Isolation of the Golgi apparatus from suspension cultured tobacco cells and preliminary observations on the intracellular transport of extensin-precursor

Suspension cultured tobacco cells were fractionated into 3 fractions by sucrose density gradient centrifugation. The Golgi stacks were recovered over the density range of 1.15 to 1.17 g·cm⁻³. The contents of the fractions were identified by electron microscopic observations, phosphotungstic acid—chromic acid staining, and assays of marker enzymes or organella. Cells were pulse labelled with¹⁴C-proline, and changes in content of¹⁴C-hydroxyproline-containing macromolecules in the fractions were followed. It was demonstrated that extensin-precursor was transported from the Golgi apparatus to the cell wall, and participation of a vesicular structure in the transport was suggested.     

Increased sensitivity of scleroderma fibroblasts in culture to stimulation of protein and collagen synthesis by serum

Serum effects on ¹⁴C-proline incorporation and ¹⁴C-hydroxyproline synthesis by normal and scleroderma fibroblasts in culture were studied. Serum resulted in 97% and 212% increases in ¹⁴C-proline incorporation in two lines of scleroderma fibroblasts while the increase in normal fibroblasts was only 53%. Effects on collagen synthesis were more pronounced. Addition of serum resulted in 124% and 445% increments in ¹⁴C-hydroxyproline synthesis in the scleroderma fibroblasts but only a 43% increment in the normal fibroblasts. The results indicate that cultured scleroderma fibroblasts have increased sensitivity to biosynthetic stimulation by serum and this mechanism may be of pathogenetic importance in the excessive collagen accumulation characteristic of the disease.     

Increased collagen synthesis in Kirsten sarcoma virus-transformed BALB 3T3 cells grown in the presence of dibutyryl cyclic AMP

Growth of Kirsten sarcoma virus-transformed BALB 3T3 (Ki-3T3) cells in the presence of dibutyryl cyclic AMP (dbcAMP) resulted in alteration of morphology, inhibition of growth, and increased collagen synthesis as measured by incorporation of ¹⁴C-proline into collagenase-digestible protein. There was an increase in incorporation of ¹⁴C-proline into collagen when expressed not only as dpm per μg DNA or protein, but also as the relative rate of collagen synthesis compared to total cellular protein synthesis, which suggests that an alteration in amino acid transport cannot totally account for the increased incorporation into collagen. The three properties studied were all affected over a concentration range of 0.10 to 1.0 mM dbcAMP, but each had a slightly different dose-response curve. At 0.5 mM dbcGMP or sodium butyrate, there was no affect on growth, morphology, or the relative rate of collagen synthesis indicating specificity for the dibutyryl analog of cAMP. Growth of the parent line, BALB 3T3, was inhibited by 0.5 mM dbcAMP, but the relative rate of collagen synthesis did not increase. These results suggest that although growth, morphology, and collagen synthesis are altered in transformed cells so that they more closely resemble those of the parent line, each property may be regulated independently.     

The Incorporation of 14C-Proline into the Proteins of Growing Cells: I. EVIDENCE OF SYNTHESIS IN DIFFERENT PROTEINS AND CELLULAR COMPONENTS

¹⁴C-proline was supplied to aerated potato disks, in which celldivision was occurring, and also to rapidly growing potato carrotexplants. It was absorbed and incorporated into all the subcellularprotein fractions examined, including the electrophoreticallydistinguishable fractions of the soluble protein of the potatodisks and explants. The ¹⁴C-proline was partially convertedto ¹⁴C- hydroxyproline in all the protein fractions, exceptfor one of the soluble protein fractions of potato explantsand the soluble proteins of one set of potato disks. Most ofthe ¹⁴C-proline and ¹⁴C-hydroxyproline contained in the tissuewas found in the soluble protein and also in the cellular fragmentsobtained by centrifugation at 500 g. The relative importanceof the soluble protein in the incorporation of ¹⁴C-proline andits conversion to ¹⁴C-hydroxyproline was greatest over a shortperiod of a few hours of contact with the ¹⁴C-proline supplied.Over a longer period (70 hours) the cellular fragments (500g) had become the most important and contained over 40 per cent.of the total ¹⁴C, and more than 60 per cent. of the ¹⁴C-hydroxyproline,in the protein of the tissues. In the soluble fraction of potatoexplants, seven protein bands were distinguishable on electrophoresis.A different but characteristic value of the ratio ¹⁴C-hydroxyprolineto ¹⁴C-proline was associated with each protein band, exceptfor the one region where ¹⁴C-hydroxyproline did not occur. Thebasic proteins (i.e. those moving towards the cathode) werethe most active in the incorporation of ¹⁴C-proline and itsconversion to ¹⁴C-hydroxyproline. The rather general distributionof the ¹⁴C-hydroxyproline is noted and the possible siginificanceof the basic proteins and the proteins associated with the cellularfragments (500 g) is considered in relation to the growth, celldivision, and cell wall formations which occurs in the rapidlygrowing tissue cultures.     

