Ultrastructure of maturing fish (Oreochromis niloticus) and snake (Waglerophis merremii) erythroid cells with regard to hemoglobin biosynthesis

• 1.1. Fish and snake immature erythrocytes were submitted to a comparative ultrastructural study, analysing changes in organelles involved in hemoglobin (Hb) biosynthesis.
• 2.2. Iron uptake occurs probably via transferrin, and ferruginous compounds accumulate as siderosomes, taken as iron sources for heme biosynthesis, later on caught by a double lamella.
• 3.3. Mitochondrial membrane of the inner camera differentiates to lamellated bodies that, sucessively, give rise to expansions for ferruginous material and globin chains captation, constituting prehemosomal vesicles, which become condensed vesicles, followed by prohemosomes.
• 4.4. Through an internal membrane rearrangement, prohemosomes change to hemosomes wherein, hypothetically, heme and the globin chains assembly may occur.
• 5.5. In both fish and snake erythroid cells, all stages for hemosomegenesis are similar to the stages found in erythroid cells of other vertebrate species, including humans, except that fish cells often present single organelles of still unknown function, void of internal membrane.
• 6.6. Through electrophoresis of the respective supernatants obtained after osmotical lysis of the organellar fractions, it was shown that fish hemosomes contain three Hb patterns, while snake hemosomes present two patterns.

     

Are lysosomes directly involved in the iron uptake by reticulocytes?

• 1.1. The mechanism by which iron is transferred from the plasma protein transferrin into erythroid precursors for incorporation in heme is not completely understood.
• 2.2. To show a direct functional role of lysosomes in the process of iron uptake we tried to isolate lysosomes from reticulocytes, which have been incubated with ¹²⁵ITf⁵⁹Fe.
• 3.3. However, with various cell fractionating techniques described for liver cells no pure lysosomes from reticulocytes could be obtained.
• 4.4. Fluorescence and electron microscopy showed that reticulocytes hardly contain well-defined lysosomes.
• 5.5. There are several indications that in reticulocytes acid vacuoles instead of lysosomes are involved in the removal of iron from endocytosed transferrin.
• 6.6. The presence of apoTf, monoferric TfFe(A), monoferric TfFe(B) in the medium after incubation of reticulocytes with diferric transferrin, together with the fact that both iron binding sites of transferrin release their iron at pH present in acid vacuoles, suggests a second mechanism of iron uptake by reticulocytes, in which acid vacuoles are not involved.

     

The role of glutamine and glucose analogues in metabolic inhibition of human myeloid leukaemia In vitro

• 1.1.Glutamine analogues l-[alphaS,5S]-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) and 6-diazo-5-oxo-l-norleucine (DON) have been shown to possess cytotoxic activity against a wide variety of animal and human xenografted solid tumours, however their potential in man has been limited by toxicity.
• 2.2.We have analysed the effects of acivicin and DON on glutamine utilization to determine whether the reason for the disappointing therapeutic profile is solely due to the inefficient inhibition of glutamine metabolism.
• 3.3.Human myeloid leukaemic cells treated with acivicin inhibited ribonucleotide biosynthesis but not energy production via glutaminolysis and had little effect on viability, whereas treatment with DON inhibited both ribonucleotide biosynthesis and glutamine oxidation and resulted in reduced viability.
• 4.4.Treatment of the myeloid leukaemic cells with the glucose analogue 2-deoxy-d-glucose in addition to DON potentiated the inhibition of de novo nucleotide biosynthesis, glutaminolysis and glycolysis, and caused a further reduction in cell viability.
• 5.5.These results provide further support for the essential role of glutamine in cellular metabolism, and indicate that use of the glutamine analogue DON in the treatment of acute myeloid leukaemia may be more clinically effective if used in combination with 2-deoxy-d-glucose.

     

Larval Musca domestica lipophorin biosynthesis

• 1.1. Larval Musca domestica lipophorin biosynthesis was studied in vitro.
• 2.2. The newly synthesized lipophorin has a density a little lower than the circulating lipophorin after 1 hr of incubation. After 3 hr of incubation the fat body cells transfer lipids to the lipophorin that attains the density of circulating lipophorin.
• 3.3. The lipophorin synthesized in vitro is identical to circulating lipophorin in density and in electrophoretical behavior.
• 4.4. However these two molecules must have differences since the circulating lipophorin transfers lipids to fat body cells while the synthesized in vitro does not.
• 5.5. The biosynthesis of Musca lipophorin shows differences with the Manduca sexta lipophorin biosynthesis.

