Search

Kaposi sarcoma (KS) herpesvirus (KSHV) infection of endothelial cells (EC) is associated with strong induction of heme oxygenase-1 (HO-1), a stress-inducible host gene that encodes the rate-limiting enzyme responsible for heme catabolism. KS is an angioproliferative tumor characterized by the proliferation of KSHV-infected spindle cells, and HO-1 is highly expressed in such cells. HO-1 converts the pro-oxidant, proinflammatory heme molecule into metabolites with antioxidant, anti-inflammatory, and proliferative activities. Previously published work has shown that KSHV-infected EC in vitro proliferate in response to free heme in a HO-1-dependent manner, thus implicating virus-enhanced HO-1 activity in KS tumorigenesis. The present study investigated the molecular mechanisms underlying KSHV induction of HO-1 in lymphatic EC (LEC), which are the likely spindle cell precursors. In a time course analysis of KSHV-infected cells, HO-1 expression displays biphasic kinetics characterized by an early transient induction that is followed by a more sustained upregulation coincident with the establishment of viral latency. A viral microRNA miR-K12-11 deletion mutant of KSHV was found to be defective for induction of HO-1 during latency. A potential mechanism for this phenotype was provided by BACH1, a cellular HO-1 transcriptional repressor targeted by miR-K12-11. In fact, in KSHV-infected LEC, the BACH1 message level is reduced, BACH1 subcellular localization is altered, and miR-K12-11 mediates the inverse regulation of HO-1 and BACH1 during viral latency. Interestingly, the data indicate that neither miR-K12-11 nor de novo KSHV gene expression is required for the burst of HO-1 expression observed at early times postinfection, which suggests that additional virion components promote this phenotype., IMPORTANCE While the mechanisms underlying KSHV induction of HO-1 remain unknown, the cellular mechanisms that regulate HO-1 expression have been extensively investigated in the context of basal and pathophysiological states. The detoxifying action of HO-1 is critical for the protection of cells exposed to high heme levels. KS spindle cells are erythrophagocytic and contain erythrocyte ghosts. Erythrocyte degeneration leads to the localized release of heme, creating oxidative stress that may be further exacerbated by environmental or other cofactors. Our previous work showed that KSHV-infected cells proliferate in response to heme and that this occurs in a HO-1-dependent manner. We therefore hypothesize that KSHV induction of HO-1 contributes to KS tumor development via heme metabolism and propose that HO-1 be evaluated as a therapeutic target for KS. Our present work, which aimed to understand the mechanisms whereby KSHV induces HO-1, will be important for the design and implementation of such a strategy.

Background BST2/tetherin is an innate immune molecule with the unique ability to restrict the egress of human immunodeficiency virus (HIV) and other enveloped viruses, including Ebola virus (EBOV). Coincident with this discovery was the finding that the HIV Vpu protein down-regulates BST2 from the cell surface, thereby promoting viral release. Evidence suggests that the EBOV envelope glycoprotein (GP) also counteracts BST2, although the mechanism is unclear. Results We find that total levels of BST2 remain unchanged in the presence of GP, whereas surface BST2 is significantly reduced. GP is known to sterically mask surface receptors via its mucin domain. Our evaluation of mutant GP molecules indicate that masking of BST2 by GP is probably responsible for the apparent surface BST2 down-regulation; however, this masking does not explain the observed virus-like particle egress enhancement. We discovered that VP40 coimmunoprecipitates and colocalizes with BST2 in the absence but not in the presence of GP. Conclusions These results suggest that GP may overcome the BST2 restriction by blocking an interaction between VP40 and BST2. Furthermore, we have observed that GP may enhance BST2 incorporation into virus-like particles. Understanding this novel EBOV immune evasion strategy will provide valuable insights into the pathogenicity of this deadly pathogen.

CD16+ monocytes represent 5-10% of circulating monocytes in healthy individuals and are dramatically expanded in several pathological conditions including AIDS and HIV-1-associated dementia (HAD). CD16+ monocytes constitutively produce high levels of pro-inflammatory cytokines and neurotoxic factors that may contribute to the pathogenesis of these disorders. Monocyte recruitment into the central nervous system (CNS) and other peripheral tissues in response to locally produced chemokines is a critical event in immune surveillance and inflammation and involves monocyte arrest onto vascular beds and subsequent diapedesis. Here we investigate the ability of CD16+ monocytes to undergo transendothelial migration (TEM) under constitutive and inflammatory conditions. CD16+ monocytes underwent TEM across unstimulated human umbilical vascular (HUVEC) and brain microvascular endothelial (BMVEC) cell monolayers in response to soluble fractalkine (FKN/CX3CL1). Stimulation with tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma) induced high and low expression of membrane-bound FKN on HUVEC and BMVEC, respectively, together with expression of VCAM-1 and intercellular adhesion molecule-1 (ICAM)-1. By contrast, only HUVEC expressed CD62E while BMVEC remained negative. Both CD16- and CD16+ monocyte subsets adhered to TNF/IFN-gamma-stimulated HUVEC with higher frequency than to unstimulated HUVEC. Monocyte chemoattractant protein-1 (MCP-1) triggered efficient TEM of CD16- monocytes across TNF/IFN-gamma-stimulated HUVEC, whereas soluble FKN failed to induce TEM of CD16+ monocytes across stimulated HUVEC. These results demonstrate that stimulation with TNF and IFN-gamma triggers expression of membrane-bound FKN on both HUVEC and BMVEC, but prevents TEM of CD16+ monocytes in response to soluble FKN. Thus, pro-inflammatory CD16+ monocytes may contribute to the pathogenesis of HAD and other inflammatory CNS diseases by affecting the integrity of the blood-brain barrier as a consequence of their massive accumulation onto inflamed brain vascular endothelial cells expressing FKN and other adhesion molecules.

