Inhibitory effect of the flavonoid silymarin on the erythrocyte hemolysis induced by phenylhydrazine

The flavonoid silymarin, which is used as a therapeutical agent in the treatment of liver diseases, can inhibit the hemolysis and lipid peroxidation induced by phenylhydrazine on erythrocytes obtained from rats treated with the flavonoid. This effect is ascribed to the antioxidant properties as a free radical scavenger exhibited by the flavonoid. Silymarin failed to inhibit the glutathione depletion induced by phenylhydrazine on erythrocytes. It is proposed that the flavonoid acts at the membrane level of the cell avoiding the lipid peroxidative and fluidizing effect of phenylhydrazine.     

Vasoactive intestinal peptide (VIP) binding to solubilized material from rat liver plasma membranes

A non-ionic detergent such as Lubrol-PX extracts in soluble form the VIP-binding structures of rat liver plasma membranes. Detergent-solubitized proteins bind specifically [¹²⁵I]VIP and the complex tracer-protein is identified by the use of Sepharose 6B columns. The interaction is only possible in the absence of detergent (below 0.001%) and is inhibited by native peptide. A molecular weight of about 80,000 was estimated for VIP-binding proteins by reference to a series of globular markers of proteins. Binding to VIP soluble proteins is specific and dependent on time as studied by the Hummel and Dreyer (Biochim. Biophys. Acta 63:530–532, 1962) assay.     

Ontogenetical development of the chick and duck subcommissural organ

Summary The ontogenetical development of the subcommissural organ (SCO) was investigated in chick embryos collected daily from the 1st to the 21st day of incubation. Some duck embryos, and adult chickens and ducks were also studied. Immunocytochemistry using an anti-Reissner's fiber (RF) serum as the primary antibody was the principal method used.In the chick embryos the events occurring at different days of incubation were: day 3 morphologically undifferentiated cells in the dorsal diencephalon displayed immunoreactive material (IRM); days 4 to 6 immunoreactive cells proliferated, formed a multilayered structure and developed processes which traversed the growing posterior commissure and ended at the brain surface; day 7 i) blood vessels penetrated the SCO, ii) scarce hypendymal cells appeared, iii) the first signs of ventricular release of IRM were noticed, iv) appearance of IRM bound to cells of the floor of the Sylvius aqueduct; day 7 to 10 the number of apical granules and amount of extracellular IRM increased progressively; day 11 RF was observed along the Sylvian aqueduct; day 12 RF was present in the lumbar spinal cord; day 13 IRM on the aqueductal floor disappeared; days 10 to 21 i) hypendymal cells proliferated, developed processes and migrated dorsally, ii) ependymal processes elongated and their endings covered the external limiting membrane. In adult specimens the ependymal cells lacked basal processes and the external membrane was contacted by hypendymal cells. The duck SCO appears to follow a similar pattern of development.Supported by Grant I/60 935 from the Stiftung Volkswagenwerk, Federal Republic of Germany, and Grant RS-82-18 from the Dirección de Investigaciones, Universidad Austral de Chile. M.H. was recipient of a personal grant from JNO (29-5-54), which is gratefully acknowledged     

The concentration and control of cytoplasmic free inorganic pyrophosphate in rat liver in vivo

The concentration of cytoplasmic free pyrophosphate was calculated in freeze-clamped livers of rats from the measured concentration of reactants and K(eq.) of the UDP-glucose pyrophosphorylase reaction (UDP-alpha-d-glucose 1-phosphate uridylyltransferase, EC 2.7.7.9). The K(eq.) of the UDP-glucose pyrophosphorylase reaction was redetermined at 38 degrees C, pH7.0, I=0.25mol/l and free [Mg(2+)]=1mm, and was 4.55 in the direction of glucose 1-phosphate formation. The activity of UDP-glucose pyrophosphorylase in rat liver was between 46 and 58mumol of glucose 1-phosphate formed/min per g fresh wt. in the four dietary conditions studied. A fluorimetric assay with enzymic cycling was developed for the measurement of glucose 1-phosphate in HClO(4) extracts of rat liver. The calculated free cytoplasmic PP(i) concentration in nmol/g fresh wt. of liver was 2.3+/-0.3 in starved, 3.8+/-0.4 in fed, 4.9+/-0.6 in meal-fed and 5.2+/-0.4 in sucrose-re-fed animals. These values agree well with recently determined direct measurements of total PP(i) in rat liver and suggest that there is not a large amount of bound or metabolically inert PP(i) in rat liver. The cytoplasmic [ATP]/[AMP][PP(i)] ratio is 10(3) times the cytoplasmic [ATP]/[ADP][P(i)] ratio and varies differently with dietary state. The reaction PP(i)+H(2)O-->2P(i) catalysed by inorganic pyrophosphatase (EC 3.6.1.1) does not attain near-equilibrium in vivo. PP(i) should be considered as one of the group of small inorganic ions which is metabolically active and capable of exerting a controlling function in a number of important metabolic reactions.     

