THE ROLE OF THE SPLEEN IN THE REPOPULATION OF THE HAEMOPOIETIC SYSTEM OF HEAVILY-IRRADIATED MICE

The respective role of the spleen or of the bone marrow in the regeneration of the haemopoietic progenitor compartment of heavily-irradiated mice has been investigated. Splenectomy was used to this end in animals injected with exogenous isogenic cells or regenerating from endogenous spleen or marrow cells. Analysis of the data as a function of time shows that the presence of the spleen affects marrow CFU repopulation only at the early post-irradiation stages. The expansion of the marrow progenitor pool proceeds, however, rather independently of the spleen and marrow CFU remain eventually as the main source of haemopoietic cells in the surviving mice. Thus the reaction of the spleen may be envisaged as a fast, important but transient contribution to the overall haemopoietic function of heavily-irradiated animals.     

STUDIES IN THE PHYSIOLOGY OF FUSARIA. THE RESPIRATORY AND FERMENTATIVE MECHANISMS

1.Fusarium tricothecoides was selected for a study of the respiratory and fermentative activities of Fusaria. "Resting cell" suspensions were investigated by the Barcroft manometric technique. 2. The results of the investigation indicate clearly that the mechanism of endogenous metabolism (respiration) is distinct from the exogenous mechanism (fermentation). Anaerobically no significant CO₂ production is apparent without added substrate. In the presence of glucose the anaerobic CO₂ evolution is practically equal to the added CO₂ evolved aerobically in the presence of added glucose. Low concentrations of iodoacetate or fluoride selectively poison the exogenous mechanism but do not affect the endogenous mechanism. Alcohol is not produced in the course of endogenous metabolism, but is produced in the presence of added glucose. 3. A study of the metabolism of the organism throughout its entire growth phase from 1 to 7 days has been made. 4. The ability of suspensions of Fusarium sp. H., obtained by growth on a variety of common substrates, to attack a large number of carbon sources with the production of exogenous CO₂ was determined. It is found that organisms grown on glucose will attack only glucose, mannose, and fructose, but none of the common intermediary metabolites except pyruvic acid. Organisms grown on galactose attack galactose, as well as the other hexoses, indicating an adaptive mechanism. 5. An identical mechanism for the dissimilation of glucose, mannose, and galactose is indicated since no additive effects with these substrates were observed. Growths on non-hexose carbon sources attack glucose slightly under the experimental conditions with the evolution of CO₂, but do not attack any other substrate. This would indicate a residual glucose-dissimilating mechanism in all growths investigated. 6. Striking similarities between the general metabolism of resting suspensions of Fusarium sp. H. and resting suspensions of yeast cells are apparent.     

THE PREADAPTIVE OXIDATION OF GALACTOSE BY YEAST

A preadaptive purely aerobic utilization of galactose by yeast cells has been demonstrated. Hence, the adaptation by yeast to galactose is not to its utilization per se, but specifically to its metabolism by a glycolytic mechanism. An examination of this preadaptive oxidation of galactose reveals that it has many characteristics in common with the endogenous metabolism of yeast. Included among these are the similarities of the R.Q. values and the response of the Q _OO2 and Q_COCO2 ^O₂ to KCN and iodoacetic acid. Further, a competitive interaction appears to exist between the endogenous respiration and the preadaptive oxidation of the galactose. The latter can replace the endogenous respiration as a source of energy for the adaptation to the fermentation of the galactose. Carbon balance studies of the galactose oxidation revealed that polysaccharide could be formed as a result of this metabolism during the preadaptive period. Non-adaptable cells were also found to possess the capacity to oxidize galactose in the complete absence of any ability to metabolize it anaerobically. The significance of these findings for the biochemistry and physiology of the adaptation is discussed.     

Role of fatty acid in the control of protein synthesis in liver cells

Concentrations of oleate (0.2-1 mM) within the physiological range of plasma free fatty acids induced a dose dependent statistically significant inhibition of protein labelling in isolated liver cells. The inhibitory effect was as high as 50% and it was not impeded when long chain fatty acid oxidation was prevented. Experiments carried out with hepatocytes from 48 h fasted rats, incubated in the absence of any exogenous energy source, show that the inhibition of endogenous long chain fatty acid oxidation induced a decreased rate of protein synthesis apparently related to changes in the cellular energy state. It is concluded that fatty acids play a dual role in the regulation of protein synthesis in liver cells: 1. endogenous fatty acids appear to be the main energy fuel for protein synthesis when no other exogenous substrate is present and the carbohydrate stores are low; 2. exogenous fatty acids seem to control protein synthesis by interacting with some key regulatory step.     