CD16+ monocytes represent 5–10% of peripheral blood monocytes in normal individuals and are dramatically expanded in several pathological conditions including sepsis, human immunodeficiency virus 1 infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines and may represent dendritic cell precursors in vivo. The mechanisms that mediate the recruitment of CD16+ monocytes into tissues remain unknown. Here we investigate molecular mechanisms of CD16+ monocyte trafficking and show that migration of CD16+ and CD16− monocytes is mediated by distinct combinations of adhesion molecules and chemokine receptors. In contrast to CD16− monocytes, CD16+ monocytes expressed high CX3CR1 and CXCR4 but low CCR2 and CD62L levels and underwent efficient transendo-thelial migration in response to fractalkine (FKN; FKN/CX3CL1) and stromal-derived factor 1α (CXCL12) but not monocyte chemoattractant protein 1 (CCL2). CD16+ monocytes arrested on cell surface–expressed FKN under flow with higher frequency compared with CD16− monocytes. These results demonstrate that FKN preferentially mediates arrest and migration of CD16+ monocytes and suggest that recruitment of this proinflammatory monocyte subset to vessel walls via the CX3CR1-FKN pathway may contribute to vascular and tissue injury during pathological conditions.

Like the other more well-characterized post-translational modifications (phosphorylation, methylation, acetylation, acylation, etc.), the attachment of the 76 amino acid ubiquitin (Ub) protein to substrates has been shown to govern countless cellular processes. As obligate intracellular parasites, viruses have evolved the capability to commandeer many host processes in order to maximize their own survival, whether it be to increase viral production or to ensure the long-term survival of latently infected host cells. The first evidence that viruses could usurp the Ub system came from the DNA tumor viruses and Adenoviruses, each of which use Ub to dysregulate the host cell cycle (Scheffner et al., 1990; Querido et al., 2001). Today, the list of viruses that utilize Ub includes members from almost every viral class, encompassing both RNA and DNA viruses. Among these, there are examples of Ub usage at every stage of the viral life cycle, involving both ubiquitination and de-ubiquitination. In addition to viruses that merely modify the host Ub system, many of the large DNA viruses encode their own Ub modifying machinery. In this review, we highlight the latest discoveries regarding the myriad ways that viruses utilize Ub to their advantage.

Kaposi sarcoma (KS) is a multi-focal angioproliferative disease driven by infection of cells by Kaposi sarcoma herpesvirus/human herpesvirus-8 (KSHV/HHV8). KSHV/HHV8 infection activates numerous sequential and parallel signaling pathways creating an angiogenic–inflammatory state that leads to the development of Kaposi sarcoma. These pathways can be grouped into KSHV/HHV8- specific pathways and cellular growth/angiogenic pathways that are “pirated” by KSHV/HHV8. Advances in our understanding of the pathogenesis of KS parallel the clinical development of signal transduction inhibitors. This chapter reviews the signaling pathways active in KSHV/HHV8-infected cells and discusses the use of clinical inhibitors of these pathways in the treatment of KS.

Kaposi sarcoma (KS) is the most common AIDS-associated malignancy and is characterized by angiogenesis and the presence of spindle cells. Kaposi sarcoma-associated herpesvirus (KSHV) is consistently associated with all clinical forms of KS, and in vitro infection of dermal microvascular endothelial cells (DMVECs) with KSHV recapitulates many of the features of KS, including transformation, spindle cell proliferation, and angiogenesis. To study the molecular mechanisms of KSHV pathogenesis, we compared the protein expression profiles of KSHV-infected and uninfected DMVECs. This comparison revealed that heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in heme catabolism, was up-regulated in infected endothelial cells. Recent evidence suggests that the products of heme catabolism have important roles in endothelial cell biology, including apoptosis and angiogenesis. Here we show that HO-1 mRNA and protein are up-regulated in KSHV-infected cultures. Comparison of oral and cutaneous AIDS-KS tissues with normal tissues revealed that HO-1 mRNA and protein were also up-regulated in vivo. Increased HO-1 enzymatic activity in vitro enhanced proliferation of KSHV-infected DMVECs in the presence of free heme. Treatment with the HO-1 inhibitor chromium mesoporphyrin IX abolished heme-induced proliferation. These data suggest that HO-1 is a potential therapeutic target for KS that warrants further study. (Blood. 2004;103: 3465-3473)

