The regulation of rat liver tryptophan pyrrolase activity by reduced nicotinamide-adenine dinucleotide (phosphate). Experiments with glucose and nicotinamide.

1. Chronic administration of glucose or nicotinamide in drinking water inhibits the activity of rat liver tryptophan pyrrolase, and subsequent withdrawal causes an enhancement. The enzyme activity is also inhibited by administration in drinking water of sucrose, but not fructose, which is capable of preventing the glucose effect. 2. The inhibition by glucose or nictinamide is not due to a defective apoenzyme synthesis nor a decreased cofactor availability. 3. The inhibition by nicotinamide is reversed by regeneration of liver NAD+ and NADP+ in vivo by administration of fructose, pyruvate or phenazine methosulphate. Inhibition by glucose is also reversed by the above agents and by NH4Cl. Reversal of inhibition by glucose or nicotinamide is also achieved in vitro by addition of NAD+ or NADP+. 4. Glucose or nicotinamide increases liver [NADPH]. [NADP+] is also increased by nicotinamide. [NADPH] is also increased by sucrose, but not by fructose, which prevents the glucose effect. Phenazine methosulphate prevents the increase in [NADPH] caused by both glucose and nicotinamide. 5. It is suggested that the inhibition of tryptophan pyrrolase activity by glucose or nicotinamide is mediated by both NADPH and NADH.     

Volume-sensitive K influx in human red cell ghosts

K influx into resealed human red cell ghosts increases when the ghosts are swollen. The influx demonstrates properties similar to volume-sensitive K fluxes present in other cells. The influx is, for the most part, insensitive to the nature of the major intracellular cation and therefore is not a K-K exchange. The influx is much greater when the major anion is Cl than when the major anion is NO3; Cl stimulates the flux and, at constant Cl, NO3 inhibits it. Increase in the influx rate is rapid when shrunken ghosts are swollen or when NO3 is replaced by Cl. The volume-sensitive K influx requires intracellular MgATP at low concentrations, and ATP cannot be replaced by nonhydrolyzable ATP analogues. The volume-sensitive influx is inhibited by Mg2+ and by high concentrations of vanadate, but is stimulated by low concentrations of vanadate. It is not modified by cAMP, the removal of Ca2+ by EGTA, substances that activate protein kinase C, or by inhibition of phosphatidylinositol kinase. The influx is inhibited by neomycin and by trifluoperazine.     

Overlapping and separate controls on the phosphate regulon in Escherichia coli K12

The physiological and genetic controls operating on phosphate-regulated promoters were studied in greater detail. This was done by defining the control for three phosphate-regulated genes: phoA, psiE, and psiO. Each is highly inducible by phosphate starvation. Individually, these phosphate-starvation-inducible, psi, genes at the same time show common and differing features in their molecular control. The phoA gene, encoding alkaline phosphatase, is specifically induced by phosphate starvation. It is negatively controlled by phoR as well as by the phosphate-specific transport (PST) system in Escherichia coli. phoA induction is positively controlled by the phoB, M, and R products; it is unaffected by the cAMP and CAP system. The psiE and psiO genes were studied by using strains with lacZ fused to their respective promoters. psiE-lacZ is induced by phosphate-, carbon- or nitrogen-limited growth. Genetically, psiE-lacZ induction is partially phoB and phoR-dependent. However, its expression is phoM-independent. This implies that phoB/phoR coupled control differs from phoB/phoM coupled control. Repression of psiE-lacZ is substantially altered in only some PST mutants, such as phoT. In addition, psiE-lacZ is negatively controlled by the cAMP and CAP system. psiO-lacZ is induced by phosphate-, carbon- or nitrogen-limited growth or by anaerobiosis. Its expression is unaffected by any pho mutation that has been previously described. A cell density-dependent induction of psiO-lacZ is observed in lon mutants. Also, psiO-lacZ is negatively controlled by the cAMP-CAP system. In summary, these results demonstrate that co-ordinately regulated promoters can have some common regulatory elements while, at the same time, not sharing other controlling factors.     

Ultrastructure of the anterior alimentary tract of a monogenean, Diclidophora merlangi

The anterior alimentary tract of Diclidophora merlangi is composed of a complex series of morphologically distinct epithelia interconnected by septate desmosomes and penetrated by the openings of numerous unicellular glands. The mouth and buccal cavity are lined by an infolding of modified body tegument, distinguished by uniciliate sense receptors, buccal gland openings, and in the buccal region by a dense, spiny appearance. The prepharynx is covered by an irregularly folded epithelium and, for part of its length, by the luminal cytoplasm of the prepharyngeal gland cells. The epithelium is syncytial and pleiomorphic, and regional variation in structure is common. A separate epithelium invests the lips of the pharynx and its free surface is greatly amplified by numerous, dense lamellae of varying dimensions. The lip epithelium is continuous with cytoplasmic processes of cells located external to the pharynx. A further, distinct epithelium borders the pharynx lumen and is composed of discrete cytoplasmic units connected by short septate desmosomes. The oesophagus is lined by a modified caecal epithelium, lacking haematin cells, and, in places, is perforated by the openings of oesophageal gland cells; it is continuous with the syncytial connecting tissue of the gut caeca.     

