Meibomian gland tissue and its inhibition by tetracycline

Rabbit Meibomian gland tissue was incubated with radioactive acetate, propionate, valine, leucine or isoleucine. The time-course of incorporation of radioactivity into total lipids from acetate and isoleucine was studied in Hanks' or Krebs medium. Incorporation of acetate or isoleucine into lipid classes was followed by TLC, and of acetate label into individual fatty acids and alcohols by GLC with radioactivity detection. Radioactivity was highest in the minor acid components C_16:1 and C_18:1. Levels in branched fatty acids fell with chain length, reflecting the time-course of successive chain elongations. Use of amino acids or propionate as precursors suggested that molecules containing a specific branched structure were preferentially incorporated; this indicates a binding preference at the level of the fatty acid synthase which might explain the very high proportion of anteiso-branched structures in the rabbit secretion. Incorporation of label into total lipids was significantly reduced by tetracycline in the medium, with 50% reduction at about 1.5 mg/ml for acetate and 0.7 mg/ml for isoleucine.     

In vitro assembly of dogfish brain tubulin and the induction of coiled ribbon polymers by calcium

It was previously shown that BHK21 cells were arrested in the G1 phase of the cell cycle when cultured in medium lacking serine. In this study the effect of serine limitation on protein synthesis was examined. Shifting cells from medium supplemented with 10% fetal calf serum to medium supplemented with 10% dialyzed serum resulted in a 50% reduction in the rate of protein synthesis. The reduced rate was attained within 4–10 min after shift-down and was restored completely within 5–15 min after shift-up to 10% dialyzed serum plus 0.05 mM serine, the same approximate concentration of serine present in 10% fetal calf serum. Exogenous serine appears to be incorporated into protein from a precursor pool which is functionally compartmentalized inasmuch as incorporation of serine into protein became linear within 10 min after the addition of label while the specific activity of serine in the acid soluble fraction did not attain a constant value during 60 min of labeling. The serine: leucine ratio in total cellular protein was determined from cells cultured in ten percent dialyzed serum plus 0.05 mM serine by amino acid analysis and was compared with the ratio of [³H]serine and [¹⁴C]leucine incorporated into protein. The results indicated that 50–60% of the serine utilized for protein synthesis under these conditions was derived from the medium while the other 40–50% was generated within the cell.     

Relations Between Coenzyme A and Presumptive Acyl Carrier Protein in Different Conditions of Streptococcal Growth

Exploration of the specific role of cystine in the postexponential growth of Streptococcus faecalis led to an inquiry into the fate of cellular coenzyme A (CoA) and acyl carrier protein (ACP), both of which depend for their biosynthesis on cystine and pantothenate as precursors. In S. faecalis cells labeled by growth in the presence of (14)C-pantothenate, the label could be separated on the basis of solubility at pH 2.1 into two fractions of sharply differing metabolic characteristics. The fractions were not purified, but the soluble (14)C behaved analytically like CoA, and the insoluble (14)C was considered to represent an ACP-like entity on the basis of circumstantial evidence. The fate of these two fractions under various conditions of growth was studied. When the medium contained an excess of the needed precursors, the cellular content of CoA and ACP appeared to remain constant during exponential growth, and in a molar ratio of about 4 CoA to 1 ACP. Cellular ACP, once formed, appeared to be stable under these conditions, but CoA was degraded and replaced at the rate of approximately 20% per division period. With restrictive levels of pantothenate in the medium, initially formed CoA disappeared during growth, as a result, apparently of being converted to ACP. However, when the resulting CoA-depleted cells were returned to a medium containing enough pantothenate, resumption of normal growth was preceded by a lag period, during which rapid conversion of ACP to CoA appeared to take place.     

The effect of corticotropin on phospholipid metabolism in isolated adrenocortical cells.