Polyphosphate synthesis in yeast

Polyphosphate synthesis was studied in phosphate-starved cells of Saccharomyces cerevisiae and Kluyveromyces marxianus. Incubation of these yeasts for a short time with phosphate and either glucose or ethanol resulted in the formation of polyphosphate with a short chain length. With increasing incubation times, polyphosphates with longer chain lengths were formed. Polyphosphates were synthesized faster during incubation with glucose than with ethanol. Antimycin did not affect the glucose-induced polyphosphate synthesis in either yeast. Using ethanol as an energy source, antimycin A treatment blocked both polyphosphate synthesis and accumulation of orthophosphate in the yeast S. cerevisiae. However, in K. marxianus, polyphosphate synthesis and orthophosphate accumulation proceeded normally in antimycin-treated cells, suggesting that endogenous reserves were used as energy source. This was confirmed in experiments, conducted in the absence of an exogenous energy source.     

METABOLIC INTERMEDIATES IN ADAPTIVE FERMENTATION OF GALACTOSE BY YEAST

Pyruvic acid, which is known to be an intermediate of glucose fermentation, was added to yeast during adaptation to galactose fermentation. It was found to neutralize the inhibition by sodium fluoride, and to decrease the apparent time of adaptation from 90 to about 45 or 60 minutes. In control experiments, it was shown that intact yeast is unable appreciably to ferment or decarboxylate alone, although it oxidizes the compound readily. Experiments in which galactose and pyruvate were added at various times and in different orders were used to eliminate the possible complications of the rates at which these compounds penetrate the cells. Under these conditions, it was not possible to reduce the time of adaptation below 45 minutes. It was concluded that the rôle of added pyruvate was to serve as a source of acetaldehyde, which in turn could accept hydrogen and be reduced to alcohol. Substances, such as triose phosphate, which could serve as hydrogen donors were not produced from galactose in appreciable quantities until 45 minutes had elapsed. This time was therefore inferred to be the true adaptation time, during which the first synthesis of adaptive enzymes occurred. Some determinations of the distribution of phosphorylated intermediates at various stages during the adaptive process were carried out. It was found that ATP, which usually serves to phosphorylate hexoses, accumulates during the preadaptive phase, diminishes rapidly after 60 minutes, and subsequently increases once more. The source of the ATP phosphate appeared to be PPA or triose phosphate initially present in the cells. It was inferred that the adaptive enzyme was concerned with the phosphorylation of galactose and the conversion of the phosphate ester to a glucose ester, which could then be fermented by the normal enzymes of the cell. Added ATP was found to stimulate adaptation to a considerable extent, but did not shorten the time of adaptation below 75 minutes. This seemed consistent with the rôle of ATP as a phosphate donor for galactose. Creatine was found to inhibit adaptation to some degree, in agreement with its known ability to act as a competitive phosphate acceptor. It was demonstrated that yeast produces, during and after adaptation, substances which shorten the apparent adaptation time of fresh samples of yeast. In agreement with our other findings, it appeared that such substances were not formed before about 45 minutes. They are probably not metabolic intermediates, and may be identical with the adaptive principle which can be extracted from adapted cells.     

Influence of exogenous substrates on the endogenous respiration of Pseudomonas aeruginosa