Changes in the concentrations of hepatic metabolites on administration of dihydroxyacetone or glycerol to starved rats and their relationship to the control of ketogenesis

1. Glycerol and dihydroxyacetone, both antiketogenic and readily metabolized, but differing in their effects on the redox state of the hepatic NAD couples, were given to starved rats and the contents of metabolites were measured in freezeclamped liver and in the blood. The object was to study the effects of changes in the redox state and of the availability of oxidizable substrates on the rate of ketone-body formation. 2. Intramuscular administration of dihydroxyacetone, glycerol or glucose to starved rats decreased the concentrations of acetoacetate and 3-hydroxybutyrate in the blood by 70-80% within 60min., whereas there was no major change in the free fatty acid concentration. 3. Dihydroxyacetone, but not glucose or glycerol, caused an immediate and sustained twofold increase in the blood lactate concentration. 4. Dihydroxyacetone and glycerol caused a rapid fall in the hepatic concentrations of ketone bodies, dihydroxyacetone being more effective. 5. This decrease was not accompanied by significant changes in the concentrations of acetyl-CoA, long-chain acyl-CoA or free CoA. 6. The hepatic glycerophosphate concentration rose about 40-fold on administration of glycerol, whereas with dihydroxyacetone the increase was only about 50%. The large increase in glycerophosphate concentration after administration of glycerol was completely prevented by pretreatment of the rats with tri-iodothyronine. Triiodothyronine-treated rats showed the same decrease in ketone-body concentrations after administration of glycerol as the untreated rats. 7. Glycerol and dihydroxyacetone caused an increase in the hepatic lactate concentration; the pyruvate concentration rose only after injection of dihydroxyacetone. 8. Both compounds increased liver glycogen. 9. Calculation of the [free NAD(+)]/[free NADH] ratios indicated that dihydroxyacetone increased the ratio in cytoplasm and mitochondria, whereas glycerol caused a prompt fall in both compartments, followed at 10min. by a slight rise in the mitochondrial compartment. 10. Dihydroxyacetone did not alter the hepatic content of ATP. 11. The findings suggest that the main reason for the antiketogenic effect of glycerol and dihydroxyacetone was a consequence of their ready metabolism and the provision of an increased supply of C(3) intermediates for conversion into oxaloacetate. Under the test conditions, neither the hepatic content of alpha-glycerophosphate nor the redox state of the NAD couples appeared to play a major role in the regulation of ketogenesis.     

A possible role for PAF in allergen-induced late responses: modification by a selective antagonist

We determined whether platelet-activating factor (PAF) plays a role in allergen-induced airway responses by studying the effects of a selective PAF antagonist WEB-2086 on antigen-induced early and late airway responses in allergic sheep. In seven sheep, inhaled Ascaris suum produced significant early (282%) and late (176%) increases in specific lung resistance (sRL). WEB-2086 (1 mg/kg iv) given 20 min before antigen challenge did not affect the early response, but the peak late increase in sRL was only 37% over base line (P less than 0.05 vs. control). To study the mechanism by which PAF contributes to antigen-induced responses, we evaluated the effects of pharmacological probes on PAF-induced bronchoconstriction. Inhaled PAF (dose range 75-700 micrograms) caused reproducible (r = 0.781, P less than 0.05) increases in sRL in eight sheep. The PAF-induced bronchoconstriction was blocked by WEB-2086 (1 mg/kg iv) and by the leukotriene antagonist FPL-55712 (30 mg by aerosol); however, neither the cyclooxygenase blocker indomethacin (2 mg/kg iv) nor the histamine H1-antagonist chlorpheniramine (2 mg/kg iv) blocked the PAF response. WEB-2086, however, did not block bronchoconstriction induced by aerosol leukotriene D4, indicating that PAF acts indirectly through leukotrienes. Finally, we determined whether PAF could induce late airway responses. Inhaled PAF produced an immediate increase in sRL in all seven sheep tested, but late airway responses were observed in only three of the seven sheep.(ABSTRACT TRUNCATED AT 250 WORDS)     

Targeted cytotoxic cells in human peripheral blood lymphocytes.

We have isolated subsets of cells from human PBL and have investigated their abilities to mediate lysis targeted by bispecific antibodies. Targeted cytotoxic cells were divided into two distinct types based on buoyant density. The low buoyant density fraction contained all of the targetable cytotoxic activity in unstimulated PBL, including both T and K cells targeted with anti-CD3 and anti-Fc gamma RIII (CD16) containing bispecific antibodies, respectively. Both types of targetable cytotoxic cells required IL-2 for maintenance of cytotoxic activity, expressed the CD56 (NKH1) marker, and mediated MHC-unrestricted lysis. The targetable T cells in low density PBL were exclusively CD8+ and represented only about 2% of the total PBL. The high buoyant density lymphocytes, depleted of NK cells, had no targetable activity, but were able to generate over several days, targetable T cell activity in the presence of a TCR cross-linking signal plus IL-2. Unlike the low-density cells, the activated high buoyant density effector T cells did not express CD56, consisted of both CD4+ and CD8+ cells, and did not mediate MHC-unrestricted lysis. These cells proliferated more rapidly and generated more total lytic activity than the low-density fraction. Our studies show that targetable cytotoxic activity in human PBL is mediated by several subsets of cells with different activation requirements. Presumably all of these activities could be directed against unwanted cells in clinical or preclinical studies involving targeted cytotoxic cells.     

Increased Resistance Against Oxidative Stress Is Observed During A Short Period of Renal Reperfusion After A Temporal Ischaemia

Reperfusion of rat kidney submitted to temporal ischaemia induces a decrease in glutathione content. Lipid peroxidation is not detected in kidney homogenates but microsomes obtained after periods of reperfusion longer than 60 minutes show increased malondialdehyde values correlated with high oxygen consumption and superoxide free radical generation. Microsomes obtained from kidneys submitted to 15 or 60 minutes of reperfusion are resistant to NADPH-induced lipid peroxidation but after 120 minutes of reperfusion an increased lipid peroxidative response is observed. Although the mechanism of the protection found in microsomes against the induction of oxidative stress in the first 60 minutes of reperfusion is unknown, it is postulated that this subcellular fraction plays an important role in the oxidative stress observed after longer periods of reperfusion.