     

The subunit structure of Daphnia pulex hemoglobin

• 1.1. The extracellular hemoglobin of Daphnia pulex has an apparent molecular weight of 430,000–470,000 by gel chromatography and an S_20,w = 16.9 at pH 7.0.
• 2.2. Purified hemoglobin contains one heme per 18,000–20,000 g protein. The polypeptide chains are heterogeneous with mol. wts between 31,000–37,000. Some high mol. wt (M_r = 53,000–86,000) material is also present.
• 3.3. The hemoglobin dissociates at pH 10.5 in EDTA into 3S material which can be digested with subtilisin into 16,000 mol wt heme-containing polypeptide chains.
• 4.4. The amino acid composition of the intact hemoglobin is identical to that of the heme-containing fragments generated by proteolytic digestion of the 3S material.
• 5.5. These results are consistent with the hypothesis that D. pulex hemoglobin is composed of subunits containig two heme groups per 35,000 mol. wt polypeptide chain.

     

Hemolymph ferritin and radula structure in the limpets Patelloida alticostata and Patella peronii (Mollusca: Gastropoda)

• 1.1. The mean concentration of total hemolymph iron was 3060μg/100 ml in Patella peronii and 2950μg/100 ml in Patelloida alticostata.
• 2.2. Ferritin was found to act as a major iron-binding protein in the hemolymph of both P. peronii and P. alticostata.
• 3.3. P. alticostata ferritin has a molecular weight of approximately 505,000, while that of P. peronii has a mol wt. of approximately 520,000.
• 4.4. The lateral radula teeth of both species are mineralized by deposits of silica (SiO₂) and iron in the form of goethite (α-FeOOH).
• 5.5. Hemolymph ferritin is suggested to act as a high capacity transport system to supply iron to the mineralizing front of the radula.

     

Unfolding and release of heme from human hemoglobins A,S and F

• 1.1. Analysis of the Soret spectra of hemoglobins A, S and F has been used to determine the extent of heme exposure and release from these hemoglobins in the presence of several solvent perturbants.
• 2.2. Oxyhemoglobin S unfolding in the presence of either urea or propyl urea resulted in greater heme exposure and release than either oxyhemoglobins A or F.
• 3.3. Methemoglobin formation resulted in lower denaturation midpoints for each hemoglobin compared to the reduced oxyhemoglobin state; methemoglobin F had the lowest denaturation midpoint under isothermal denaturing conditions.
• 4.4. Rate of heme exposure was greater for oxyhemoglobin S than oxyhemoglobin A in the presence of 200 μM the anionic detergent sodium dodecyl sulfate.
• 5.5. Evidence for increased levels of heme release in hemoglobin S may be related to the greater tendency of sickled red cell membranes to undergo lipid oxidation.

     

Characteristics of Crassostrea virginica crystalline style chitin digestion

• 1.1. Fundamental chitin digestion characteristics of Crassostrea virginica crystalline style were investigated.
• 2.2. Optimum temperature and pH were 34°C and 4.8. respectively.
• 3.3. The colloidal regenerated chitin (0.56mol/0.5 ml: GlcNAc equivalents) was saturating under all enzyme levels encountered.
• 4.4. There was no evidence of end product inhibition, even after 100 hr incubation.
• 5.5. Calculated K_m for the chitinase complex was 1.19mM when determined using a 30 min assay, but was only 0.70 mM when determined using a 4.6 hr assay.
• 6.6. Both K_m values are lower than reported for similar assays in other molluscs and for most bacteria.
• 7.7. Effect of substrate preparation on the kinetics are discussed.
• 8.8. Eight peaks of chitinase activity were resolved by DEAE-Fractogel ion exchange chromatography.