Kaposi sarcoma–associated herpesvirus (KSHV) is associated with KS, primary effusion lymphoma (PEL), and multicentric Castleman disease. Reactivation of KSHV in latently infected cells and subsequent plasma viremia occur before the development of KS. Intracellular signaling pathways involved in KSHV reactivation were studied. In latently infected PEL cells (BCBL-1), KSHV reactivation in single cells was determined by quantitative flow cytometry. Viral particle production was determined by electron microscope analyses and detection of minor capsid protein in culture supernatants. Agents that mobilized intracellular calcium (ionomycin, thapsigargin) induced expression of KSHV lytic cycle-associated proteins and led to increased virus production. Calcium-mediated virus reactivation was blocked by specific inhibitors of calcineurin-dependent signal transduction (cyclosporine, FK506). Similarly, calcium-mediated virus reactivation in KSHV-infected dermal microvascular endothelial cells was blocked by cyclosporine. Furthermore, retroviral transduction with plasmid DNA encoding VIVIT, a peptide specifically blocking calcineurin-NFAT interactions, inhibited calcium-dependent KSHV reactivation. By contrast, chemical induction of lytic-phase infection by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate was blocked by protein kinase C inhibitors, but not by calcineurin inhibitors. In summary, calcineurin-dependent signal transduction, an important signaling cascade in vivo, induces calcium-dependent KSHV replication, providing a possible target for the design of antiherpesvirus strategies in KSHV-infected patients.

1. The growth of Escherichia coli proline auxotrophs on medium containing L-proline (50 microgram/ml) induces catabolic enzymes. A bioradiological assay system for proline, using proB cells of E. coli, might give erroneous results owing to proline catabolism by the proline auxotrophs on which the assay depends. 2. Differential utilization of proline and 1-pyrroline-5-carboxylate by the proB cells for the synthesis of protein, and failure of the method to distinguish between these two possible products of the proline-biosynthetic enzymes, might also give rise to error. 3. The proline-dependent incorporation of [14C]phenylalanine into the protein of proline-starved proB auxotrophs was to some degree directly influenced by the presence of crude cell extract from E. coli, even though this was not supplied with substrate and cofactors, and could thus not itself synthesize proline. 4. The kinetics of proline biosynthesis by cell-free extracts were linear and biphasic, only the last phase being affected by the concentrations of substrate and extract. This phenomenon is not understood. 5. Proline biosynthesis is inhibited, not only by high concentrations of ATP, but also by aspartate, glycine, alanine and serine, aspartate having the greatest effect. 6. Attempts at complementation in vitro between extracts of proline auxotrophic mutants were not successful, suggesting the possibility that strain X680 (proA) and/or X278 (proB) may be a double mutant. 7. The enzymes of proline biosyntehsis are co-eluted from a column of Bio-Gel A1.5M in a position corresponding to a mol.wt. of 350000. 8. Comparisons between rates of proline biosynthesis in vivo and in vitro were made.

The conversion of glutamic acid to proline by an Escherichia coli extract was studied. The activity was dependent upon the presence of ATP and NADPH and was largely unaffected by the presence of NH 3 or imidazole. The first two pathway enzymes appear to exist as a complex which stabilizes a labile intermediate postulated as γ-glutamyl phosphate. Attempted synthesis of this compound was unsuccessful due to its spontaneous cyclization to 2-pyrrolidone 5-carboxylate. Dissociation of the enzyme complex upon dilution of the extract is presumed responsible for an experimentally observed “dilution effect”. E. coli pro A − and pro B − auxotroph extracts failed to complement one another in the biosynthesis of proline. This is attributed to the lack of a dynamic equilibrium between the complex and its constituent enzymes. In vivo studies with E. coli showed no evidence for metabolic channeling in the final reaction of proline synthesis, the reduction of Δ 1 -pyrroline 5-carboxylate.

Differential centrifugation of osmotically lysed lysozyme-EDTA spheroplasts from Escherichia coli sedimented 50–70% of the glycolytic activities examined in a low speed pellet; the remaining activity, occurring in a high speed supernatant, contained the soluble enzymes of the cell. The distribution pattern of the enzymes could be altered by extrusion of the spheroplasts through the French Press or by lysis at different pH values. Electron micrographs of the pellet fraction revealed lysed spheroplasts mostly devoid of cellular constituents but consisting of cytoplasmic membranes surrounded by partially degraded cell wall fragments. Washing of the pellet showed that the enzymes were not all bound to the same degree to the membrane fraction. Throughput activity of the glycolytic pathway was demonstrated for the membrane fraction, but none was observed for the soluble fraction of the cell (i.e. for enzymes present in the supernatants) unless these were first concentrated by ultrafiltration. The supernatant from the lysed spheroplasts, together with a further supernatant obtained by washing the membrane pellet, was concentrated by ultrafiltration and chromatographed on a Bio-Gel column. The eluate contained glycolytic activities both in fractions corresponding to relatively high and relatively low molecular weight material The high molecular weight species, containing a proportion of all the enzymes studied, had a molecular weight of at least 1.2 × 10 6 . A multienzyme aggregate containing one each of the glycolytic enzymes would have a molecular weight of ∼ 1.3 × 10 6 . The specific rate of pyruvate formation from glucose by the high molecular weight species was similar to that obtained from a preparation in which the fractions containing all the low molecular weight material enzyme activities were pooled and concentrated by ultrafiltration. Using the high molecular weight material, studies were made of the ability of added unlabelled glycolytic intermediates to compete for catalytic sites with intermediates produced endogenously from [ 14 C 6 ] glucose. The relatively weak competition observed indicated a high degree of protection afforded the labelled intermediates derived from [ 14 C 6 ] glucose.