Gronlund, Audrey F. (University of British Columbia, Vancouver, Canada), and J. J. R. Campbell. Influence of exogenous substrates on the endogenous respiration of Pseudomonas aeruginosa. J. Bacteriol. 91:1577-1581. 1966.-The influence of growth conditions, ammonium ions, and glucose concentration on endogenous respiration in Pseudomonas aeruginosa was determined by measuring C(14)O(2) evolution from uniformly labeled cells that had previously been grown on C(14)-glucose. A 93% suppression of endogenous C(14)O(2) evolution was evident under growth conditions, and a 66% suppression was observed in the presence of excess glucose. Increasing exogenous glucose concentrations supported decreasing levels of endogenous C(14)O(2) evolution. Ammonium ions slightly suppressed endogenous activity and enhanced the decrease in C(14)O(2) release observed with exogenous glucose. In addition, the effect of exogenous glucose, alpha-ketoglutarate, 2-ketogluconate, aspartic acid, and adenosine selectively on both endogenous ribonucleic acid (RNA) and protein oxidation was followed by measuring C(14)O(2) evolution from cells grown with C(14)-uracil or C(14)-proline. The five exogenous substrates examined suppressed endogenous RNA oxidation, and the degree of suppression appeared to be correlated with the amount of oxygen consumption and, hence, energy gained during the oxidation of these substrates. Oxidation of endogenous protein was decreased when cells were incubated with glucose, aspartate, and adenosine, but was increased when alpha-ketoglutarate and 2-ketogluconate were the exogenous substrates. The influence of the oxidizable exogenous compounds appeared to be related, in part, to the ammonium ion requirement imposed upon the cells for assimilation of the individual exogenous substrate.     

Endogenous respiration and regulation in a primitive marine fungus

The relationship between the respiration and the presence and utilization of endogenous and exogenous substrates was studied in the non-filamentous obligately marine fungus Thraustochytrium aureum. Using isotopic and manometric methods, it was shown that almost all exogenous glucose is assimilated, whilst almost all the oxygen consumption in the presence of exogenous glucose was due to oxidation of endogenous reserves. In contrast, exogenous glutamate, which cannot serve as the sole carbon source for growth, inhibits respiration of endogenous materials, and is itself rapidly oxidized. The uncoupler 2,4-dinitrophenol stimulates the oxidation of endogenous reserves without affecting the uptake and use of exogenous glucose. These data strongly support the idea of physiologic compartmentation in this organism.     

Glucuronide transport across the endoplasmic reticulum membrane is inhibited by epigallocatechin gallate and other green tea polyphenols

Toxic endogenous or exogenous compounds can be inactivated by various conjugation reactions. Glucuronidation (i.e. conjugation with glucuronate) is especially important due to the large number of drugs and chemical carcinogens that are detoxified through this pathway. Stable and harmless glucuronides can be reactivated by enzymatic hydrolysis thus inhibitors of glucuronidase activity reduce the risk of chemical carcinogenesis. The aim of this study was to reveal whether this mechanism contributes to the anti-cancer effect of green tea flavanols, which has been shown in various animal models. Therefore, we investigated the effect of these polyphenols on deglucuronidation in rat liver microsomes and in Hepa 1c1c7 mouse hepatoma cells, using 4-methylumbelliferyl glucuronide as model substrate. Tea flavanols inhibited beta-glucuronidase in intact vesicles, where glucuronide transport across the microsomal membrane is rate-limiting, but were almost ineffective in permeabilized vesicles. Epigallocatechin gallate, the major green tea flavanol was shown to have a concentration-dependent inhibitory effect on both beta-glucuronidase activity and glucuronide transport in native vesicles. Epigallocatechin gallate also inhibited beta-glucuronidase activity in native Hepa 1c1c7 mouse hepatoma cells, while failed to affect the enzyme in alamethicin-permeabilized cells, where the endoplasmic membrane barrier was eliminated. Our findings indicate that tea flavanols inhibit deglucuronidation in the endoplasmic reticulum at the glucuronide transport stage. This phenomenon might potentially contribute to the cancer-preventing dietary or pharmacological effect attributed to these catechins.     

Do purely capital layers exist among flying birds? Evidence of exogenous contribution to arctic-nesting common eider eggs