The Relationship between Growth and Proline Incorporation after Auxin Treatment of Mung Bean Hypocotyls

Indoleacetic acid (IAA) stimulates the incorporation of ¹⁴C-proline into both the cyloplasmic and the cell wall fractions of the hypocotyl of mung bean (Phaseolus aureus Roxb. cv. Black). It neither stimulates the transfer of ¹⁴C-proline from the cyloplasmic fraction into the cell wall fraction, nor the retention of ¹⁴C-proline in the wall or cytoplasmic fractions. Moreover, the stimulation of growth caused by IAA parallels the stimulation of the incorporation of proline into the cytoplasmic fraction, but does not parallel the stimulation into the cell wall fractions. The stimulation of the incorporation into the cyloplasmic fraction seems to appear within 30 minutes after auxin treatment, at about the same time the increase in the growth is observed in response to IAA, suggesting a connection between these effects. On the other hand, the stimulation of the proline incorporation into the cell wall fraction seems to require more than 90 minutes after auxin treatment, suggesting no close connection between growth and proline incorporation into the cell wall fraction.     

Energy substrates for flight in the Colorado beetle,Leptinotarsa decemlineata Say

Colorado beetles could be induced to fly when suspended on a roundabout. The group which showed the greatest tendency to fly when suspended and flew for the longest period were females 10–12 days after diapause break. Flights lasted for 20–30 min and were occasionally longer. No change in haemolymph carbohydrate levels occurred during flight but significant increases in haemolymph total lipid levels and significant decreases in haemolymph proline levels were observed. ¹⁴C-proline injected into the haemolymph was utilised very rapidly in flown insects but disappeared only slowly in non-flown insects.     

The Incorporation of 14C-Proline into the Proteins of Growing Cells: II. A NOTE ON EVIDENCE FROM CULTURED CARROT EXPLANTS BY ACRYLAMIDE GEL ELECTROPHORESIS

The method of acrylamide gel electrophoresis has been appliedto the separation of the proteins of growing carrot explantswhich are soluble in a buffer solution at pH 8.3. At least ninedistinct bands were detectable in this way. When carrot tissuehad absorbed ¹⁴C-proline it entered into the composition ofall these bands and, in all except one, it was converted tohydroxyproline to different degrees which characterized theband in question. Thus, in the soluble protein the ¹⁴C-hydroxyproline:¹⁴C-proline ratio varied from 0 to 0.53. The bulk of the proteinin the tissue was insoluble in buffer at pH 8.3; it containedthe bulk of the radioactivity absorbed from proline (84.8 percent.); and its average ¹⁴C-hydroxyproline : ¹⁴C-proline ratiowas the highest of all (1.28). A particulate protein preparation,separated at 25,000 g. from the soluble protein, had an intermediateratio (0.643) of ¹⁴C-hydroxyproline to ¹⁴C-proline. Therefore,there are in cultured carrot explants many distinct proteinmoieties which incorporate ¹⁴C-proline, and they convert itto ¹⁴C-hydroxyproline to very different degrees. The evidenceis consistent with the incorporation of the proline, first intothe various soluble proteins which are electrophoretically separableand subsequently, with progressively greater hydroxylation,into the more insoluble protein that constitutes the bulk ofthe protein of the cell and its organelles. It is, therefore,quite incorrect to say (as some have done) that all of the hydroxyproline-containingprotein is in very close association with the cell wall, forpart of it is present in the cell in soluble and electrophoreticallyseparable forms.     