Moses , V. (University of California, Berkeley), and M. Calvin . Lifetime of bacterial messenger ribonucleic acid. J. Bacteriol. 90: 1205–1217. 1965.—When cells from a stationary culture of Escherichia coli were placed in fresh medium containing inducer for β-galactosidase, growth, as represented by increase in turbidity and by total protein synthesis, started within 30 sec. By contrast, β-galactosidase synthesis was greatly delayed compared with induction during exponential growth. Two other inducible enzymes ( d -serine deaminase and l -tryptophanase) and one repressible enzyme (alkaline phosphatase) showed similar lags. The lags were not due to catabolite repression. They could not be reduced by pretreatment of the culture with inducer, or by supplementing the fresh medium with amino acids or nucleotides. The lag was also demonstrated by an i − mutant constitutive for β-galactosidase synthesis. An inhibitor of ribonucleic acid (RNA) synthesis, 6-azauracil, preferentially inhibited β-galactosidase synthesis compared with growth in both inducible and constitutive strains. Puromycin, an inhibitor of protein synthesis, acted as an inhibitor at additional sites during the induction of β-galactosidase synthesis. No inhibition of the reactions proceeding during the first 20 sec of induction was observed, but puromycin seemed to prevent the accumulation of messenger RNA during the period between 20 sec and the first appearance of enzyme activity after 3 min. It is suggested that these observations, together with many reports in the literature that inducible enzyme synthesis is more sensitive than total growth to some inhibitors and adverse growth conditions, can be explained by supposing that messenger RNA for normally inducible enzymes is biologically more labile than that for some normally constitutive proteins. The possible implications of this hypothesis for the achievement of cell differentiation by genetic regulation of enzyme synthesis are briefly discussed.

A study has been made of the experimental errors inherent in investigations of intermediary metabolism in a rat liver preparation, using C 14 -labeled substrates, chromatography, and radioautography as analytical tools. Assessed on the basis of absolute amount of C 14 incorporated into each metabolic product, the variation between experiments using samples of tissue from different animals was considerably greater than that using several samples of tissue from one animal. However, if the C 14 incorporated into each metabolite was expressed as a percentage of the total C 14 incorporated into all the metabolites, the variation between replicate samples of tissue from one animal was approximately the same as that using tissue from several animals. In both cases the experimental variation was such that, unless the effect of an alteration in the environment resulted in a change in the amount of C 14 in any particular compound of at least 80–85%, it could not be regarded as significant. This applied only to compounds containing more than 2% of the total incorporated C 14 ; substances containing less C 14 showed an even greater variation. The main sources of variation were probably slight uncontrolled differences in the incubation conditions, together with inaccuracies in the determinations of radioactivity in those compounds containing very small amounts of the radioisotope.

The relation between chemical structure and uncoupling activity in congeners of salicylate has been studied by means of their effects on the oxygen consumption of a suspension of baker's yeast utilising a limited quantity of glucose. Salicylic acid, salicylaldehyde, 2-hydroxyacetophenone, salicylamide, 3-methylsalicylic and 1-hydroxy-2-naphthoic acids were found to show uncoupling activity.

SUMMARY: Cultures of the fungus Zygorrhynchus moelleri were grown in a glucose-acetate + ammonia + salts medium in the presence of either radioactive carbon dioxide or acetate labelled in either the methyl- or the carboxyl-positions. After growth the protein was hydrolysed and the pattern of incorporation of radiocarbon into the amino acids was determined by chromatography and radioautography; activity from carbon dioxide appeared in glutamie and aspartic acids, methionine, threonine, isoleucine, proline and arginine. With labelled acetate lysine and leucine were also radioactive. A pool of free amino acids was present in this fungus. Several amino acids were isolated from the three samples of hydrolysed protein and their specific activities were determined. Assuming that the tricarboxylic acid cycle operated in this organism, a number of predictions concerning the distribution of radiocarbon in certain amino acids which could be made were largely confirmed by the experimental findings. By partial degradation of glutamie and aspartic acids, followed by assay of the radioactivity in the products, it was possible to calculate that about 60% of the oxalacetate used for citrate synthesis was recycled C4-dicarboxylic acid; 40% was synthesized from pyruvate and carbon dioxide. Approximately 25% of the total respiratory carbon dioxide evolved could be accounted for in terms of decarboxylations occurring within the citric acid cycle.

Prevost , C. (University of California, Berkeley), and V. Moses . Action of phenethyl alcohol on the synthesis of macromolecules in Escherichia coli . J. Bacteriol. 91: 1446–1452. 1966.—A kinetic study of the effects of various concentrations of phenethyl alcohol on the synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), protein, and β-galactosidase in Escherichia coli has confirmed that RNA synthesis, rather than DNA synthesis, is first and most affected by phenethyl alcohol. The presence of inducer did not protect β-galactosidase synthesis from inhibition by phenethyl alcohol. Little preferential inhibition of β-galactosidase synthesis was observed; this is in contrast to the severe catabolite repression which results from partial inhibition of total protein synthesis caused by chloramphenicol or starvation for a required amino acid. We found no evidence that messenger RNA synthesis was inhibited to a greater extent than total RNA synthesis.

