Proteolysis during long‐term glucose starvation in Staphylococcus aureus COL

A combination of pulse‐chase experiments and 2‐D PAGE revealed that protein degradation appears to play a crucial role for the cell physiology of Staphylococcus aureus COL during extended periods of glucose starvation. The synthesis rate of virtually all cytosolic and radioactively labeled proteins from growing cells seemed dramatically reduced in the first 3.5 h of glucose starvation. The stability of proteins synthesized in growing cells was monitored by a pulse‐chase approach on a proteome wide scale. Especially, enzymes involved in nucleic acid and amino acid biosyntheses, energy metabolism and biosynthesis of cofactors were found rather rapidly degraded within the onset of the stationary phase, whereas the majority of glycolytic and tricarboxylic acid cycle enzymes remained more stable. Furthermore, single enzymes of biosynthetic pathways were differentially degraded. A metabolite analysis revealed that glucose completely depleted from the medium in the transient phase, and amino acids such as alanine and glycine were taken up by the cells in the stationary phase. We suggest that vegetative proteins no longer required in non‐growing cells and thus no longer protected by integration into functional complexes were degraded. Proteolysis of putative non‐substrate‐bound or “unemployed” proteins appears to be a characteristic feature of S. aureus in order to access nutrients as an important survival strategy under starvation conditions.     

Stimulation of hepatic biogenesis of sterols on administration of adenosine compounds.

The glycosylations of hydroxylysine during collagen biosynthesis in isolated chick-embryo tendon cells were studied by using pulse-chase labelling experiments with [14C]-lysine. The hydroxylation of lysine and the glycosylations of hydroxylysine continued after a 5 min pulse label for up to about 10 min during the chase period. These data differ from those obtained previously in isolated chick-embryo cartilage cells, in which, after a similar 5 min pulse label, these reactions continued during the chase period for up to about 20 min. The collagen synthesized by the isolated chick-embryo tendon cells differed markedly from the type I collagen of adult tissues in its degree of hydroxylation of lysine residues and glycosylations of hydroxylysine residues. When the isolated tendon cells were incubated in the presence of L-azetidine-2-carboxylic acid, the degree of glycosylations of hydroxylysine during the first 10 min of the chase period was identical with that in cells incubated without thcarboxylic acid for at least 60 min, whereas no additional glycosylations took place in the control cells after the 10 min time-point. As a consequence, the collagen synthesized in the presence of this compound contained more carbohydrate than did the collagen synthesized by the control cells. Additional experiments indicated that azetidine-2-carboxylic acid did not increase the collagen glycosyltransferase activities in the tendon cells or the rate of glycosylation reactions when added directly to the enzyme incubation mixture. Control experiments with colchicine indicated that the delay in the rate of collagen secretion, which was observed in the presence of azetidine-2-carboxylic acid, did not in itself affect the degree of glycosylations of collagen. The results thus suggest that the increased glycosylations were due to inhibition of the collagen triple-helix formation, which is known to occur in the presence of azetidine-2-carboxylic acid.     

Effect of High Glucose Concentration on Collagen Synthesis and Cholesterol Level in the Phenotypic Modulation of Aortic Cultured Smooth Muscle Cells of Sand Rat (Psammomys obesus)

To simulate diabetic conditions, the effects of high glucose concentration on collagen synthesis and cholesterol level in cultured aortic smooth muscle cells of Psammomys were investigated. For collagen biosynthesis, smooth muscle cells (SMCs) were incubated in synthetic proliferative phase and in postconfluent phase with ³H-proline. Cellular cholesterol was determined by enzymatic method. Under high glucose concentration, the results showed morphological modifications characterized by morphometric cellular, nuclear, and nucleolar changes. In biochemical studies, the authors observed an increase of free and esterified cellular cholesterol as well as of total proteins, collagen biosynthesis, and α1 (I+III) and α2 (I) chains of collagen contained in the SMCs and in the extracellular matrix. These results showed the sensitivity of Psammomys aortic SMCs to high glucose concentration and would constitute an interesting cellular model to study atherosclerosis pathogeny in experimental diabetes.     