Trypsin-dispersed cat adrenocortical cells were incubated at 37 degrees C in modified Eagle's medium containing [14C]arachidonic acid of sodium [14C]-acetate and then in non-radioactive medium. Radioactive incorporation was obtained in all phospholipids, with the greatest amount of radioactivity in phosphatidylcholine, followed by phosphatidylethanolamine, phosphatidyl-serine, and phosphatidylinositol. Concentrations of individual phospholipids generally paralleled the relative amounts of corresponding radiolabeled phospholipids, although the percentage of phosphatidylinositol was considerably lower than its radioactive counterpart, resulting in a high specific activity of this particular phospholipid. Although a potently steroidogenic concentration of corticotropin failed to enhance release of label from any particular phospholipid, analysis of specific activity showed that corticotropin stimulation was accompanied by an increased turnover of phosphatidylinositol and phosphatidic acid. These studies demonstrate that isolated cortical cells have the capacity to synthesize phospholipids from radioactive precursors. The finding that the acute effects of corticotropin are associated with changes in specific phospholipids, including phosphatidylinositol and phosphatidic acid, conforms to the general pattern observed in other secretory systems.     

Incorporation of polyunsaturated fatty acids into bis(monoacylglycero)phosphate and other lipids of macrophages and of fibroblasts from control and Niemann-Pick patients

On the basis of earlier studies of rabbit pulmonary alveolar macrophages, the incorporation of 14C-labelled polyunsaturated fatty acids into the lipids of human fibroblasts from patients with various phenotypes of Niemann-Pick disease was examined in order to define further the disturbance in metabolism of bis(monoacylglycero)phosphate occurring in these disorders. Docosahexaenoic acid, which had not been studied previously, was found to be incorporated by macrophages into bis(monoacylglycero)phosphate in a highly selective fashion and was therefore used along with arachidonic acid for studies of fibroblasts. Following incubation of fibroblasts in serum-free medium for 60 min, the distribution of arachidonic acid label in lipids was: phosphatidylcholine, 51%; phosphatidylethanolamine, 12%; phosphatidylinositol, 9.5%; and bis(monoacylglycero)phosphate, 2.3%; and of docosahexaenoic acid label was 36, 20, 2.6 and 10.3% respectively. Phosphatidylinositol had the highest specific activity of arachidonic acid label and bis(monoacylglycero)phosphate of docosahexaenoic acid label. Prolongation of incubation to 21 h, with or without removal of label remaining in the medium at 1 h, resulted in proportional redistributions with phosphatidylcholine decreasing and phosphatidylethanolamine increasing. In bis(monoacylglycero)phosphate and phosphatidylinositol, the proportions of arachidonic acid label decreased and increased respectively, whereas the proportions of docosahexaenoic acid label in these lipids were unchanged. As virtually all label taken up by cells was esterified, these redistributions are taken to reflect transacylations. In Niemann-Pick cells, the expected redistribution of arachidonic acid label in bis(monoacylglycero)phosphate failed to occur with cell types A and B which are deficient in sphingomyelinase-phospholipase C, and excess label accumulated after a 21-h incubation. Excess docosahexaenoic acid label also accumulated in the bis(monoacylglycero)phosphate of these cells. The highly selective incorporation of docosahexaenoic acid in two cell types suggests a special role for bis(monoacylglycero)phosphate in the metabolism of n-3 polyunsaturated fatty acids. A high specific activity found early in incubations of macrophages suggests that polyunsaturated fatty acids may be incorporated into phospholipids during de novo synthesis of phosphatidic acid.     