The nematode, Caenorhabditis briggsae, was cultured axenically in a mixture of chick embryo extract, autoclaved liver extract and sodium acetate-2-14C. A protein hydrolysate was prepared from the worms and the eggs which were collected from the cultures. Chromatography and radioautography were carried out in a study of the amino acid composition of the hydrolysate. The following amino acids were found labelled : aspartic acid, glutamic acid, alanine, proline, glycine, serine. Cystein and cystine were oxidized to cysteic acid which was also labelled. The following amino acids were not labelled: arginine, histidine, lysine, methionine, threonine, tyrosine, valine and the combined spot representing phenylalanine, leucine and isoleucine. Tryptophane would have been destroyed by our method of hydrolysis. Since the labelled amino acids are synthesized by the worm, it is suggested, tentatively, that they are not required in an otherwise adequate diet. So far as the unlabelled amino acids are concerned, it is suggested, on the basis of the results of certain culture experiments (published separately) that, with the probable exception of tyrosine, they are essential in the diet of C. briggsae.

When glucose was added to carbohydrate-starved cells of Zygorhyncus moelleri the rate of oxygen uptake did not immediately rise to a constant value, but there was a lag period of 2 or 3 hours before it reached its maximum level. The length of this lag period increased from a few minutes for short periods of starvation to 2-3 hours after 12 hours in a carbohydrate-free medium. Factors believed to affect cellular permeability (a cationic detergent, adjustments of the pH, and of the potassium/calcium ratio) reduced the length of the lag period by not more than 40 per cent, of the original value without affecting the final rate of oxygen uptake. Investigation of the entry of glucose into the cells showed that the rate of oxygen uptake was not limited by the concentration of intra-cellular glucose for more that about 11 per cent, of the lag period in starved cells. The reasons for this difference in the percentage of the lag period apparently due to a permeability barrier are tentatively discussed in connexion with the route by which glucose enters the cells.

SUMMARY: The presence or absence of regulatory control mechanisms has been studied for the enzymes 6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase and hexokinase in Bacillus subtilis. The cellular levels of these enzymes have been investigated during steady exponential growth in various media and following growth shifts. Comparison has been made of the behaviour of these enzymes with that of β-galactosidase in a strain of Escherichia coli in which the regulator and operator genes are deleted. Criteria for recognizing truly constitutive enzymes are discussed, and it is tentatively concluded that while the synthesis of the dehydrogenases is controlled, that of hexokinase is probably constitutive.

The effects on a number of parameters of transferringEscherichia coli between protonated and deuterated media were studied; these included growth, oxygen consumption and the synthesis of DNA, RNA, total protein, and β-galactosidase. Similar measurements were made on cells fully adapted to growth on deuterated media. The amino acid compositions of deuterated and protonated cellular protein were similar, but in deuterated cells the ratio protein: DNA was doubled. Deutero- and protio-β-galactosidase had similarK M values and turnover numbers in D2O and H2O. The kinetics of β-galactosidase synthesis were not changed by deuteration, but it was found that lower concentrations of inducer were required to achieve particular levels of induction. Brief exposure to inducer in one medium, followed by removal of inducer and expression of enzyme-forming-potential in either D2O or H2O, showed that mRNA synthesized by deuterated cells was translated equally well in both media. mRNA synthesized by protonated cells was translated about twice as efficiently in H2O. Inducible strains (but not a regulator constitutive) lost the capacity to synthesize enzymically active β-galactosidase after more than 100 generations in D2O-acetate. The defect persisted when such cells were grown in H2O-acetate, but enzyme activity was restored by growth in H2O-glycerol. The failure to produce active enzyme was not due to a failure of the induction mechanism; gel electrophoresis revealed the presence of an inactive protein species. The nature of adaptation to deuteration is discussed.

Although the functions of the essential major elements in plant metabolism have been studied for many years, little work has been done concerning the effect of these elements during short-term incorporation of radioactive carbon dioxide. This may be of some importance as it has been the general custom during photosynthesis studies in this laboratory to suspend algae in various dilute buffer solutions or in distilled water alone assuming that the salt remaining within the cells from the time of growth in nutrient solution are sufficient in quantity for the cells not to become deficient in one or more of the essential elements during the course of the experiment. There are some indications, however, that the addition of salts to algae suspended in distilled water may have a rapid, pronounced effect on some metabolic system within the plant. Thus, Clendenning, Brown and Eyster (1956) have reported that Nostoc muscorum, if rinsed and resuspended in distilled water, loses most of its photosynthetic capacity, which can, however, be completely restored by the addition of potassium ion in concentrations no greater than a few parts per million. Also, K. Baalorud (personal communication) found that the photosynthetic rate of a marine diatom, when suspended in synthetic magnesium-free water, can be greatly increased by the addition of magnesium salts. In view of these observations it appeared worthwhile to investigate the effects of the addition of the essential elements in those photosynthesis experiments in which the cells are kept in distilled water for varying periods of time.

In view of the wide application of radioactive carbon dioxide in tracer experiments together with a reported anomalous effect of the isotope content on the tracer behavior, we have investigated the effect of varying specific activity of the carbon dioxide on both the rate and pattern of photosynthetic carbon fixation. We have found no effect of the carbon-14 other than the ordinary mass effect in both algae and higher plants.