The strategy of relying extensively on stored resources for reproduction has been termed capital breeding and is in contrast to income breeding, where needs of reproduction are satisfied by exogenous (dietary) resources. Most species likely fall somewhere between these two extremes, and the position of an organism along this gradient can influence several key life-history traits. Common eiders (Somateria mollissima) are the only flying birds that are still typically considered pure capital breeders, suggesting that they depend exclusively on endogenous reserves to form their eggs and incubate. We investigated the annual and seasonal variation in contributions of endogenous and exogenous resources to egg formation in eiders breeding at the East Bay colony in the Canadian Arctic. We collected prey items along with females and their eggs during various stages of breeding and used two complementary analytical approaches: body reserve dynamics and stable isotope [δ(13)C, δ(15)N] mixing models. Indices of protein reserves remained stable from pre-laying to post-laying stages, while lipid reserves declined significantly during laying. Similarly, stable isotope analyses indicated that (1) exogenous nutrients derived from marine invertebrates strongly contributed to the formation of lipid-free egg constituents, and (2) yolk lipids were constituted mostly from endogenous lipids. We also found evidence of seasonal variation in the use of body reserves, with early breeders using proportionally more exogenous proteins to form each egg than late breeders. Based on these results, we reject the hypothesis that eiders are pure capital layers. In these flying birds, the fitness costs of a strict capital breeding strategy, such as temporary loss of flight capability and limitation of clutch and egg size, may outweigh benefits such as a reduction in egg predation rate.     

Overexpression of enzymes that repair endogenous damage to DNA.

A significant contribution to human mutagenesis and carcinogenesis may come from DNA damage of endogenous, rather than exogenous, origin. Efficient repair mechanisms have evolved to cope with this. The main repair pathway involved in repair of endogenous damage is DNA base excision repair. In addition, an important contribution is given by O6-alkylguanine DNA alkyltranferase, that repairs specifically the miscoding base O6-alkylguanine. In recent years, several attempts have been carried out to enhance the efficiency of repair of endogenous damage by overexpressing in mammalian cells single enzymatic activities. In some cases (e.g. O6-alkylguanine DNA alkyltransferase or yeast AP endonuclease) this approach has been successful in improving cellular protection from endogenous and exogenous mutagens, while overexpression of other enzymatic activities (e.g. alkyl N-purine glycosylase or DNA polymerase beta) were detrimental and even produced a genome instability phenotype. The reasons for these different outcomes are analyzed and alternative enzymatic activities whose overexpression may improve the efficiency of repair of endogenous damage in human cells are proposed.     

Enterobacter sp. VKGH12 growing with n-butanol as the sole carbon source and cells to which the alcohol is added as pure toxin show considerable differences in their adaptive responses

The solvent-tolerant bacterium Enterobacter sp. VKGH12 is able to grow in toxic concentrations of n-butanol up to 1.5 % (volume in volume) as the sole carbon and energy source. Morphology changes in the cells growing on increasing concentrations of n-butanol were observed. The size of the bacteria decreased with increasing concentrations of n-butanol, also leading to an enhanced ratio between the surface and volume of the cells. This is in complete contradiction to the reaction of glucose-grown cells to which n-butanol had been added as a toxin. Similar differences were found in typical adaptive responses to toxic organic compounds, namely changes in fatty acid composition of membrane lipids and the activity of catalase. In both cases, reactions depending on the n-butanol concentrations could be observed when the toxin was added to glucose-grown cells, whereas no reaction was observable when the cells were growing in n-butanol as the sole carbon and energy source. This is another proof for the observation that there are certain differences between the adaptive strategies of cells when adapting to high concentrations of a growth substrate and those when adapting to a toxin added to growing cells.     

Interactions between enzyme-forming systems during adaptation

Experiments on enzymatic adaptations in yeast to galactose and maltose under various conditions are examined. The pertinent facts established may be summarized as follows:- 1. The presence of exogenous nitrogen stimulates the rate of adaptation and raises considerably the attainable level of enzyme activity. 2. This stimulation is absent if the cells are unable to assimilate the added nitrogen. 3. Competitive interactions can be exhibited between two adaptive enzyme systems induced either serially or simultaneously in the same cell. 4. A similar kind of interaction was observed between an adaptive and a so called "constitutive" enzyme. 5. The presence of exogenous nitrogen modifies greatly the nature and extent of the interaction between the enzyme-forming systems. The significance of these results to our understanding of the mechanism of the modification and maintenance of cellular enzymatic constitution is discussed. The validity of the distinction between "constitutive" and "adaptive" enzymes is reexamined in the light of the data presented.     