Stimulation of low Km cyclic AMP phosphodiesterase by sulphydryl modification

Low concentrations of the organic mercurials, p-chloromercuribenzoate or p-chloromercuriphenylsulphonate activate the particulate low Km phosphodiesterase from adipose tissue and liver. Higher concentrations are inhibitory. Enzyme which has been activated by treatment of adipocytes with insulin, is not activated by the organic mercurials although inhibition by higher concentrations is seen. Enzyme from non-insulin treated adipocytes is activated and solubilised by mild trypsin treatment. Enzyme activated by either insulin treatment, or by p-chloromercuribenzoate is not further activated by trypsin, but it is solubilised.     

Effects of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate on phosphofructokinase from chicken erythrocytes

1. Phosphofructokinase (EC 2.7.1.11) from chicken erythrocytes is activated by fructose 2,6-bisphosphate, glucose 1,6-bisphosphate and AMP, and it is inhibited by 2,3-bisphosphoglycerate and inositol hexaphosphate. 2. The stimulatory effects produced by the two bisphosphorylated hexoses are additive and the effects produced by fructose 2,6-bisphosphate and by AMP are synergistic. 3. The activatory effect produced by fructose 2,6-bisphosphate is counteracted by fructose 1,6-bisphosphate. 4. The inhibition produced by both 2,3-bisphosphoglycerate and inositol hexaphosphate is released by fructose 2,6-bisphosphate. 5. It is concluded that, like phosphofructokinase from mammalian tissues, the enzyme from chicken erythrocytes can be modulated by the relative concentrations of those metabolites.     

Purification and polyamine activation of a high-affinity 3',5'-cAMP phosphodiesterase from rabbit muscle

A high-affinity phosphodiesterase, termed PDE II, has been purified about 1400-fold from rabbit skeletal muscle. This enzyme is activated by treatment with proteases. It is also activated specifically by polyarginine and arginine-rich histones, but not by other polyanions. The activation is counteracted nonspecifically by polycations, such as heparin and chondroitin sulphate. When the enzyme is fully activated by polyarginine it is no longer susceptible to activation by proteases. A conformational or structural change must thus occur in the enzyme by the binding of the polyanions.     

Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii

Azotobacter beijerinckii possesses the enzymes of both the Entner-Doudoroff and the oxidative pentose phosphate cycle pathways of glucose catabolism and both pathways are subject to feedback inhibition by products of glucose oxidation. The allosteric glucose 6-phosphate dehydrogenase utilizes both NADP(+) and NAD(+) as electron acceptors and is inhibited by ATP, ADP, NADH and NADPH. 6-Phosphogluconate dehydrogenase (NADP-specific) is unaffected by adenosine nucleotides but is strongly inhibited by NADH and NADPH. The formation of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate by the action of the Entner-Doudoroff enzymes is inhibited by ATP, citrate, isocitrate and cis-aconitate. Glyceraldehyde 3-phosphate dehydrogenase is unaffected by adenosine and nicotinamide nucleotides but the enzyme is non-specific with respect to NADP and NAD. Citrate synthase is strongly inhibited by NADH and the inhibition is reversed by the addition of AMP. Isocitrate dehydrogenase, a highly active NADP-specific enzyme, is inhibited by NADPH, NADH, ATP and by high concentrations of NADP(+). These findings are discussed in relation to the massive synthesis of poly-beta-hydroxybutyrate that occurs under certain nutritional conditions. We propose that synthesis of this reserve material, to the extent of 70% of the dry weight of the organism, serves as an electron and carbon ;sink' when conditions prevail that would otherwise inhibit nitrogen fixation and growth.     

Oxidation of glycine by Phaseolus leghaemoglobin with associated catabolic reactions at the haem.

Leghaemoglobin from the root nodules of kidney bean (Phaseolus vulgaris) reacts in alkaline glycine solutions as a glycine oxidase in a reaction that may also be regarded as a coupled oxidation. Leghaemoglobin is reduced to the ferrous form by glycinate, the oxygen complex is formed, and finally the haem is attacked to yield a green reaction product. Glycine is simultaneously oxidized to glyoxylate, and hydrogen peroxide is generated. The initial velocity of the formation of the green product is proportional to the concentrations of leghaemoglobin and glycine, and the optimum pH for the reaction is 10.2. The green product is not formed if carbon monoxide, azide of imidazole is bound to the haem, whereas oxidation of glycine to glyoxylate is not inhibited by azide and not essentially by carbon monoxide. Haem breakdown is activated by digestion of leghaemoglobin by carboxypeptidase, and partly inhibited by catalase and superoxide dismutase.     