Metabolic shift from lipogenesis to glycogenesis in the last instar larval fat body of the silkworm,Bombyx mori

When [¹⁴C]glucose was injected into the last instar larvae of the silkworm, Bombyx mori, the label was incorporated into various tissues at varying degrees depending on the developmental stages. Fat body exhibited high incorporation rates throughout the feeding periods. Silk glands became active in incorporation but midgut decreased toward larval maturation. The pulse labeling experiment clearly demonstrated that the metabolic shift from lipogenesis to glycogenesis occurred in fat body at the middle of the last instar; a predominant incorporation was found in lipids when [¹⁴C]glucose was injected at the early stage, while at the late stage glycogen synthesis became most active. Incorporation into fat body proteins was not a major factor throughout the instar. Extirpation of silk glands enhanced incorporation into glycogen and proteins at the late stage but did not affect lipid synthesis. Long-term chase showed that fat body lipids and proteins synthesized at the early stage were totally carried over into the pupal fat body, while much glycogen produced at the late stage was used during the larval-pupal transformation with the remainder carried over into the pupa.From these results the metabolic shift from lipogenesis to glycogenesis in fat body is discussed in relation to the storage function of the fat body for pupal metamorphosis.     

Involvement of osteopontin upregulation on mesangial cells growth and collagen synthesis induced by intermittent high glucose

Glucose fluctuations are strong predictor of diabetic vascular complications. We explored the effects of constant and intermittent high glucose on the proliferation and collagen synthesis of cultured rat mesangial cells. Furthermore, the possible involvement of osteopontin (OPN) was assessed. In rat mesangial cells cultured in 5, 25, or 5 mmol/L alternating with 25 mmol/L glucose in the absence or presence of neutralizing antibodies to OPN, β3 integrin receptor and β5 integrin receptor, the cell proliferation, collagen synthesis, and the expression of OPN and type IV collagen were assessed. In cultured mesangial cells, treatment with constant or intermittent high glucose significantly increased [³H]thymidine incorporation in a time‐dependent manner. A modest increase was observed at 12 h, and further deteriorated afterwards, and reached the maximum incorporation at 48 h. Treatment with constant high glucose for 48 h resulted in significant increases in [³H]thymidine incorporation, cell number, [³H]proline incorporation, mRNA, and protein levels of type IV collagen and OPN compared with mesangial cells treated with the normal glucose, which were markedly enhanced in cells exposed to intermittent high glucose medium. In addition, neutralizing antibodies to either OPN or its receptor β3 integrin but not neutralizing antibodies to β5 integrin can effectively prevented proliferation and collagen synthesis of mesangial cells induced by constant or intermittent high glucose. Intermittent high glucose exacerbates mesangial cells growth and collagen synthesis by upregulation of OPN expression, indicating that glycemic variability have important pathological effects on the development of diabetic nephropathy, which is mediated by the stimulation of OPN expression and synthesis. J. Cell. Biochem. 109: 1210–1221, 2010. © 2010 Wiley‐Liss, Inc.     

The production of protease by dense suspensions ofBacillus megaterium KM Sp−

The synthesis and secretion of extracellular protease was demonstrated during the incubation of dense susponsions of the asporogenicBacillus megaterium KM. The overall production of the enzyme by cells incubated with glucose in a nitrogen-free medium was found to be only slightly lower than that in the presence of an inorganic nitrogen source. The capacity to form protease decreased exponentially with increasing density of the bacterial suspension. The synthesis of the enzyme was interrupted after the exhaustion of glucose. A repeated exchange of the medium made it possible to reach relatively high and continuous production of protease for several hours. The total amount of extracellular proteins synthesized during incubation of the dense suspension in media with or without a nitrogen source was less than 2% of a total of newly formed proteins. The amount of these extracellular proteins was slightly lower in the absence of Ca²⁺ being considerably decreased when the dense suspension was incubated with chloramphenicol.     

Kinetics of intracellular degradation of newly synthesized collagen

The objective of this work was to determine the time dependence of the basal component of intracellular degradation of newly synthesized collagen. Chick embryo tendon fibroblasts were incubated with [14C]proline, and degradation was quantified by measuring hydroxy[14C]proline in a low molecular weight fraction. When cultures were pulse labeled for 15 min and then incubated under chase conditions for 105 min, the amount of degraded collagen attained a value equal to approximately 20% of the amount synthesized during the labeling period; the data were fit with a simple exponential function that had a 40-min rise time and a 12-min lag time. In continuously labeled cultures, the rates of collagen synthesis and secretion reached constant values within 15 and 45 min, respectively. Degradation products were first detected 6-9 min after collagen synthesis began and were transported out of the cells more rapidly than intact collagenous molecules; however, percent degradation increased slowly and did not reach a constant value even after 240 min of incubation. Since collagen degradation lags collagen synthesis, it follows that degradation is a posttranslational, rather than a cotranslational, process, and since degradation and secretion are kinetically distinguishable, it follows that they occur in parallel pathways. A simple nonlinear model for posttranslational processing of collagen is proposed.     