Studies by Lynch and Calvin (1952,1953) have established the nature of the compounds incorporating C{sup 14} nonphotosynthetically from C{sup 14}O{sub 2} in thirteen microorganisms: a yeast, a protozoan, two water moulds, one slime mould, three algae, three bacteria, and the green flagellate Euglena Gracilis. With the exception of H. gracilis, and of Lactobacillus cassi which fixed no detectable amounts of carbon dioxide, all these organisms fixed carbon dioxide into amino and organic acids derived from the tricarboxylic acid cycle, and into a few other compounds in individual cases (tyrosine, phenylalanine, polysaccharides [probably glucose polymers], acetic acid and butyric acid). The authors concluded that the presence of C{sup 14} in almost all these compounds could be accounted for by the carboxylation of pyruvate to yield oxalacetic or malic acids, followed by transaminase reactions. In E. gracilis, however, considerable quantities of activity also appeared in phosphorylated compounds in the dark, especially in the sugar monophosphates, phosphoglyceric acid, and phosphoenolpyruvic acid. Only with this organisms was a kinetic study performed to determine the identity and degree of labeling of the compounds containing C{sup 14} after varying periods of time. It was not stated definitely by which route carbon dioxide entered the photosynthetic intermediates,more » but it was implied that it was incorporated directly into phosphoglyceric acid, and that the energy for this process, which in photosynthesis is derived from sunlight, and was provided by respiration or fermentation. Certain aspects of these presumptions however, do not adequately account for all the experimental data.« less

1. Repression by glucose of beta-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i(-)) and permease-less (y(-)) cells as well as in the corresponding i(+) and y(+) strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-beta-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of beta-galactosidase synthesis (e.g. isopropyl thio-beta-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-beta-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.

SUMMARY: The intra- and extracellular concentrations of a number of phosphorylated intermediary metabolites in Escherichia coli were measured radiochemically during growth in low phosphate (0.75 mM) media containing [32P]Pi. Distribution studies showed that most of the monophosphate metabolites were present in the supernatant medium outside the bacteria. This was much less marked for the di- and triphosphates, and was not due to bacterium lysis. Adenosine 3′,5′-cyclic monophosphate was present solely in the medium under all conditions tested. 3′,5′-Cyclic-AMP added to the medium was only partly effective in reversing repression of β-galactosidase synthesis by gluconate. This was not due to degradation of the nucleotide. In another series of strains, chemical determinations were made of the intra-and extracellular concentrations of fructose 1,6-diphosphate and triose phosphate under varying conditions of growth in high phosphate (0.1 M) media; while most of the fructose 1,6-diphosphate remained inside the bacteria, triose phosphate was present largely in the medium. In all of these studies there was no correlation between the severity of catabolite repression and the concentrations of metabolites either inside the bacteria, or in the medium.

Escherichia coli, strain 300U, was grown for 450 generations in 6 different media. Each line was then subcultured into each of the 6 media, and allowed to grow for about 12 generations. The growth rates and rates of induced β-galactosidase synthesis were, as expected, highly dependent on the medium in which the cells were growing at the time of sampling. Both parameters, and particularly β-galactosidase synthesis, also showed some influence of the medium in which the cells had previously been grown. Of 26 soluble protein components separated by gel electrophoresis in the same experiment, 9 were highly dependent on the current medium, with insignificant effects of the previous growth conditions; 4 were significantly affected by the previous medium but were not influenced by the current medium; 3 were influenced both by current and earlier growth conditions; and 10 components showed no variation. In further experiments, the kinetics of labelled phenylalanine incorporation into soluble proteins were studied by pulse-labelling during growth upshifts and downshifts. The responses of the various protein components to the growth shift were kinetically variable; some responded immediately while in others the response was delayed. In other cases, a series of oscillations were observed after an initial response, while a final category showed little response to the growth shift. The proportional incorporation of phenylalanine into the different bands was still changing 1–2 generations after the shift. It is concluded that a period of adaptation of at least 12 generations is required for cells to reach a new equilibrium after a growth shift.

Using radioactive carbon dioxide, an attempt has been made to distinguish the various pools of intermediary metabolism which may be physically or chemically separate within the cell. Some correlation between the structural elements of the cells and these pools appears possible.

Contemplation of the sheer complexity of cellular metabolism, coupled with the realization that in bacterial cells it all takes place in a minute volume exhibiting a minimum of cellular architectural detail, soon begins to prompt questions about the nature of intracellular organization in prokaryotic cells. The very absence of membrane-bounded organelles suggests that, aside from reactions localized in or about the cell membrane, organizational structure is likely to be based on direct macromolecular interactions. These would yield macro structures which in some cases might also act in a more-or-less compartmentalized fashion, leading to the separate channelling of whole series of sequential reactions. There is some variation in the way in which the terms “compartment” and “channel” are used. In the present contribution a “channel” is defined as a structure or a facet of organization which acts as a constraint to free chemical diffusion. A “compartment” is a sequestered volume; it may be similar to a channel. Any defined aspect of biochemical organization may represent a compartment so long as it remains chemically inactive. For example, a ligand bound to an enzyme is in a compartment until it undergoes a reaction.

