The differential effects of the carcinogen dimethylnitrosamine on isocitrate and 6-phosphogluconate metabolism in single intact cells

A microspectrofluorimetric study is made of the influence of dimethylnitrosamine on NADP reduction, following sequential microinjections into the same L cell, of two substrates: (1) isocitrate, with activity of isocitrate dehydrogenase both in the extramitochondrial and intramitochondrial compartments, (2) 6-phosphogluconate, with activity of the dehydrogenase in the extramitochondrial compartment. In control L cells a two-step reduction of NAD(P) is obtained followed by relatively slow reoxidation. In the minutes which follow addition of carcinogen, e.g., dimethylnitrosamine, to the cell medium the isocitrate and 6-phosphogluconate-induced transient NADP reoxidation is decreased in magnitude compared to control, while the rate constant of NADPH reoxidation is considerably accelerated, possibly due to requirements at the level of the microsomal metabolizing system. Observations within the first hour of carcinogen addition suggest an interesting system for evaluating the immediate actions of carcinogens at extranuclear sites: i.e., a comparative study of NADP reduction-reoxidation rate constants via injection of substrates for extra- vs. intramitochondrial pathways.     

Hormones and liver mitochondria: Influence of growth hormone,thyroxine, testosterone,and insulin on thermotropic effects of respiration and fatty acid composition of membranes

The effects of hypophysectomy and subsequent administration of growth hormone, thyroxine, insulin, and testosterone were examined in rat liver for the relationship between the thermotropic effects on State 3 respiration (ADP induced) and fatty acid composition of the phospholipid fraction of intact mitochondria as well as of inner membrane vesicles. The Arrhenius profile for energy-linked (succinate) State 3 respiration of mitochondria from hypophysectomized rats lacked the discontinuity at 23.5 °C seen with mitochondria from normal rats. After injections of the hormones the discontinuity representing the transition temperature from gel to liquid crystalline state of lipids occurred at different temperatures: 18.5 °C for growth hormone, 26.0 °C for thyroxine, 19.5 °C for growth hormone + thyroxine, 27.6 °C for insulin, and 25.3 °C for testosterone. The energy of activation between 37.5 and 23.5 °C was 1.9 times greater for hypophysectomy than for controls. Growth hormone was the most effective in restoring the energy of activation to normal, above as well as below transition temperature. The effect of thyroxine appears to be due to a larger stimulation of the State 4 respiration than that of growth hormone, insulin, or testosterone, especially at higher temperatures. Phospholipids extracted from intact mitochondria or inner membrane vesicles of hypophysectomized rats contained less arachidonic acid (20:4) and more linoleic acid (18:2) than those of normal rats. In addition, the contents of some of the minor fatty acids were also changed. Calculated unsaturation index showed an 18.8 and 14.9% depletion in unsaturation in whole mitochondria and inner membranes, respectively. Among the different hormones used to treat the hypophysectomized rats, growth hormone was the most effective in restoring the transition temperature and fatty acid composition to normal levels and increasing the gain in body weight. Although the other hormones increased total unsaturation index to some extent, some of the individual fatty acids were affected differently. Good correlation exists between the unsaturation index of mitochondrial fatty acids and transition temperature of State 3 respiration. These results strongly suggest a role for the hormones, particularly growth hormone, in the control of mitochondrial membrane fluidity of hypophysectomized rat liver, through fatty acid composition of phospholipids.     

Thermographic visualization of cell death in tobacco and Arabidopsis

Pending cell death was visualized by thermographic imaging in bacterio‐opsin transgenic tobacco plants. Cell death in these plants was characterized by a complex lesion phenotype. Isolated cell death lesions were preceded by a colocalized thermal effect, as previously observed at sites infected by tobacco mosaic virus (TMV) ( Chaerle et al. 1999 Nature Biotechnology 17, 813–816). However, in most cases, a coherent front of higher temperature, trailed by cell death, initiated at the leaf base and expanded over the leaf lamina. In contrast to the homogenous thermal front, cell death was first visible close to the veins, and subsequently appeared as discrete spots on the interveinal tissue, as cell death spread along the veins. Regions with visible cell death had a lower temperature because of water evaporation from damaged cells. In analogy with previous observations on the localized tobacco–TMV interaction ( Chaerle et al. 1999 ), the kinetics of thermographic and continuous gas exchange measurements indicated that stomatal closure preceded tissue collapse. Localized spontaneous cell death could also be presymptomatically visualized in the Arabidopsis lsd2 mutant.     

Early Inhibition of Acetylcholinesterase and Choline Acetyltransferase Activity in Herpes simplex Virus Type 1 Infection of PC12 Cells

Abstract: Early in the course of productive Herpes simplex virus type 1 (HSV-1) infection of PC12 cells, activities of both acetylcholinesterase (AChE) and choline acetyltransferase (CAT) fell. Studies using metabolic inhibitors and a temperature-sensitive mutant of the virus suggested that the decline in activities of both enzymes was associated with events occurring early in the replicative cycle related to expression of the immediate-early (α) group of viral polypeptides. HSV-1 gene products thus may alter specialized cell functions well before the production of viral progeny and initiation of cell lysis. The early clinical manifestations of nervous system viral infection may reflect focal metabolic disturbance rather than, or in addition to, simple cell death.     