Alterations of phospholipid metabolism by phorbol esters and fatty acids occur by different intracellular mechanisms in cultured glioma, neuroblastoma, and hybrid cells

Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and turnover of phosphatidylcholine (PtdCho) and other phospholipids have been studied in glioma (C6), neuroblastoma (N1E-115), and hybrid (NG108-15) cells in culture using [methyl-3H]choline, [32P]Pi, [1,2-14C]ethanolamine, or 1-14C-labeled fatty acids as lipid precursors. 100-500 microM oleic acid stimulated PtdCho synthesis 3- to 5-fold in all three cell lines, but had little influence on chase of choline label following a 24-h pulse. Phorbol ester (50-200 nM) stimulated PtdCho synthesis 1.5- to 3-fold in C6 cells, was without effect in N1E-115 cells, and had intermediate effects on NG108-15 cells. Phorbol ester stimulated both uptake of extracellular choline and synthesis of PtdCho, whereas fatty acid stimulated only synthesis. Release of radioactivity from 24-h pulse-labeled PtdCho to the medium was enhanced by phorbol ester in C6 cells. Incorporation of [32P]Pi, primarily into PtdCho, was stimulated, whereas utilization of [1,2-14C]ethanolamine or 1-14C-fatty acid was little altered by phorbol ester. C6 cells "down-regulated" with phorbol ester lost the stimulatory response of subsequent treatment with phorbol esters on PtdCho synthesis, but the response to fatty acid was enhanced. Fatty acid had little influence on the relative binding of phorbol ester or "translocation" of phorbol ester binding sites. Accordingly, metabolism of phospholipids in these cultured cells of neural origin is markedly influenced by cell type, phospholipid class, condition of incubation medium, and nature of stimulator. Phorbol esters and fatty acids appear to enhance phospholipid synthesis and turnover by distinct intracellular mechanisms.     

Non-specific incorporation of nucleic acid precursors in Blastomyces dermatitidis and Histoplasma capsulatum

Incorporation of thymidine, thymidine monophosphate (TMP), thymidine triphosphate (TTP), uridine and orotic acid into DNA, RNA and protein in Blastomyces dermatitidis and Histoplasma capsulatum was studied utilizing a specific acid hydrolysis technique developed for these fungi. Thymidine was incorporated to the greatest extent (approximately 0.5 % of added label) followed by uridine, orotic acid, TMP and TTP. In Blastomyces, uridine and orotic acid labeled primarily RNA. TMP and TTP labeled RNA, DNA and protein at nearly the same level. In Histoplasma RNA was labeled poorly by any of these precursors. TMP and TTP labeled DNA predominately and protein to a slightly lower level. Deoxyadenosine or uridine media supplements of 250 g/ml did not enhance incorporation. All precursors tested were found to be nonspecific in that RNA, DNA and protein were labeled. All data indicate that neither RNA nor DNA synthesis can be specifically measured in whole cells or acid precipitates by any of these precursors. Specific radiometric monitoring with these isotopes therefore requires the separation of these macromolecules.     

Biosynthesis of 14C- and 35S-lincomycin using different sources for the label]

The sources of radioactive labels were chosen for biosynthesis of labeled lincomycin. The levels of the label incorporation into lincomycin were high with all the sources used when the lincomycin-producing organism was cultivated on the synthetic medium as compared to the complex medium. Incorporation of the label into lincomycin was most effective when I14C- or 214C-thyrosine was used as the precursors. These precursors were effective in both the complex and the synthetic media. In production of significant amounts of the labeled lincomycin I14C- or 214C-sodium propionate, I14C- or 142C-sodium acetate and 14C-protein hydrolysate may be used as the sources of the radioactive carbon. In production of 35S-lincomycin K235SO4 may be used as the source of the label. The optimal conditions for biosynthesis of 14C- and 35S-lincomycin were developed (concentration of some components of the medium, time of the label addition and others).     

Incorporation of fucose and glucosamine into cell bound and medium released macromolecules.