Incorporation of [6-3H] glucose into lipid, protein, RNA and DNA of slices of differentiating rat cerebral cortex

Abstract— An assay in vitro utilizing [6^?3H)glucose as precursor for synthesis of lipids, proteins, RNA and DNA was developed for incubated slices of rat cerebral cortex. The developmental changes in synthesis of macromolecules were followed during differentiation of rat cerebral cortex. The incorporation of glucose into lipids and proteins decreased 10-fold in incubated slices of cerebral cortex during progression from foetal to adult ages. In contrast, the specific radioactivities of RNA and DNA in incubated slices increased from the values at 3 days prepartum to peaks at 2–4 weeks postpartum.     

NEUROSECRETORY CELL PROTEIN METABOLISM IN THE LAND SNAIL,OTALA LACTEA

—Protein synthesis in an identified molluscan neurosecretory cell of the land snail, Otala lactea was examined using three different types of polyacrylamide gel electrophoresis. Cells taken from active snails synthesized specific low molecular weight proteins while those from aestivated snails did not. Most of the newly synthesized low molecular weight proteins in the active snails were lost from the cell body when the preparations was chased for 19 h in label-free enriched medium in the presence of anisomycin, an inhibitor of protein synthesis. If colchicine, a blocker of axonal transport, was included in the chase medium, the proteins present following a pulse were largely replaced by smaller molecular weight species. The results suggest that specific low molecular weight proteins are converted to smaller species and then transported from the cell body.     

Carbohydrate metabolism during cold-induced sweetening of potato tubers

The aim of this work was to discover the effects of lowering the temperature from 25° to 2° on the metabolism of glucose [U-¹⁴C] by tubers of Solanum tuberosum. Isotope was applied to tubers via a 50-μl hole made with a capillary pipette. Tubers were incubated for 2 hr, the pulse; then the glucose- [U-¹⁴C] was replaced with glucose, and incubation was continued for 18 hr, the chase. The detailed distribution of ¹⁴C was determined at the end of the pulse and at the end of the chase at 2°, and compared with those found at 25°. Lowering the temperature reduced the proportion of metabolized ¹⁴C that entered the respiratory pathways. At 2°, but not at 25°, hexose phosphates were the most heavily labelled fraction after the pulse: during the chase at 2° much of this label was metabolized to sucrose. We conclude that lowering the temperature preferentially restricts glycolysis and diverts hexose phosphates to sucrose. We suggest that this is an important cause of cold-inducing sweetening of the tubers and is due to cold-lability of key glycolytic enzymes.     

Modulation of albumin-like protein and lysozyme production by bovine tracheal gland serous cells. Dependence on culture conditions

Bovine tracheal submucosal gland serous cells were cultured in medium supplemented with either 10% fetal calf serum or 2% Ultroser G, a commercial serum substitute for cell culture. The proteins synthesized and secreted into the culture medium during [35S]methionine pulse, chase and isoproterenol-stimulated periods were analyzed. Marked differences in the patterns of secretory radiolabeled proteins with Mr values ranging from 15,000 to 95,000 were observed between pulse and chase media of cells cultured in fetal calf serum and Ultroser G. In the presence of Ultroser G, albumin-like protein production was inhibited 95% as compared to cultures incubated with fetal calf serum. A bovine lysozyme-type enzymatic activity was detected only in medium from stimulated cells cultured in Ultroser G. The results suggest that bovine tracheal serous cells synthesize different proteins according to the composition of culture medium and release certain proteins when adrenergically stimulated.     

Products of rat liver mitochondrial protein synthesis: electrophoretic analysis of the number and size of these proteins and their solubility in chloroform: methanol