     

The regulation of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax (bass) liver

• 1.1. Kinetic constant values of the reaction catalyzed by bass liver glucose 6-phosphate dehydrogenase show to be modified between 10 and 40°C.
• 2.2. The Arrhenius plot between 10 and 50°C shows two slopes with different activation energies.
• 3.3. These results suggest a regulation of this enzyme by environmental temperature.
• 4.4. Kinetics of ATP inhibition were examined between pH 6.2 and 7.8: patterns and K_i values obtained are affected by the pH variation.
• 5.5. NADH is an effective inhibitor of bass glucose 6-phosphate dehydrogenase but this enzyme does not show NAD-linked activity.
• 6.6. Kinetics of pyridoxal 5′-phosphate inhibition have indicated the presence of a lysine in the catalytic site for NADP⁺.

     

Studies on the nutrition of the supralittoral isopod Ligia pallasii using chemically defined artificial diets: Assessment of vitamin,carbohydrate, fatty acid,cholesterol and mineral requirements

• 1.1. An artificial diet, consisting of a dry aggregate of 59 chemical substances, was used to assess the requirements of the sea slater Ligia pallasii for vitamins, carbohydrates, fatty acids, cholesterol and minerals.
• 2.2. Good growth and survival of L. pallasii was obtained on the diet, comparable to that on seaweeds and to that shown by a field population.
• 3.3. No dietary requirements for vitamins, fatty acids or cholesterol were shown for periods of 40 weeks or more for L. pallasii.
• 4.4. Carbohydrates were shown to be required by L. pallasii in its diet, in the order: starch, lactose > maltose, glucose > sucrose, cellulose.
• 5.5. Dietary requirements for minerals were, in order: calcium, magnesium, phosphorus > copper, nickel, zinc > iron, manganese, sulphur > iodine, silicon.
• 6.6. The results are discussed in relation to the role of gut bacteria in supplying required nutrients to their isopod hosts and the enhancement of this process through coprophagic behaviour.

     

Light-induced effects of several enzymes of carbohydrate metabolism in Phycomyces blakesleeanus

• 1.1. The photoregulation shown by glyceraldehyde 3-phosphate dehydrogenase and glucose 6-phosphate dehydrogenase appears to be independent of the mad gene product(s) and also independent of carotene biosynthesis regulation.
• 2.2. The photoregulation of malate dehydrogenase appeared to be dependent on the mutation of the mad and car S genes.
• 3.3. Pyruvate kinase and lactate dehydrogenase may be classified as light-independent.
• 4.4. The action of ATP and fructose 1,6-bisphosphate on the enzymes studied was generally independent of light/dark grown conditions.
• 5.5. However, the effect of fructose 1,6-bisphosphate on Phycomyces pyruvate kinase appears to be light-dependent.

     

Analysis of peptide biosynthesis in the neurointermediate lobe of Xenopus laevis using high-performance liquid chromatography: occurrence of small bioactive products

• 1.1. Ppeitde biosynthesis in neurointermediate lobes of black adapted Xenopus laevis was studied using pulse-chase incubation and reversed-phase, high-performance liquid chromatographic analysis.
• 2.2. During the pulse period one major product was synthesized, which was subsequently converted to 12 chase peptides, suggesting a precursor-product mode of biosynthesis for this tissue.
• 3.3. Chase peptides I, II and IV possessed high melanotropic activity. Alpha-MSH did not appear to be among the chase peptides. Peptide II had also high corticotropic activity.
• 4.4. Peptides I and II are probably small, since they were TCA-soluble and ran faster on acid-urea gels than α-MSH. They may, however, well be structurally related to this latter hormone.

     

Growth and hemolymph trehalose levels of Heliothis zea larvae fed diets containing various sugars

• 1.1. The amount of sugar required for growth of Heliothis zea larvae on a chemically defined diet was determined.
• 2.2. Larvae grew well on fructose, galactose, sucrose, trehalose and raffinose diets but not on diets containing more than 0.5% glucose.
• 3.3. A starch diet did not promote rapid larval growth.
• 4.4. Hemolymph trehalose levels in 12-day-old larvae ranged from none to 45μmoles/ml.
• 5.5. A method for analysis of hemolymph trehalose by gas chromatography is described.