Effects of anisotonicity on pentose-phosphate pathway,oxidized glutathione release and t-butylhydroperoxide-induced oxidative stress in the perfused liver of air-breathing catfish,<Emphasis Type="Italic">Clarias batrachus</Emphasis>

Both hypotonic exposure (185 mOsmol/l) and infusion of glutamine plus glycine (2 mmol/l each) along with the isotonic medium caused a significant increase of¹⁴CO₂ production from [1-¹⁴C]glucose by 110 and 70%, respectively, from the basal level of 18.4 ± 1.2 nmol/g liver/min from the perfused liver ofClarias batrachus. Conversely, hypertonic exposure (345 mOsmol/l) caused significant decrease of¹⁴CO₂ production from [1-¹⁴C]glucose by 34%.¹⁴CO₂ production from [6-¹⁴C]glucose was largely unaffected by anisotonicity. The steady-state release of oxidized glutathione (GSSG) into bile was 1.18 ±0.09 nmol/g liver/min, which was reduced significantly by 36% and 34%, respectively, during hypotonic exposure and amino acid-induced cell swelling, and increased by 34% during hypertonic exposure. The effects of anisotonicity on¹⁴CO₂ production from [1-¹⁴C]glucose and biliary GSSG release were also observed in the presence of t-butylhydroperoxide (50 (Amol/1). The oxidative stress-induced cell injury, caused due to infusion of t-butylhydroperoxide, was measured as the amount of lactate dehydrogenase (LDH) leakage into the effluent from the perfused liver; this was found to be affected by anisotonicity. Hypotonic exposure caused significant decrease of LDH release and hypertonic exposure caused significant increase of LDH release from the perfused liver. The data suggest that hypotonically-induced as well as amino acid-induced cell swelling stimulates flux through the pentose-phosphate pathway and decreases loss of GSSG under condition of mild oxidative stress; hypotonically swollen cells are less prone to hydroperoxide-induced LDH release than hypertonically shrunken cells, thus suggesting that cell swelling may exert beneficial effects during early stages of oxidative cell injury probably due to swelling-induced alterations in hepatic metabolism.     

Influence of age and caging upon protein metabolism,hypopharyngeal glands and trophallactic behavior in the honey bee (Apis mellifera L.)

Summary Different-aged honey bees were either kept in a cage together with young sisters for eight days or lived in their colony. Following an injection of¹⁴C-phenylalanine (Phe) we measured incorporation of¹⁴C-Phe into head protein and total protein, as well as the size of the hypopharyngeal glands. While confined in a cage for four hours, injected bees (from colony or cage) dispensed the¹⁴C-labelled protein-rich products of their hypopharyngeal glands to recipients. Eight-day-old colony bees had well developed hypopharyngeal glands, whereas at the age of sixteen days the glands had already decreased in size. Young caged bees had smaller hypopharyngeal glands. Colony bees had higher incorporation rates into total protein and head protein than bees living in a cage. Bees of different age classes, irrespective of caging, fed the same number of recipients; but the amount of¹⁴C-labelled protein-rich jelly distributed by caged bees was significantly smaller than that distributed by colony bees. Our results indicate that trophallaxis between young donor workers and newly emerged recipient worker bees is not the key factor for regular development and activity of the hypopharyngeal glands.Dedicated to Achim Lass's daughter Katrin.     

Effects of proline and carbohydrates on the metabolism of exogenous proline by excised bean leaves in the dark

Proline was metabolized when vacuum infiltrated into starved bean (Phaseolus vulgaris L.) leaves from plants previously in the dark for 48 hours, but an equivalent increase in protein proline was not observed. When ¹⁴C-proline was infiltrated into starved leaves, a large percentage of the ¹⁴C was recovered in other amino acids, organic acids, and CO₂, in addition to that recovered as protein proline. However, extensive oxidation of proline was observed only if enough proline was added to increase substantially the endogenous concentration of proline. Increasing the endogenous concentration did not affect the amount of proline that was incorporated into protein.     

The effect of rifampicin onMycobacterium smegmatis

Rifampicin was found to inhibit the growth and incorporation of¹⁴C-adenine,¹⁴C-leucine and¹⁴C-glycine in exponentially growing cells ofM. smegmatis cultivated in Merrill’s synthetic medium. Increasing concentrations of the antibiotic inhibited respiration in resting cells, in the presence of glucose or 2-oxoglutarate as substrates in particular. In addition to the well-known interference of rifampicin with the biosynthesis of RNA, the effect on the energy metabolism should also be considered.     

