Catabolism of myo-Inositol to Precursors Utilized for De Novo Glycerolipid Biosynthesis

Systemic injection of [2-3H]myo-inositol into frogs resulted in the incorporation of more than half of the label into glycerolipid classes other than phosphoinositides in retinal rod outer segment membranes. Following methanolysis and differential extraction of isolated lipid classes, radioactivity was recovered primarily in the aqueous phase. After phospholipase C hydrolysis of the total membrane lipids, 97% of the radioactivity was extractable with organic solvents, and 70% of the label in lipids was in 1,2-diglycerides. These results indicate that the label was incorporated primarily into the glyceryl moiety of the membrane glycerolipids. Intraocular injection of frog eyes or in vitro incubation of frog retinas with [2-3H]myo-inositol resulted in the incorporation of radioactivity almost exclusively into phosphoinositides in rod outer segment membranes. Incubation of retinas with [U-14C]glucuronic acid did not result in the formation of labeled retinal lipids. These results suggest that myo-inositol can be catabolized systemically to precursors utilized for glycerolipid biosynthesis in the retina.     

In vitro establishment of human fibroblasts of lysosomal diseases,GM1-gangliosidosis and Sandhoff disease,by transformation with origin-minus SV40 DNA

The permanent human cell lines preserving defects of lysosomal enzymes, G_M1-1019-SV and SA-1077-SV, were established from the respective fibroblasts from patients with G_Ml-gangliosidosis and Sandhoff disease by transfection with replication origin-minus simian virus 40 DNA. These ceils grow rapidly without entering senescence during more than 120 population doublings. The activity of -galactosidase in G_M1-1019-SV and of B-N-acetylhexosaminidase in SA-1077-SV was respectively 40- and 180-fold lower than that of normal fibroblasts.     

Stomatal response to air humidity and its relation to stomatal density in a wide range of warm climate species

The gas exchange of 19 widely different warm climate species was observed at different leaf to air vapour pressure deficits (VPD). In all species stomata tended to close as VPD increased resulting in a decrease in net photosynthesis. The absolute reduction in leaf conductance per unit increase in VPD was greatest in those species which had a large leaf conductance at low VPDs. This would be expected even if stomata of all species were equally sensitive. However the percentage reduction in net photosynthesis (used as a measure of the relative sensitivity of stomata of the different species) was also closely related to the maximal conductance at low VPD. Similarily the relative sensitivity of stomata to changes in VPD was closely related to the weighted stomatal density or crowding index.The hypothesis is presented that stomatal closure at different VPDs is related to peristomatal evaporation coupled with a high resistance between the epidermis and the mesophyll and low resistance between the stomatal apparatus and the epidermal cells. This hypothesis is consistent with the greater relative sensitivity of stomata on leaves with a high crowding index.The results and the hypothesis are discussed in the light of selection, for optimal productivity under differing conditions of relative humidity and soil water availablility, by observation of stomatal density and distribution on the two sides of the leaf.Visiting scientist, plant physiologist and research assitant of the Cassava Program     

Arterial perfusion of frog tongue for intracellular recording of taste cell receptor potential

The frog tongue was perfused through its artery with a Ringer solution using a peristaltic pump, and a method was developed to record stable intracellular receptor potentials of taste cells. Perfusing at 0.05 ml/min with a Ringer solution containing 5% dextran did not cause tongue edema, but perfusing at the same rate with Ringer without dextran caused edema. After perfusion at 0.05 ml/min with 100 mM K Ringer, the membrane potential of taste cells gradually decreased and reached a constant level in about 30 min, indicating that the intercellular fluid of the tongue could be replaced within this time period. While the artery of the frog tongue was perfused at 0.05 ml/min with Ringer containing 5% dextran, intracellular receptor potentials of taste cells elicited by four basic taste stimuli (1 M NaCl, 10 mM quinine-HCl (Q-HCl), 1 mM acetic acid and 1 M galactose) were similar to those obtained from the control taste cells under normal blood flow.     

Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells during development: Effect of malnutrition

Incorporation of [³H]thymidine into DNA and of [³⁵S]sulfate into sulfatides of oligodendroglial cells isolated from brain slices incubated with the radioactive precursor was studied in normal and malnourished rats at different ages. The pattern and the values of incorporation of [³H]thymidine into DNA were similar in both groups of animals. The maximum value of incorporation was observed at 7 days of age decreasing rapidly thereafter and leveling off between 18–21 days. In both groups of animals labeling of sulfatides attained a maximum at 18 days of age, showing similar values of incorporation up to that age. However, at 21 days of age; the values corresponding to malnourished rats were found to be 40% lower in comparison to controls. The results suggest that (a) proliferation of oligodendroglial cells stops at similar ages in normal and malnourished rats, (b) expression of sulfatide synthesis by oligodendroglial cells is similar in both groups of animals up to 18 days, and (c) the starved rats seem to be unable to maintain normal synthesis of these galactolipids throughout the entire period of active myelinogenesis.     

Neonatal malnutrition in the rat affects the delivery of sulfatides from microsomes and their entry into myelin

Brain slices from 18 day old normal and malnourished rats were incubated in the presence of [³⁵S]sulfate to explore its incorporation into sulfatides of a total brain homogenate and the appearance of labeled sulfatides in different subcellular fractions. While the incorporation of label into sulfatides of the total homogenate was similar in both groups of animals, in subcellular fractions separated on a linear sucrose density gradient, labeling of sulfatides in malnourished animals was relatively higher in the region corresponding to the microsomal fraction. Time course incorporation and pulse-chase experiments were carried out to explore the kinetics of labeling of microsomal and myelin sulfatides. In pulse-chase experiments, normal controls showed a decrease in the specific radioactivity of sulfatides in the microsomal fraction after the chase, which was not observed in malnourished animals, while the appearance of labeled sulfatides in the myelin fraction of the latter group of animals was found to be lower than in normals. These results suggest that in neonatal malnutrition there is a defect in the transport of de novo synthesized sulfatides towards myelin or/and a problem in the assembly of these lipids into the myelin membrane.     

Correlation of in vitro properties of Rhodococcus (Corynebacterium) equi with virulence for mice

To study the virulence of Rhodococcus (Corynebacterium) equi, seven ATCC strains of different serotypes were tested for their LD50 in mice, clearance of the organism from the lungs and spleen following intravenous or intratracheal inoculation, and in vitro interaction with murine peritoneal macrophages. Strains ATCC 33704 and 33705 were virulent for mice and multiplied in the lungs and spleen, resulting in death of the animal in 5 days. The other five strains were avirulent for mice. The number of bacteria in the lungs and spleen of mice given these five strains decreased immediately. Pulmonary clearance of strains ATCC 33703, 33706, and 33707 was significantly more rapid than that of the virulent strains ATCC 33704 and 33705 12 hr after inoculation. Complete clearance of the avirulent strain ATCC 33707 occurred by day 14, while that of virulent ATCC 33704 and 33705 strains occurred by day 30. The virulent strains ATCC 33704 and 33705 were resistant not only to phagocytosis but also to intracellular killing by macrophages. Strains ATCC 33702 and 33706 were rapidly killed by macrophages although they were rather resistant to phagocytosis. Strain ATCC 33703 was easily phagocytized though resistant to killing by macrophages. The most avirulent strains, ATCC 33707 and 6939, were easily phagocytized and rapidly killed by macrophages. These results indicate that virulence appeared to be related to the ability of the organisms to resist clearance from the lungs and spleen and to resist phagocytosis and intracellular killing by macrophages.