Analytical characterization and purification of plasma membrane from cultured hepatoma cells (HTC cells)

The plasma membrane of the hepatoma cell line, HTC cells, has been characterized and purified by cell fractionation techniques. In the absence of true 5′-nucleotidase in HTC cells, alkaline phosphodiesterase I has been used as a marker enzyme, following conclusions gained from differential and isopycnic centrifugation studies (Lopez Saura, P., Trouet A. and Tulkens P. (1978) Biochim. Biophys. Acta 543, 430–449). To confirm this localization, HTC cells were exposed to anti-plasma membrane IgG at 4°C and fractionated. Alkaline phosphodiesterase I and IgG showed super imposable distribution patterns in linear sucrose gradients. Alkaline phosphodiesterase I is, however, only poorly resolved from enzyme markers of other organelles, especially NADPH-cytochrome $\text{c}$ reductase (endoplasmic reticulum) and galactosyltransferase (Golgi complex). Maximal purification from the homogenate is only 13-fold, on a protein basis, even when using a microsomal fraction (67 and 13% of alkaline phosphodiesterase I and protein, respectively) as the starting material. Improved resolution can be obtained after the addition of small quantities of digitonin (equimolar with respect to the cholesterol content). Digitonin increases the buoyant density of alkaline phosphodiesterase I by approx. 0.05 g/cm³, whereas the buoyant densities of galactosyltransferase and NADPH-cytochrome $\text{c}$ reductase are increased only by 0.03 and 0.015 g/cm³, respectively. Accordingly, a procedure has been designed which yields a fraction containing 22.8% of alkaline phosphodiesterase I with a purification of 21-fold on a protein basis. The content of NADPH-cytochrome $\text{c}$ reductase and galactosyltransferase is 1.2 and 2.1%, respectively. Electron microscopy shows smooth surface membrane elements and vesicles, with only occasional other recognizable elements.     

A mutant of Halobacterium halobium with constitutive production of bacteriorhodopsin

Abstract A purple mutant of Halobacterium halobium was isolated in a previous study. The 'in vitro' absorption spectra of the cells gave a broad shoulder around 570 nm. The amounts of bacteriorhodopsin were high under any growth condition (including aerobic) inhibitory for the wild-type strain. The mutant grew faster under illuminated microaerophilic conditions and showed faster proton extrusion than the wild-type strain. This evidence shows that the mutant has a constitutive bacteriorhodopsin production not influenced by the oxygen concentration in the medium. However, some stimulation by light was found.     

In vivo and in vitro sugar transport in frog intestine

In the frog intestine, both in vitro and in vivo, experiments were carried out in order to increase knowledge of the mechanism of sugar exit across the basolateral membrane of the enterocyte. The frog intestine was chosen because it lacks crypt cells and, consequently, any external fluid circuit mechanism during sugar transport can be avoided. Therefore, the sugar concentration in the absorbate collected on the serosal side is likely to be similar to that present underneath the basolateral membrane of the enterocyte. Under this condition, cell and absorbate sugar concentrations are similar; yet there is a concomitant net transintestinal sugar transport. Moreover, in in vivo experiments a net transintestinal sugar transport takes place even against a concentration difference. These results suggest that sugar exit across the basolateral membrane is not simply due to a chemically facilitated diffusion.     

Plasma catecholamines: effects of footshock level and hormonal modulators of memory storage

Plasma levels of norepinephrine (NE) and epinephrine (EPI) were measured in male Sprague-Dawley rats before and at several times after training injections of agents known to enhance or to impair later retention performance for a one-trial inhibitory (passive) avoidance task. Two days before testing, each animal was surgically prepared with a chronic tail artery catheter that allows for repeated blood sampling in unhandled rats. Exposure to a single, intense training footshock (3.0 mA, 2.0 sec duration) resulted in an immediate but transient increase in plasma levels of EPI and to a lesser extent NE. Plasma levels of both catecholamines did not differ between unshocked controls and animals that received a weak training footshock (0.6 mA, 0.5 sec duration). An injection of EPI at a dose that enhances retention performance (0.1 mg/kg, sc) resulted in increments in plasma EPI levels of 0.8-1.9 ng/ml from 5 to 40 min after injection. An injection of EPI (0.5 mg/kg, sc) at a dose that produces retrograde amnesia resulted in increments in plasma EPI ranging from 3.7 to 4.5 ng/ml during the 40 min after injection. Plasma NE levels were not significantly altered following an EPI injection. A single injection of adrenocorticotropin (ACTH, 0.3 or 3.0 IU per rat) did not alter the plasma catecholamine responses to training with a weak footshock. Similarly, the synthetic ACTH analog, Organon 2766 (125 or 250 mg/Kg) did not affect plasma catecholamines in untrained (unshocked) rats.These results demonstrate that significant increments in plasma levels of NE and EPI occur shortly after inhibitory avoidance training. Furthermore, an injection of EPI that enhances retention of an inhibitory avoidance task mimics the magnitude, though not the temporal characteristics, of the endogenous adrenal medullary response to a training footshock. Other hormonal treatments (ACTH and Organon 2766) which enhance memory storage do not affect plasma levels of NE and EPI.