Conjugative Plasmid Transfer between Bacteria under Simulated Marine Oligotrophic Conditions

Marine Vibrio S14 strains and an Escherichia coli strain were starved in artificial seawater (NSS) with no added carbon, nitrogen, or phosphorus. The broad-host-range plasmid RP1 was transferred between the starving S14 strains and also from the E. coli donor to the S14 recipient under oligotrophic conditions, in which mixtures of donor and recipient cells were held on Nuclepore filters either floated on NSS or held such that NSS flowed through the filter. Transconjugants were obtained from S14 donors and recipients starved for at least 15 days before being mixed together for conjugation, whereas transconjugants were recovered from the E. coli donor and S14 recipient for up to 3 days of prestarvation, but not after 5 days. Transconjugants were obtained when there were as few as about 10⁵ and 10⁴ cells of starving S14 donors and recipients, respectively, per ml held on the filters. Starved donor and recipient mixtures incubated at 4 or 26°C, as well as those allowed to mate for 2, 5, or 24 h, all yielded numbers of transconjugants which were not significantly (P > 0.05) different.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Separation of rabbit epiphyseal chondrocytes in various stages of differentiation

Summary Separation of fractions enriched in hypertrophic cells and proliferative cells has been achieved by density gradient centrifugation of cells from collagenase digests of rabbit epiphyseal cartilage. Concentrated suspensions of cells are centrifuged on a continuous Percoll density gradient. Hypertrophic cells remain in the upper part of the gradient and proliferative zone cells move to the lower regions. The resultant fractions show differences in mean cell diameter, alkaline phosphatase activity, morphology and synthetic activity in culture. Fractions rich in hypertrophic cells contain larger cells and more alkaline phosphatase activity than those enriched in proliferative cells. In culture the hypertrophic cells flatten as large irregular polygonal cells, whereas proliferative fractions form smaller spindle-shaped cells. In micromass culture hypertrophic fractions incorporate less ³⁵S-sulphate and ¹⁴C-proline, and less tritiated thymidine than do proliferative fractions. These results suggest a general reduction in matrix and DNA synthesis with the attainment of the fully differentiated hypertrophic state, coincident with the expression of alkaline phosphatase activity and mineralisation of the cartilage matrix.     

Metabolism ofAedes aegypti cells grown in vitro. 1. Incorporation of3H-uridine and14C-leucine

Summary Metabolic activity ofA. aegypti cells grown in vitro has been studied by incorporation of³H-uridine and¹⁴C-leucine. “Chase” experiments with unlabeled precursors, and the use of actinomycin D and puromycin, showed that³H-uridine was incorporated into cellular RNA, and that¹⁴C-leucine was incorporated into protein of these cells. Incorporation of³H-uridine was inhibited when actinomycin D was used at a concentration of 10 μg/ml, and¹⁴C-leucine incorporation was inhibited to the same extent by puromycin at a concentration of 100 μg/ml medium. Contribution No. 148.     

Soil Strain of Bacillus subtilis Harboring a Large Plasmid That Mediates High-Frequency Conjugal Mobilization

The ability of a soil strain of Bacillus subtilis harboring a large plasmid, p19, to mobilize a small staphylococcal plasmid, pUB110, was studied. The latter plasmid was transferred to the recipient cells of Bacillus subtilis168 at a high frequency (about 10^–2 per recipient cell) both on the filter surface and in liquid medium. Mobilization was initiated 40 min after the beginning of the contact between donor and recipient cells.     

Hydroxyproline-rich cell wall protein (extensin): Biosynthesis and accumulation in growing pea epicotyls

Plant cell walls contain a glycoprotein component rich in the otherwise rare amino acid hydroxyproline. We examined the synthesis and accumulation of wall hydroxyproline during different states of elongation growth in pea epicotyls. Light-grown peas contained more wall hydroxyproline than their taller, dark-grown counterparts. When elongation was studied by marking growing stems in situ, there was a marked accumulation of wall hydroxyproline coincident with the cessation of elongation. Dividing and elongating regions of the epicotyl showed less wall hydroxyproline than did regions where elongation was no longer occurring.Hydroxyproline biosynthesis was examined by incubation of excised sections of tissues in various growth states in ¹⁴C-proline. The extent of conversion of these residues to ¹⁴C-hydroxyproline served as a measure of the rate of hydroxyproline synthesis. This rate was highest in tissues which had ceased elongation. The low rate of hydroxyproline synthesis in dividing and elongating cells was probably not due to the inability to hydroxylate peptidyl proline or to secrete proteins.These data show a positive correlation between the synthesis and accumulation of cell wall hydroxyproline and the cessation of cell elongation in pea epicotyls.     