微小泰泽球虫内生阶段虫体内多糖的细胞化学的研究

采用纯种微小泰泽球虫(Tyzzeria parvula)卵囊,人工感染四日龄雏鹅,定时剖杀,取肠道组织进行石蜡切片,细胞化学染色,观察微小泰泽球虫内生阶段虫体内多糖的分布。结果表明:滋养体、多核体、小配子体以及早期的大配子体内均不含多糖。裂殖子PAS反应阳性,但也有少数第二代裂殖子阴性。随着大配子体的发育,多糖逐渐在其体内合成,合子时期达到高峰。提示:微小泰泽球虫体内的多糖是一个合成—积累—消耗的过程。     

Role of L-lactate as an energy substrate in primary rat podocytes under physiological and glucose deprivation conditions

Lactate has long been acknowledged to be a metabolic waste product, but it has more recently been found as a fuel energy source in mammalian cells. Podocytes are an important component of the glomerular filter, and their role in maintaining the structural integrity of this structure was established. These cells rely on a constant energy supply and reservoir. The utilization of alternative energy substrates to preserve energetic homeostasis is a subject of extensive research, and lactate appears to be one such candidate. Therefore, we investigated the role of lactate as an energy substrate and characterize the lactate transport system in cultured rat podocytes during sufficient and insufficient glucose supplies. The present study, for the first time, demonstrated the presence of lactate transporters in podocytes. Moreover, we observed modified the amount of these transporters in response to limited glucose availability and after l-lactate supplementation. Simultaneously, exposure to l-lactate preserved cell survival during insufficient glucose supply. Interestingly, during glucose deprivation, lactate exposure allowed the steady flow of glycolysis and prevented glycogen reserves depletion. Summarizing, podocytes utilize lactate as an energy substrate and possess a developed system that controls lactate homeostasis, suggesting that it plays an essential role in podocyte metabolism, especially during fluctuations of energy availability.     

On the nature of sporogenesis in some aerobic bacteria

Washed vegetative cells of various species of aerobic spore-forming bacteria sporulate abundantly when shaken in distilled water in air. The spores thus formed possess the same heat resistance as spores formed in a complete growth medium. Various factors influencing sporogenesis in water are described. Glucose in low concentration completely suppresses sporogenesis under these conditions and the suppression is relieved by the presence of ammonia as an exogenous source of nitrogen. Various amino acid and purine antimetabolite analogues inhibit sporogenesis and their inhibitory effects are completely reversed by much smaller amounts of the corresponding metabolites. Sporogenesis is thus regarded as a de novo synthesis of spore proteins from preexisting endogenous (enzyme) proteins. Cells low in protein fail to sporulate and the capacity of the cell to adaptively attack maltose and trehalose is strongly interfered with after the cell is irreversibly committed to sporulation, but not before that. Evidence is advanced supporting the hypothesis that sporogenesis is an endogenous process which commences when the supply of exogenous energy and carbon is depleted. It utilizes low molecular weight nitrogenous substances liberated by the degradation of preexisting enzyme proteins of the vegetative cell. Sporogenesis and adaptive enzyme formation are regarded as competitive synthetic processes, both utilizing endogenous enzyme proteins. The events of sporogenesis suggest that this process may be an adaptive protein synthesis, analogous to adaptive enzyme synthesis.     