Rat liver mitochondria were incubated in vitro with radioactive leucine, and submitochondrial particles prepared by several methods. Analysis of the labeled mitochondrial membrane fractions by sodium dodecylsulfate gel electrophoresis revealed three labeled bands of molecular weights corresponding to 40,000; 27,000; and 20,000 daltons. Electrophoresis for longer times at higher concentrations of acrylamide revealed eight labeled bands, ranging in molecular weights from 48,000 to 12,000.Mitochondria were incubated for 5 min with [³H]leucine followed by a chase of unlabeled leucine. Gel electrophoresis of the membranes obtained after labeling for 5 min indicated significant synthesis of polypeptides in the 40,000 M_r, range and very little labeling of low molecular-weight polypeptides. After addition of the chase, increased synthesis of the high molecular-weight polypeptides was observed; however, no significant increase or decrease of radioactivity in the bands of low molecular-weight was observed, suggesting that rat liver mitochondria have the ability to synthesize complete proteins in the M_r 27,000–40,000 range.Approximately 16% of the total leucine incorporated into protein by isolated rat liver mitochondria in vitro could be extracted by chloroform: methanol. Gel electrophoresis of the chloroform: methanol extract revealed several bands containing radioactivity with the majority of counts in a band of 40,000 molecular weight. Gel electrophoresis of the chloroform: methanol extract of lyophilized submitochondrial particles indicated label in two broad bands in the low molecular-weight region of 14,000-10,000 with insignificant counts in the higher molecular-weight regions of the gel.Yeast cells were pulse labeled in vivo with [³H]leucine in the presence of cycloheximide and the submitochondrial particles extracted with chloroform:methanol. The extract separated after gel electrophoresis into four labeled bands ranging in molecular weight from 52,000 to 10,000. Preincubation of the yeast cells with chloramphenicol prior to the pulse labeling caused a 6-fold stimulation of labeling into the band of lowest molecular weight of the chloroform: methanol extract. These results suggest that the accumulation of mitochondrial proteins synthesized in the cytoplasm, when chloramphenicol is present in the medium, may stimulate the synthesis of certain specific mitochondrial proteins which are soluble in chloroform: methanol.     

The synthesis of acidic chromosomal proteins during the cell cycle of HeLa S-3 cells. II. The kinetics of residual protein synthesis and transport

The kinetics of acidic residual chromosomal protein synthesis and transport were studied throughout the cell cycle in HeLa S-3 cells synchronized by 2 mM thymidine block and selective detachment of mitotic cells. Pulse labeling the cells with leucine-³H for 2 min and then "chasing" the radioactive proteins for up to 3 hr showed that the amount of protein synthesized, transported, and retained in the acidic residual chromosomal protein fraction is greater immediately after mitosis and later in G₁ than in the S or G₂ phases of the cell cycle. During S, only 20–25% of the proteins synthesized and transported to the acidic residual chromosomal protein fraction are chased during the first 2 hr after pulse labeling, whereas up to 40% of the material entering the residual nuclear fraction in mitosis, G₁, and G₂ leaves during a 2 hr chase. Polyacrylamide gel electrophoretic profiles of these proteins, at various times after pulse labeling, reveal that the turnover of individual polypeptides within this fraction has kinetics of synthesis and turnover which are markedly different from one another and undergo stage-specific changes.     

Programmed synthesis of collagen during postembryonic development of the nematode Panagrellus silusiae.

The relative rate of collagen synthesis in the free-living nematode Panagrellus silusiae during postembryonic development was found to be discontinuous by measuring either the incorporation of tritium into material extracted as collagen or the amount of collagen-bound tritiated proline and hydroxyproline after 2-hr incubations of whole worms with [³H]proline. A peak of collagen production preceded each of the three molts that were examined. Moreover, protocollagen prolyl hydroxylase activity during each intermolt period paralleled the pattern of collagen synthesis. On the other hand, a triphasic pattern was not observed when noncollagenous proteins were labeled with either [³H]tryptophan or [³H]leucine. In addition, the level of soluble radioactive proline that accumulates in whole organisms after 2-hr incubation periods did not fluctuate appreciably during postembryonic development. The mean ratio of hydroxy-proline to proline in a number of collagen samples extracted at various times during the maturation phase was 0.113 ± 0.040. Pulse and chase experiments with [³H]proline indicated that most of the collagen synthesized during a peak period is lost after the second ecdysis following the labeling interval. In contrast, a considerable proportion of the collagen synthesized during nonpeak periods is retained throughout the postembryonic period. It is postulated that the modulated pattern of collagen biosynthesis in Panagrellus reflects, for the most part, a quantitative regulation of the production of cuticular collagen during postembryonic development.     

Mechanism of synthesis of D-glucans by D-glucosyltransferases from Streptococcus mutans 6715