(1) Determinations were carried out on the incorporation of fucose-6-(3H) and glucosamine-6-(3H) into trichloracetic acid insoluble macromolecules which remained bound to the cells or were released into the medium of chick embryo muscle cell cultures. The radioactivity determined in the medium was corrected for unspecific binding of label to components of the medium. (2) During an incorporation period of six hours the incorporation per microgram DNA with fucose as label into cell bound macromolecules is about twice as high as the incorporation into macromolecules released into medium. With glucosamine about twice as much is incorporated into medium released into the cell bound macromolecules. (3) The incorporation per microgram DNA increased during a culture period of three days but the increase ceases at different times during this culture period when determined with fucose or glucosamine or for cell bound and medium released material. (4) An increase in cell density increases the incorporation per DNA of fucose and to a much slighter extent that of glucosamine. Reduction of cell density by addition of cytosine arabinoside to the medium does not increase the incorporation per microgram DNA. (5) The effect of changes of fibroblast/myoblast ratios on the incorporation of fucose and glucosamine were examined. No significant effect was observed for a ratio of 10-30% fibroblasts when control cultures or cultures after cell sedimentation were maintained in complete medium. Marked changes were observed after culture in medium without protein components. Under these conditions an increase in the fibroblast/myoblast ratios were observed as well as an increase in the incorporation of label into medium released and a decrease into cell bound macromolecules.     

Heterogeneous distribution of the precursor of type I and type III collagen and fibronectin in the rough endoplasmic reticulum of palatal mesenchymal cells of the mouse embryo cultured in ascorbate-depleted medium

Summary In order to examine the intracellular distribution of precursors of type I and type III collagen and fibronectin in the palatal mesenchymal (MEPM) cells of the mouse embryo cultured under ascorbate-deficient conditions, immuno-electron-microscopic studies were carried out by use of affinity purified antibodies for these proteins. MEPM cells were obtained from the palatal shelves of 14-day-old mouse fetuses and cultured for 3–7 days in medium, either with or without 50 ng/dish/day ascorbic acid. Results obtained were as follows: (1) Although the rough endoplasmic reticulum (rER) of MEPM cells cultured for 5 days in ascorbate-supplemented medium was flattened, that in cells cultured in ascorbate-deficient medium had a distended or vesicular appearance. (2) Vesicular or distended rER showed heterogeneous staining for both type I and type III collagen, namely, some parts of rER showed positive staining for both types of collagen, while others showed negative staining. (3) Both type I and type III collagen showed codistribution in the same vesicular rER. (4) Vesicular rER showed negative or very faint labelling for fibronectin. These results may suggest regional differences in the function of rER.     

Radiolabeling of mammalian cells in tissue culture by use of acetic anhydride. Potential value for studying the dynamics of protein turnover in living cells.

When Chinese hamster ovary cells, growing in monolayer culture, were treated with trace quantities of radioactive acetic anhydride, label was very rapidly introduced into both the plasma membrane and intracellular components, all of which reached similar specific radioactivities. The cells themselves showed no measurable loss in viability. The incorporated label was recovered predominantly in the form of acetyl groups on proteins. Since tyrosyl hydroxyls were not extensively acetylated, it seems likely that substitution occurred mainly on amino groups. There was no indication that the acetyl groups once introduced were labile or that the modified proteins were discriminated against by the cells, since, in dividing cells, the acetylated proteins were as long-lived as those labeled with radioactive l-leucine. Nor was there any evidence for reutilization of labeled acetate following protein turnover in cells maintained in a nonproliferating state on a medium lacking the essential amino acid l-isoleucine. We suggest that acetylation could provide an ideal means for introducing a very short “pulse” of radioactivity into proteins of living cells. In addition, the method should be extremely useful for studying processes such as enzyme induction and protein turnover where the problem of amino acid reutilization has long been recognized as a possible basis for artifacts.     

Coordinated incorporation of nascent peptidoglycan and teichoic acid into pneumococcal cell walls and conservation of peptidoglycan during growth.