EFFECT OF THE ANTIBIOTIC,CYCLOHEXIMIDE, ON THE METABOLISM AND GROWTH OF SACCHAROMYCES PASTORIANUS

Coursen, B. W., and H. D. Sisler (U. Maryland, College Park.) Effect of the antibiotic, cycloheximide, on the metabolism and growth of Saccharomyces pastorianus. Amer. Jour. Bot. 47(7): 541–549. Illus. 1960.—Studies were made of the toxicity of cycloheximide and certain of its derivatives to Saccharomyces pastorianus Hansen. The ED₅₀ values for cycloheximide, its semicarbazone derivative and its oxime derivative are 0.018, 0.37, and 12.0.p.p.m., respectively. In auxanographic and liquid culture tests involving 160 organic and biochemicals, only certain methylated ring ketones and vitamin A alcohol or acetate showed appreciable antagonistic activity to the toxicity of cycloheximide. Yeast cells exposed to 3.16 p.p.m. of cycloheximide and incubated for 30 min. with uniformly labeled ¹⁴C glucose remove about 10% less activity from the medium than untreated cells. Measurements of radioactivity in compounds extracted from cells with 80% ethanol showed the presence of appreciable activity in the glutamine from untreated cells but no measurable activity in this compound from treated cells. Activity in glutamic acid from treated cells was reduced while activity in alanine and aspartic acid was increased when compared with the activity in these compounds from untreated cells. There were other differences, also, especially in the levels of activity in organic phosphorus compounds, but, in many cases, the activity in compounds from treated cells was similar to that in the corresponding compounds from untreated cells. It is possible that the antibiotic interferes with the metals involved in the enzymatic reaction leading to the synthesis of glutamic acid and glutamine or it may act as an inhibitory analog in the synthesis of these or similar compounds. The apparent interference of cycloheximide with the formation of a CO-NH bond in the synthesis of glutamine suggests also that peptide bond formation in protein synthesis may be similarly affected. A block of glutamine synthesis by cycloheximide may be sufficient to account for the toxicily of the antibiotic, but the failure of exogenous sources of glutamine to reverse the toxicity indicates that other reactions in cell metabolism may be as sensitive to cycloheximide as the synthesis of glutamine.     

beta-oxidation - strategies for the metabolism of a wide variety of acyl-CoA esters

Living organisms are exposed to a number of different fatty acids and their various derivatives arising either via endogenous synthesis or from exogenous sources. These hydrophobic compounds can play specific metabolic, structural or endocrinic functions in the organisms before their elimination, which can be metabolism to CO(2) or to more polar lipid metabolites allowing their excretion. Quantitatively, one of the major pathways metabolizing fatty acids is beta-oxidation, which consists of a set of four reactions operating at the carbons 2 or 3 of acyl-CoA esters and shortening of the acyl-chain. To allow the beta-oxidation of acyl groups with various steric variants to proceed, different strategies have been developed. These strategies include evolution of beta-oxidation enzymes as paralogues showing specificity with respect to either chain-length or modified acyl-chain, metabolic compartmentalization in eukaryotic cells, controlling of substrate transport across membranes, development of auxiliary enzyme systems, acquisition of enzymes with adaptive active sites and recruiting and optimizing enzymes from non-homologous sources allowing them to catalyze a parallel set of reactions with different substrate specificities.     

THE DETERMINATION OF PICOMOLE AMOUNTS OF ACETYLCHOLINE IN MAMMALIAN BRAIN

Abstract— In any assay for the determination of acetylcholine based on the conversion of choline to a product, the immediate problem is the removal of endogenous choline. Other published enzymatic assays have taken advantage of electrophoresis to accomplish this goal. In the assay to be described, this is accomplished by the enzymatic phosphorylation of endogenous choline by choline kinase. Once this reaction is complete, endogenous acetylcholine is simultaneously hydrolysed and then phosphorylated with [³²P]ATP. The labelled product [³²P]phosphorylcholine is separated from the labelled substrate by precipitation of the ATP and further separation is accomplished on microcolumns of ion exchange resin. Using this methodology, picomole amounts of acetylcholine, derived from tissue, can be measured.     

Energy requirements and process design considerations in compression-milling pretreatment of cellulosic wastes for enzymatic hydrolysis

The effectiveness of compression-milling pretreatment of lignocellulosics for enzymatic hydrolysis has been demonstrated for a wide variety of substrate sources. Reductions in the degree of crystallinity and the degree of polymerization of cellulose and partial destruction of the structural integrity of lignocellulosics brought about by compression milling significantly increase the susceptibility of cellulose to enzymatic hydrolysis. The enzymatic hydrolysis yield was found to be directly related to the specific energy input to the cellulosic substrate (kWh/1b substrate) by compression milling, and the energy input can be controlled by the milling time. The enzymatic hydrolysis yeilds from cellulosic materials pretreated by compression milling also vary significantly depending on the source and kind, the composition milling also vary significantly depending on the source and kind, the composition (contents of lignin and other components), and the structure. The power requirements for compression milling which renders equivalent hydrolysis yields also depend on the source and kind of lignocellulosics to be pretreated. For newspaper, the specific energy input required for 55% sugar yield is estimated as 0.3 kWh/lb substrate including 15% power loss. The additional sugar yield gained from the enzymatic hydrolysis of compression-milled newspaper (over and above the sugar yield of untreated substrate) is determined as 453 g sugar/kWh energy input.