Different enzymes involved in NADH- and NADPH-dependent respiration in the cyanobacterium Anabaena variabilis

Abstract Filaments of N₂-grown Anabaena variabilis exhibit soluble NADPH- and membrane-bound NADH-oxidizing activities. The NADPH-specific enzyme has been identified as ferredoxin-NADP oxidoreductase (FNR; EC 1.18.1.2) by the thionicotinamide-NADP transhydrogenase test, a ferredoxin-dependent hydrogenase assay, and by diaphorase systems. The FNR is easily removed by washing of French-press-prepared membranes. Concurrently, a loss of NADPH-dependent respiration is apparent, which is not reconstitutable by addition of Anabaena cytochrome c -553. The NADH-oxidizing activity, however, is only slightly affected by the washing procedure, and is completely reconstituted by cytochrome c -553. NADPH-dependent oxygen uptake is strongly inhibited by NADP, whereas inhibition of NADH-dependent oxygen uptake by NAD is less pronounced. The data give evidence that NADH and NADPH oxidations linked to the respiratory chain are mediated by two different enzymes.     

Antagonistic effects of hexose 1,6-bisphosphates and fructose 2,6-bisphosphate on the activity of 6-phosphofructokinase purified from honey-bee flight muscle.

6-Phosphofructokinase purified from honey-bee flight muscle is inhibited by ATP and, unusually, by glucose 1,6-bisphosphate and fructose 1,6-bisphosphate. The inhibition by either of the bisphosphates is not relieved by AMP, but is relieved by fructose 6-phosphate and especially by fructose 2,6-bisphosphate. Lack of effect by AMP is consistent with a low activity of adenylate kinase in this muscle.     

Correlation between transglutaminase activity and polyamine levels in human neuroblastoma cells. Effect of retinoic acid and alpha-difluoromethylornithine

The human neuroblastoma cell line SK-N-BE can be induced to differentiate by retinoic acid (RA) or by alpha-difluoromethylornithine (DFMO). The former inducer produces neurite outgrowth, 60% reduction of growth rate, overexpression of neural antigens, and enhanced gamma-aminobutyric acid (GABA) and acetylcholinesterase levels. In contrast, DFMO causes cell body elongation, complete growth inhibition, and higher binding of antibodies directed against neuroectodermal antigens. Polyamine metabolism is also differently affected by the two agents. In particular a large spermine catabolism is induced by RA, while DFMO treatment leads to a small increase in the level of this compound. The neural differentiation induced by RA is accompanied by a marked increase in transglutaminase activity and its induction is paralleled by a transient increase of putrescine and spermidine. The putrescine and spermidine depletion determined by DFMO is accompanied instead by a large inhibition of transglutaminase activity. The inhibiting effect of DFMO treatment on transglutaminase is reversed by the addition of 1 mM putrescine to the culture medium. In the presence of both RA and DFMO a mixed morphological and biochemical pattern is observed. The possibility that the expression of transglutaminase associated to cellular differentiation may be modulated by the level of its substrates is also discussed.     

Glutamine transport in brain mitochondria

Gln is transported into rat brain synaptic and non-synaptic mitochondria by a protein catalyzed process. The uptake is significantly higher in synaptic than in non-synaptic mitochondria. The transport is inhibited by the amino acids Glu, Asn and Asp, and by the TCA cycle intermediates succinate, malate and 2-OG. The inhibition by 2-OG is counteracted by AOA and is therefore assumed to be due to transamination of 2-OG, whereby Glu is formed. This presumes that Glu also binds to an inhibitory site on the matrix face of the inner membrane. The transport is complex and cannot be explained by the simple uniport mechanism which has been proposed for renal (Schoolwerth and LaNoue, 1985), and liver mitochondria (Soboll et al., 1991). Thus, Gln transport is stimulated by respiration and by the proton electrochemical gradient. Since it is indicated that both the neutral Gln zwitterion and the Gln anion are transported, there are probably different uptake mechanisms, but not necessarily different carriers. Gln may be transported by an electroneutral mechanism as a proton compensated anion, as well as electrophoretically as a zwitterion with a proton, and probably also by diffusion as a zwitterion. The properties of the brain mitochondrial Gln uptake mechanisms are also not identical with those of a purified renal Gln transporter. It is possible that the Gln transport is controlled by more than one protein, which may be situated on distinct species in a heterogeneous mitochondrial population. Since Gln is assumed to participate in energy production as well as in the synthesis of nucleic acid components and proteins in brain mitochondria, the control of Gln uptake in these organelles may be important.     