Choline-containing pneumococcal cell wals are sensitive to autolysin, whereas ethanolamine-containing walls are not. Bacteria were labeled with radioactive peptidoglycan precursors while growing either in choline- or in ethanolaminecontaining media. Subsequently, the labeled cells were allowed to grow for four to five generations in nonradioactive medium supplemented with the alternative amino alcohol source (i.e. cells labeled in choline medium yields ethanolamine; cells labeled in ethanolamine medium yields choline). The autolysin sensitivity of the isotope label in cell walls prepared from such bacteria indicates that nascent peptidoglycan and teichoic acid units that are synthesized at the same time are attached to one another, incorporated into the cell surface at the cellular equator, and remain conserved during growth the division of the bacteria.     

The synthetic potential of immobilised cells of Capsicum frutescens Mill cv. annuum

Cells of Capsicum frutescens Mill. cv. annuum, immobilised in reticulate polyurethane foam, produced higher yields of capsaicin, the pungent principle of Chilli pepper fruits, than did freely-suspended cells, when batch-cultured in a medium conducive to culture growth. In the absence of specific precursors to capsaicin, immobilised cells produced between two and three orders of magnitude higher yields than did suspended cells over 5-d or 10-d culture periods (typically up to 4 or 5 mg capsaicin g^-1 dry weight l^-1 medium compared with up to 30 g g^-1l^-1, respectively). These results were reflected by an increased rate and extent of incorporation of L-[U-¹⁴C]phenylalanine into capsaicin in immobilised as compared with freely-suspended cells, and evidence is presented for an inverse relationship between incorporation of [¹⁴C]phenylalanine into protein and capsaicin. The accumulation of capsaicin can be experimentally manipulated and increased by supplementing the medium with precursors of capsaicin such as phenylalanine and isocapric acid and by reducing the growth rate of immobilised cells by omitting growth regulators from the medium. The importance of these observations is discussed.Abbreviations HPLC high-performance liquid chromatography - Phe phenylalanine - TLC thin-layer chromatography     

The biosynthesis of hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Alignment of the butylamine segment and source of the secondary amino nitrogen

The unusual amino acid hypusine is produced in a single protein of mammalian cells by a novel posttranslational event in which a lysine residue is conjugated with the four-carbon moiety from the polyamine spermidine to form an intermediate deoxyhypusine, and in which this intermediate is subsequently hydroxylated. Specifically isotopically labeled precursors of hypusine were used to identify the biosynthetic origin of some of the atoms of hypusine and thus to provide further insight into the mechanism of this in vivo chemical modification reaction. Radiolabel from [1,4-3H] putrescine, [1,8-3H]spermidine, and [5-3H]spermidine entered hypusine during growth of Chinese hamster ovary cells. The occurrence of this label at positions 1 and 4, at position 4, and at position 1, respectively, in the 4-amino-2-hydroxybutyl portion of hypusine revealed an alignment of atoms identical to that in the butylamine segment of spermidine. Growth of cells with [epsilon-15N]lysine as the source of lysine yielded hypusine enriched in 15N, whereas only isotope-free hypusine during growth by [4-15N]spermidine. These was found in cells whose spermidine was replaced during growth by [4-15N]spermidine. These findings are in accordance with a proposal that the first phase of hypusine biosynthesis, the production of intermediate deoxyhypusine, occurs through transfer of the butylamine moiety from spermidine to the epsilon-amino nitrogen of protein-bound lysine. The technique of thermospray high-performance liquid chromatography/mass spectrometry provided positive identification of 15N in hypusine through final separation and on-column direct analysis of this amino acid. Methods of preparation are given for spermidine of high specific radioactivity, labeled specifically at position 5 with 3H, and for spermidine with 15N at the 4-position.     