1. By using Bio-Gel A1.5M and Sephadex G-150 columns, crude cell-free extracts of Escherichia coli were fractionated to demonstrate the existence of a proline-biosynthetic aggregate. 2. Sephadex G-150 resolves two glutamyl kinases that are inhibited by proline, with mol.wts. of 125000 and 38000, the reactions of which are Mg2+-dependent. The heavier species is more sensitive to inhibition by proline. 3. Gamma-Glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase (EC 1.5.1.2) have mol.wts. of approx. 125000 and 190000 respectively, the specific activity of the latter being 5 × 10(3)-fold greater than either of the other two biosynthetic enzymes or of the total pathway in vivo. 4. Bio-Gel A1.5M chromatography gave a single glutamyl kinase of mol.wt. 250000, and the possibility of this being a constituent of an enzyme complex is discussed.

1. The intermediary metabolism of two strains of Escherichia coli has been examined. One strain (Q22) exhibits acute transient repression of beta-galactosidase synthesis when glucose is supplied to cells growing on glycerol; the other strain (W3110) does not. The two strains do not differ genetically in their lac operons. 2. Strain Q22 uses about twice as much glucose as strain W3110 per unit of cell mass produced. 3. Pentose phosphate-cycle activity in the presence of glucose is much stronger in strain Q22 than in strain W3110. 4. In strain Q22 the pool sizes of glucose 6-phosphate, 6-phosphogluconate, fructose 1,6-diphosphate and NADPH increase when glucose is added to cells growing on glycerol, and beta-galactosidase synthesis is severely inhibited. After about 1hr. the synthesis of beta-galactosidase is partly resumed, and the pool sizes of the four compounds fall. ATP, NADH and several other phosphorylated compounds show no concentration changes. 5. These concentration changes do not occur in strain W3110, in which beta-galactosidase synthesis is only rather weakly repressed by glucose. 6. It is suggested that repression of enzyme synthesis by glucose requires the rapid operation of the pentose phosphate cycle, and is mediated by one of the four substances whose concentration rises and later falls in strain Q22. A definite choice of effector from among these four possibilities cannot at present be made.

1. Both permanent and transient catabolite repression of β-galactosidase synthesis in Escherichia coli are abolished by 5mm-3′:5′-cyclic-AMP when elicited by glucose, but not when caused by a mixture of glucose, glucose 6-phosphate, gluconate and casein hydrolysate (casamino acids). 2. Glucose uptake is slightly increased by 3′:5′-cyclic-AMP. 3. No significant effects of the nucleotide were found on the synthesis of protein and RNA, either in exponential growth on one substrate, or during a growth shift from glycerol to glycerol plus glucose. 4. Marked changes in the soluble-protein profiles of cells growing in glycerol and glucose were caused by the presence of 3′:5′-cyclic-AMP. 5. Measurements of 14CO2 release from specifically-labelled glucose showed that 3′:5′-cyclic-AMP greatly stimulated glycolytic activity while having a minor depressing effect on the metabolic flow through the pentose phosphate cycle. 6. The concentrations of several metabolic intermediates, particularly fructose 1,6-diphosphate, were greatly affected by the presence of 3′:5′-cyclic-AMP. 7. Several metabolites partially relieved glucose repression of β-galactosidase synthesis in EDTA-treated cells; three out of five of these metabolites reversed the effect more effectively than did 3′:5′-cyclic-AMP. 8. The evidence for and against a direct role for 3′:5′-cyclic-AMP is discussed. It is concluded that the evidence for indirect action is at least as strong as that for direct action.

Merodiploid derivatives bearing an F-linked lac operon ( i + , o + , z + , y + , a + ) from Escherichia coli were prepared from a Proteus mirabilis strain unable to utilize lactose and from a lac deletion strain of E. coli . A suitable growth medium was found in which the episomal element in the P. mirabilis derivative was sufficiently stable to allow induction of the episome-borne lac operon and thus to permit a comparison of the activities and properties of E. coli lac products in the intracellular environments of P. mirabilis and E. coli . In both derivatives the episomal lac operon was shown to be repressed in the absence of inducer. Kinetics of induction with gratuitous inducer (isopropyl-1-thio-β- d -galactoside) were similar for both β-galactosidase activity (β- d -galactoside galactohydrolase, EC 3.4.1.23) and β-galactoside transport activity in both derivatives, although the ratio of galactoside transport to β-galactosidase activity was approximately 1.6-fold higher in the E. coli derivative. Comparison of β-galactosidase and M-protein ( lac y gene product)-specific activities indicated coordinate expression of the induced lac operon in both derivatives. Quantitatively, the maximal β-galactosidase specific activity was two or three times higher for the E. coli derivative. A significant sodium azide inhibition (65% inhibition by 10 mM sodium azide) of lactose permease-mediated transport of o -nitrophenyl-β-galactoside from an outside region of high concentration to an inside region of very low concentration (“downhill transport”) was observed for the P. mirabilis derivative. Identical conditions for the E. coli derivative yielded only about 15% inhibition. Active transport of thiomethyl-β-galactoside was similar for both derivatives, the major difference being that active transport was more sensitive to azide poisoning in the P. mirabilis derivative. Preliminary examination of the thiomethyl-β-galactoside derivatives following active transport did not demonstrate the accumulation of a phosphorylated product in either strain but did reveal an unidentified derivative present in the P. mirabilis merodiploid extract which was not detectable in the E. coli merodiploid.