Two glucosyltransferases from Streptococcus mutans 6715 were purified and separated. One of the glucosyltransferases synthesized an insoluble glucan, and the other, a soluble glucan. The enzymes were immobilized on Bio-Gel P-2 beads, and the mechanism of glucan synthesis was studied by pulse and chase techniques with 14C-sucrose. Label was associated with the immobilized enzymes. The label could be quantitatively released by heating at pH 2. Analysis of the labeled products from the pulse experiment showed labeled glucose and labeled glucan; the chase experiment showed labeled glucan and a significant decrease in labeled glucose. The glucans from the pulse and the chase experiments were separated from glucose by chromatography on Bio-Gel P-6. They were reduced with sodium borohydride, and the products hydrolyzed with acid. Analysis of the labeled products from the reduced and hydrolyzed, pulsed glucans showed labeled glucose and labeled glucitol; label in the glucitol was greatly decreased in the chase experiment. These experiments showed that glucose and glucan were covalently attached to the active site of the enzymes during synthesis, and that the glucose was being transferred to the reducing end of the glucan chain. A mechanism for the synthesis of the glucans is proposed in which there are two catalytic groups on each enzyme that holds glucosyl and glucanosyl units. During synthesis, the glucosyl and glucanosyl units alternate between the two sites, giving elongation of the glucans from the reducing end. The addition of increasing amounts of B-512F dextran to the insoluble-glucan-forming glucosyltransferase produced a decrease in the proportion of insoluble glucan formed and a concomitant increase in a soluble glucan. The total amount of glucan synthesized (soluble plus insoluble) was increased 1.6 times over the amount of insoluble glucan formed when no exogenous dextran was added. It is shown that the addition of B-512F dextran affects the solubility of the synthesized alpha-(1 to 3)-glucan by accepting alpha-(1-3)-glucan chains at various positions along the dextran chain, to give a soluble, graft polymer.     

Establishment of Histone Modifications after Chromatin Assembly

Every cell has to duplicate its entire genome during S-phase of the cell cycle. After replication, the newly synthesized DNA is rapidly assembled into chromatin. The newly assembled chromatin ‘matures’ and adopts a variety of different conformations. This differential packaging of DNA plays an important role for the maintenance of gene expression patterns and has to be reliably copied in each cell division. Posttranslational histone modifications are prime candidates for the regulation of the chromatin structure. In order to understand the maintenance of chromatin structures, it is crucial to understand the replication of histone modification patterns. To study the kinetics of histone modifications in vivo, we have pulse-labeled synchronized cells with an isotopically labeled arginine (¹⁵N₄) that is 4 Da heavier than the naturally occurring ¹⁴N₄ isoform. As most of the histone synthesis is coupled with replication, the cells were arrested at the G1/S boundary, released into S-phase and simultaneously incubated in the medium containing heavy arginine, thus labeling all newly synthesized proteins. This method allows a comparison of modification patterns on parental versus newly deposited histones. Experiments using various pulse/chase times show that particular modifications have considerably different kinetics until they have acquired a modification pattern indistinguishable from the parental histones.     

Synthesis and renewal of proteins in duck anterior hypophysis in organ culture

In cultures of duck anterior pituitaries, the synthesis and renewal of the specific secretory protein prolactin and of total newly synthesized tissue proteins were studied. As concerns prolactin, assay of the tissue and culture media hormone content demonstrates de novo synthesis of prolactin in vitro at a constant rate during at least 2 wk. The prolactin content after 1 wk and after 2 wk of culture is the same and is similar to the initial content. The renewal time of this prolactin can be estimated at 28 or 48 hr. As concerns total proteins, the use of a chase after a short pulse of 5 min in the presence of tritiated L-leucine demonstrated that newly synthesized proteins are excreted into the culture medium from 30 min to 1 hr after the beginning of the chase. Therefore, the synthesis and excretion of proteins are two discontinuous phenomena. The migration rate of the total proteins was slower than that of prolactin, indicating that this hormone does not represent more than about half of the newly synthesized proteins. These conclusions are in good agreement with those based on high resolution radioautographic data previously obtained on the same material.     

Changes in the C-Labeled Cell Wall Components with Chase Time after Incorporation of UDP[C]Glucose by Intact Cotton Fibers

Intact, in vitro-grown cotton fibers will incorporate [¹⁴C]glucose from externally supplied UDP[¹⁴C]glucose into a variety of cell wall components including cellulose; this labeled fraction will continue to increase up to 4 hours chase time. In the fraction soluble in hot water there was no significant change in total label; however, the largest fraction after the 30 minute pulse with UDP[¹⁴C]glucose was chloroform-methanol soluble (70%) and showed a significant decrease with chase. The lipids that make up about 85% of this fraction were identified by TLC as steryl glucosides, acylated steryl glucosides, and glucosyl-phosphoryl-polyprenol. Following the pulse, the loss of label from acylated steryl glucosides and glucosylphophoryl-polyprenol was almost complete within 2 hours of chase; steryl glucosides made up about 85% of the fraction at that chase time. The total loss in the lipid fraction (about 100 picomoles per milligram dry weight of fiber) with chase times of 4 hours approximates the total gain in the total glucans.