14C]methylammonium transport by Frankia sp. strain CpI1

We describe an NH4+-specific transport system in the N2-fixing symbiotic actinomycete Frankia sp. strain CpI1. [14C]methylammonium was used as an NH4+ analog. No specific transport process was detected when cells were grown on high concentrations of NH4+. A transport system with a high affinity for CH3NH3+ was synthesized after 3 to 4 h of nitrogen starvation. Methylammonium transport was not significantly inhibited by a variety of amino acids, primary amines, and polyamines. Ammonium completely eliminated CH3NH3+ transport. The Km for CH3NH3+ transport was around 2 +/- 1.8 microM with a Vmax of 4 to 5 nmol/min per mg of protein. The electron transport inhibitors cyanide and azide eliminated uptake, as did the uncoupler carbonyl cyanide-m-chlorophenylhydrazone. The sulfydryl reagent p-chloromercuribenzoic acid and the heavy metal thallium also inhibited uptake, suggesting the presence of an NH4+-specific permease. Concentration of CH3NH3+ across the membrane was demonstrated by conducting uptakes at low temperature to slow the metabolism of CH3NH3+ by glutamine synthetase. At 7 degrees C most of the label was concentrated inside the cells in a form that could be chased from the cells by adding excess NH4+ to the medium. At 30 degrees C most of the label was present as an impermeant metabolite. Thin-layer chromatography of cell extracts confirmed that the radioactivity inside the cells was mainly in the form of CH3NH3+ at 7 degrees C but was present as an unidentified metabolite at 30 degrees C. These studies demonstrate that Frankia sp. strain CpI1 has a high-affinity NH4+ transport system that is synthesized in response to NH4+ starvation.     

Biosynthesis of respiratory-tract mucins. A comparison of canine epithelial goblet-cell and submucosal-gland secretions

1. A modified canine tracheal organ culture system was used to investigate differences between mucous secretions of epithelial goblet cells and the submucosal glands. 2. Denuded explants were prepared by removing goblet, ciliated and basal cells from the surface epithelium leaving an intact basement membrane and viable submucosa. 3. Denuded explants actively incorporated radioactive precursors into secreted macromolecules when cultured in medium 199 containing label. 4. Chromatography on Bio-Gel A-150m and electrophoresis on 1% agarose gels indicated that epithelial goblet cell secretions were relatively more sulphated than submucosal glandular secretions. 5. The glandular structures were shown to respond to a parasympathomimetic agent.     

Microbial synthesis of L-[15N]leucine L-[15N]isoleucine, and L-[3-13C]-and L-[3'-13C]isoleucines studied by nuclear magnetic resonance and gas chromatography-mass spectrometry

The preparation of leucine and isoleucine labeled with 15N and of site-specific 13C-labeled isoleucines is described. This method is based on the induction of the biosynthetic pathways specific for branched chain amino acids in glutamic acid producing bacteria, and controlled provision of stable isotope labeled precursors. Corynebacterium glutamicum (ATCC 13032), a glutamic acid overproducer, was incubated in leucine production medium which consisted of a basal medium supplemented with [15N]ammonium sulfate, glucose, and sodium alpha-ketoisocaproate. production of L-[15N]leucine reached 138 mumol/ml at an isotopic efficiency of 90%. It was purified and checked by proton NMR and GC-MS. The electron impact (EI) spectrum showed 95 atom% enrichment. The cultivation of C. glutamicum in a similar medium containing alpha-ketobutyrate yielded L-[15N]isoleucine at a concentration of 120 mumol/ml. The GC-MS EI and chemical ionization (CI) spectra confirmed enrichment of 96 atom% 15N as that of the labeled precursors. The biosynthesis of L-[13C]isoleucine was carried out by induced cells which were transferred to a similar medium in which [2-13C]- or [3-13C]pyruvic acid replaced glucose. 13C NMR of the product isoleucine revealed single-site enrichment at C-3 or at C-3' respective to the precursor [13C]pyruvate; i.e., C-3 was labeled from [2-13C]pyruvate and C-3' from [3-13C]pyruvate. Mass spectrometric analysis confirmed that all molecules were labeled only in one carbon. This site-specific incorporation of [13C]pyruvate is contrasted with the labeling pattern obtained when producing cells were supplied with [2-13C]acetate, instead of pyruvate, when most label was incorporated into carbons 3 and 3' of the same isoleucine molecule.