     

A novel inhibitor of glycogen phosphorylase from crayfish hepatopancreas

• 1.1. A novel glycogen phosphorylase inhibitor was partially purified from crayfish hepatopancreas.
• 2.2. The inhibitor was found only in two species of crayfish examined, and not in lobster, fresh and salt water clams, mussels or cockroaches.
• 3.3. The inhibitor is a small protein (M_r = 23,000) which did not show proteolytic activity.
• 4.4. Preliminary kinetic analysis of the inhibitory mechanism indicated that it bound to both glycogen and the glycogen phosphorylase protein.
• 5.5. Inhibitor binding to glycogen resulted in a competitive inhibition pattern with respect to glycogen phosphorylase (inhibition constant of ca 10 μg/ml).
• 6.6. The inhibitor also bound glycogen phosphorylase directly with a binding coefficient of 100 μg/ml resulting in a partially non-competitive inhibition pattern with respect to phosphate.

     

Effect of lindane on phosphatidylinositol synthesis by cerebral cortex after acute and subchronic treatment

• 1.1. The incorporation of myo-[2-³H]inositol into phosphatidylinositols was unmodified in brain cortex miniprisms from convulsant rats.
• 2.2. However, the incorporation had increased by 300–400% in non convulsant rats which had received the same amount of lindane at a lower concentration.
• 3.3. This result suggests that phosphatidylinositols are implicated in the convulsion syndrome.
• 4.4. Experiments with lindane added in vitro were performed with both subchronically lindane intoxicated and untreated rats.
• 5.5. The results show an interesting lack of parallelism.
• 6.6. This might indicate the development of some resistance to the effects of lindane, possibly as the result of complex compensatory changes in inositol lipid biosynthesis.

     

Deleterious effects of disulfiram on the respiratory electron transport system of liver mitochondria

• 1.1. The mechanism of action of disulfiram on the respiratory electron transport system of the liver mitochondria was studied in vitro.
• 2.2. Disulfiram inhibited the respiration supported by malate-glutamate as well as succinate.
• 3.3. Mitochondrial respiration inhibition was dependent upon alteration of —SH groups.
• 4.4. The inhibitory action of disulfiram might be related to the crosslinking of several proteins of the inner mitochondrial membrane.
• 5.5. The effects described above could be attributed to disulfiram per se and not to the main metabolite diethyldithiocarbamate.

     

Main kinetic characteristics of acetylcholinesterase from brain of Hypostomus punctatus,a Brazilian bentonic fish (cascudo)

• 1.1. A potentiometric method for the assay of cholinesterase has been proposed and compared with a colorimetric assay.
• 2.2. Main kinetic parameters of cholinesterase from Hypostomus punctatus brain were determined indicating that true acetylcholinesterase is by far the predominant enzyme in the brain of this fish.
• 3.3. We have compared our data with published results described from other fish species.
• 4.4. The enzyme inhibition achieved after 3 hr incubation of brain homogenates with ethyl-parathion have indicated that this enzyme shows a characteristic organophosphorous sensitive behavior.

     

Quantification of iron deposits in several body tissues of lampreys (Petromyzon marinus L.) throughout the life cycle

• 1.1. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used to measure iron concentration in several body tissues throughout the life cycle of the lamprey, Petromyzon marinus L.
• 2.2. Iron concentration in the liver rises sharply during metamorphosis, decreases in parasitic adults, and falls to the lowest value in upstream migrants.
• 3.3. In the intestine, the concentration of this metal is highest in the larval stage, but values decline steadily through transformation to their lowest levels in parasitic adults.
• 4.4. Dorsal skin has, on average, three times the iron content of ventral skin and it is only in upstream migrants that the levels of both regions increase significantly over those of other stages.
• 5.5. Differences in iron concentration in tissues of larval and adult lampreys reflect changes which take place at metamorphosis.

     

ScCobB2-mediated Lysine Desuccinylation Regulates Protein Biosynthesis and Carbon Metabolism in Streptomyces coelicolor,

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Highlights

• •Identification of the first evolutionary divergent sirtuin ScCobB2 in bacteria.
• •Implementing a global quantitative succinylome between ΔScCobB2 and WT cells.
• •ScCobB2 regulates S. coelicolor protein biosynthesis and carbon metabolism pathways.
• •The divergent sirtuin enzymes are prevalent in other groups of Actinobacteria.