Radioactive tracer techniques, coupled with chemical analysis by paper chromatography, became important in biochemical research in the late 1940’s, and today many investigations into intermediary and other aspects of metabolism rely on these methods. Soon after the marriage of chromatography to the use of tracers, various instruments were constructed which were able simultaneously to locate the positions of radioactive compounds on paper chromatograms and to determine the amount of radioactivity in each separated compound. Such devices are now available commercially in many different designs, and are intended for the scanning and counting of one-dimensional chromatograms only.

SUMMARY: The incorporation pattern of 14C from [14C]glucose by the fungus Zygorhynchus moelleri has been investigated. The following substances became labelled in incubation periods of 6 sec. to 30 min.: the monophosphates of glucose, fructose, sedoheptulose, ribose, maltose, glycerol, gluconic acid, glyceric acid, enolpyruvic acid and guanosine; the diphosphates of fructose, glucose, glyceric acid, adenosine, uridine, inosine and guanosine; the triphosphates of adenosine and uridine; uridinediphosphoglucose and uridinediphosphoribose; free maltose and fructose; aspartic acid, glutamic acid, alanine, valine, tyrosine, proline, histidine, threonine, citrulline and glutamine; malic acid, citric acid, succinic acid, fumaric acid and glyceric acid. A small percentage of the 14C was present in unidentified substances. The kinetics of the incorporation of 14C, first into uridinediphosphoglucose, then into maltose phosphate, and finally into free maltose, suggests this sequence of compounds for the biosynthetic pathway of maltose formation. The main effects of starvation of the cells by shaking them in phosphate buffer for 24 hr. were an increased % incorporation of 14C into maltose and the sugar phosphates, and a decreased % incorporation into amino acids, organic acids and nucleotides. The addition of a small amount of ammonia to starved cells increased the absolute rate of incorporation of 14C from labelled glucose into soluble cellular constituents; however, while the % of the total soluble 14C found in the nucleotides was markedly increased when ammonia was added, the % in the sugar phosphates decreased. In the presence of azide, the incorporation of 14C by starved cells into maltose and the nucleotides was severely inhibited and the incorporation into amino acids was somewhat increased. Thus, one of the most important results of starvation was to prevent incorporation of 14C into the nucleotides, organic acids and amino acids, while the addition of ammonia overcame the effects of starvation and promoted nucleotide synthesis. Ammonia had very little effect on the cells in the presence of azide. A kinetic study of the sequence in which 14C was detected in these compounds indicated that glucose was metabolized by both the glycolysis and the oxidative pathways, and later by the tricarboxylic acid cycle.

SUMMARY: Compartmentation phenomena were studied in the course of the simultaneous metabolism of glucose, galactose and lactose by organisms of Escherichia coli which were induced for either the lac operon, the gal operon, both, or neither. Metabolic patterns were investigated in each phenotype by incubating parallel identical cultures with the three sugars in equal chemical concentration but labelled differently with 14C. The four labelled substrates were glucose, galactose and lactose labelled either exclusively in the glucose moiety or exclusively in the galactose moiety. The metabolites from free glucose in the medium equilibrated with those from free galactose in the medium, but did not equilibrate with metabolic products derived from glucose generated endogenously by the hydrolysis of lactose. Similarly, metabolic products derived from galactose formed in the hydrolysis of lactose equilibrated with those from glucose from the same source, but not with metabolic intermediates formed from either free glucose or free galactose in the medium. Other interpretations of these results, not involving metabolic compartmentation, have been considered and found inadequate to account for the observed results. Some of the implications of compartmentation in bacteria are discussed.

DNA extracted with certain commercial brands of phenol is resistant to hydrolysis by the endonuclease DNAase I, while DNA extracted with other brands, or prepared by sodium chloride extraction, is susceptible to hydrolysis. The agent responsible for inhibition has been shown to be an oxidation product produced in some phenols. The inhibitor has been separated from other impurities in phenol by paper chromatography, and, by means of infrared and ultraviolet spectroscopy, it has been identified as o-hydroxybiphenyl. The kinetics of inhibition have been studied, and it was found that inhibition arises from direct action on the DNA rather than on the enzyme. Several hydroxybiphenyls and related compounds have been tested for inhibition, and a theory of molecular structure versus inhibitory effectiveness is suggested from this data. From studies on the chemical reversal of inhibition, as well as from ultraviolet spectral studies (in both absorption and circular dichroism), it appears that the mode of action of the inhibitors is hydrogen bonding to, and intercalation between, the basis of the nucleic acid.

Two new compounds, tentatively identified, have been isolated from chromatograms of ethanol-water extracts of cells of Chlorella pyrenoidosa which had been allowed to carry on photosynthesis for 3 minutes in the presence cf o compounds were 1,5 diphosphate esters of 2-carboxy-4ketopentitol and of the lactone of 2-curboxy-3-ketopentitol. The latter has been postulated as an intermediate in the photosynthetic carboxylation of ribulose-1,5-diphosphate to yield two molecules of 3-phosphoglyceric acid. While the former might be an artifact resulting from the techniques used to kill, extract, and analyze the cells, possible biological roles for it have been suggested. (auth)