Anaerobic dechlorination and mineralization of pentachlorophenol and 2,4,6-trichlorophenol by methanogenic pentachlorophenol-degrading granules

Anaerobic granules developed for the treatment of pentachlorophenol (PCP) completely minearilized¹⁴C-labeled PCP to¹⁴CH₄ and¹⁴CO₂. Release of chloride ions from PCP was performed by live cells in the granules under anaerobic conditions. No chloride ions were released under aerobic conditions or by autoclaved cells. Addition of sulfate enhanced the initial chloride release rate and accelerated the process of mineralization of¹⁴C-labeled PCP. Addition of molybdate (10 mM) inhibited the chloride release rate and severely inhibited PCP mineralization. This suggests involvement of sulfate-reducing bacteria in PCP dechlorination and mineralization. Addition of 2-bromoethane sulfonate slightly decreased the chloride release rate and completely stopped production of¹⁴CH₄ and¹⁴CO₂ from [¹⁴C]PCP. 2,4,6-trichlorophenol was observed as an intermediate during PCP dechlorination. On the basis of experimental results, dechlorination of 2,4,6-trichlorophanol by the granules was conducted through 2,4-dichlorophenol, 4-chlorophenol or 2-chlorophenol to phenol at pH 7.0–7.2.     

The cytoplasmic ribonucleoprotein particles of rat sarcoma cells

Summary The metabolism of the cytoplasmic ribosomal RNP-particles from rat sarcoma cells have been studied after intraperitoneal injection in animals of¹⁴C-leucine alone or together with H₃P³²O₄. By investigating the change of the specific activity of the ribosomes with respect to the time after injection of¹⁴C-leucine we have demonstrated that the half-life of ribosomes is 35 hours.To study the metabolic activity of ribosomes not taking part in protein synthesis, the sucrose gradient centrifugation method was used for their separation from polyribosomes. Four, seven, twelve hours after injection of the isotopes free ribosomal subunits and monoribosomes were isolated and their radioactivity was determined. As expected the label was found in the ribosomal subunits at the beginning and later on in the monoribosomal fraction. At the same time it was observed that the incorporation of H₃P³²O₄ into ribosomal subunits occurred at a much greater rate as compared with the incorporation of¹⁴C-leucine. These results indicate the existence of a pool of ribosomal proteins in sarcoma cells whose role deserves attention.an invited article     

MORPHOLOGICAL AND CHEMICAL STUDIES OF COLLAGEN FORMATION : II. Metabolic Activity of Collagen Associated with Subcellular Fractions of Guinea Pig Granulomata

Electron micrographs of thin sections of nuclear, microsomal, and mitochondrial fractions obtained from a carrageenin-induced granuloma showed considerable contamination of the heavier by the lighter fractions. Striated collagen fibrils could be identified in the nuclei + debris fraction. Only a few striated fibrils occurred in the mitochondrial fraction; very fine filaments (diameter 50 A) could be seen in this fraction, but could not be distinguished with certainty from fibrillar material derived from broken nuclei. 35 per cent of the mitochondrial and 80 per cent of the microsomal collagen was extractable by 0.2 M NaCl and could be purified by the standard methods of solution and reprecipitation. The amino acid composition of these collagen fractions determined by ion exchange chromatography was within the range normally found for collagen and gelatin from other mammalian species, allowing for 10 to 20 per cent of some non-collagenous contaminant of the microsomal collagen. Hydroxyproline and proline were isolated by chromatography on paper from hydrolysates of the nuclear, mitochondrial, and microsomal collagen fractions, after incubation of tissue slices with L-¹⁴C-proline. The specific activities of the hydroxyproline from these collagens were in the approximate ratio 1:2:6, while that of bound hydroxyproline derived from the supernatant was only 1, indicating primary synthesis of collagen in the microsomes. Attempts to demonstrate incorporation of L-¹⁴C-proline into collagen or into free hydroxyproline in cell free systems were unsuccessful, nor was it possible to demonstrate non-specific incorporation of L-¹⁴C-valine into TCA-insoluble material by various combinations of subcellular fractions.