Extrinsic allergic alveolitis

Extrinsic allergic alveolitis is caused by the inhalation of small organic allergen particles by non-atopic subjects which provoke an allergic reaction, thought to be chiefly due to a type III mechanism, in the peripheral respiratory tissues. The clinical features are determined by the nature of exposure, the immunopathological mechanism(s) involved and the site of reaction in the lung. When the exposure is intermittent and intensive, febrile episodes with respiratory symptoms beginning after four to six hours are prominent, but when it is more continuous and less intensive they are not and the features are those of a chronic fibrosing lung disease. The diagnosis is important to make because management by the avoidance of exposure is followed by improvement. It is made by recognizing the clinical presentation, by identifying the source of allergen exposure and by obtaining supportive evidence from precipitin and skin tests, or from allergen inhalation tests or lung biopsy.     

Arginine modifiers as energy transfer inhibitors in photophosphorylation.

Photophosphorylation by spinach chloroplasts is inhibited after they have been incubated in the dark with either phenylglyoxal or butanedione. Inhibition by phenylglyoxal is strongest when N-ethylmorpholine is the buffer used during the incubation; that by butanedione requires the presence of borate as buffer. The inhibitions are not reversed by simply washing out the inhibitor, suggesting that a covalent modification of one or more arginine residues is responsible. This is supported by the reversibility of the butanedione inhibition if both the inhibitor and borate buffer are removed. ATPase of the chloroplasts, and of extracted protein, is inhibited, whether activated by trypsin or by heating. This indicates that arginine residues of the coupling factor are the probable major site(s) for attack by these modifiers, leading to the observed inhibitions.     

Activation of macrophages for tumor cell cytotoxicity: identification of indomethacin sensitive and insensitive pathways

Macrophages can be activated for tumor cell cytotoxicity by endotoxin- or lymphokine-containing solutions, and in both cases activation can be blocked by the addition of indomethacin. In contrast, the activation of macrophages by nonadherent or inflammatory peritoneal cells or antibody-coated tumor cells is not affected by indomethacin. These results demonstrate that there are at least 2 distinct pathways of macrophage activation, only 1 of which is affected by the addition of the drug. Activation by endotoxin that is indomethacin sensitive requires 2 steps. At present, the first is undefined and not blocked by indomethacin, but the second is inhibited by the drug and appears to require the production of E series prostaglandins. Our results also suggest that neither step alone is sufficient to activate macrophages for cytotoxicity.     

Influence of extracellular calcium on cell permeabilization and growth regulation by the lymphokine leukoregulin

Permeablization of human K562 leukemia cells was measured in the presence and absence of extracellular ionic calcium to examine the relationship of ionic calcium to increased membrane permeability and the inhibition of cell proliferation by this lymphokine. In the absence of extracellular calcium, the ability of leukoregulin to permeabilize the cell membrane is diminished but is fully restored by addition of 1 mM extracellular Ca++ as shown flow cytometrically by loss of intracellular fluorescein. Membrane permeability is also increased by calcium ionophore A23187 but permeablization is completely blocked in calcium-free medium despite the intramembrane presence of the calcium ionophore. Membrane permeablization by the lectin phytohemagglutinin, in contrast, is independent of extracellular calcium. A similar divergence in cell proliferation activity of the three modulators of calcium flux and membrane permeability occurs in the absence of extracellular calcium. Leukoregulin inhibition of cell proliferation is abolished, inhibition by calcium ionophore A23817 is greatly reduced, and inhibition by phytohemagglutinin is unchanged. Leukoregulin permeabilized K562 cells isolated by fluorescence activated cell sorting resume proliferation after 72 h. In contrast cells permeablized by calcium ionophore A23187 or phytohemagglutinin fail to resume proliferation by 7 days. The membrane permeablizing action of leukoregulin is, therefore, partially dependent upon extracellular calcium. It is also effected through a mechanism other than calcium ionophore transport or lectin type transmembrane signaling, and is accompanied by a reversible inhibition of cell proliferation.     

Antidiuretic mechanism in Rhodnius prolixus (Stal, 1859) (Hemiptera, Reduviidae)

An antidiuretic mechanism is proposed for Rhodnius prolixus, whose activity is manifested when the diuretic phase has terminated and is maintained until the insect has fed again. Presumably this mechanism acts at the level of the proximal rectal sphincter and is inhibited by ingestion, mechanical distension of the gut and central disinhibition by decapitation. It is suggested that the antidiuretic activity is maintained by the nervous system and is modified when there is distention of the gut or when the nervous signal is interrupted by decapitation. It is demonstrated that the excretion of urine in R. prolixus is not controlled exclusively by the diuretic hormone, but rather that factors not linked to the haemolymph maintain the insects in a diuretic or nondiuretic state.