Subcellular location of enzymes involved in oxidation of n-alkane by Cladosporium resinae

More than 70% of n-hexadecane-grown cells of Cladosporium resinae ATCC 22711 were converted to spheroplasts when they were treated with chitinase and lytic enzyme from Trichoderma harziamum. The light mitochondrial fraction, containing microbodies, mitochondria and vacuoles, was isolated from spheroplasts. Vacuoles in cells were demonstrated by the inability of acridine orange to stain organelles previously treated with 2.5 μM Bafilomycin A₁, a vacuolar ATPase inhibitor. Microbodies, mitochondria and vacuoles were separated from the light mitochondrial fraction by self-generated density-gradient ultracentrifugation using iodixanol as gradient medium. NADH-dependent n-alkane monooxygenase activity and fatty alcohol oxidase activity were located in the cytoplasm and mitochondrial fractions respectively. Received: 21 September 1998 / Received revision: 21 January 1999 / Accepted: 31 January 1999     

Glucose, pyruvate and lactate efflux by the perfused liver of a teleost, Clarias batrachus during aniso-osmotic exposure

Glucose, lactate and pyruvate efflux by the perfused liver of the walking catfish, Clarias batrachus was studied during aniso-osmotic exposure. During hypo-osmotic exposure (−80 mOsmol l⁻¹, maintained with NaCl), glucose, lactate and pyruvate efflux by the perfused liver significantly decreased by 55, 19 and 16%, respectively. During hyper-osmotic exposure (+80 mOsmol l⁻¹, maintained with NaCl), efflux increased by 57, 12 and 18%, respectively. Similar effects of glucose, lactate and pyruvate efflux by the perfused liver was also seen when the anisotonicity of the medium was adjusted with mannitol instead of NaCl. The decrease of glucose, lactate and pyruvate efflux during hypo-osmotic exposure was correlated with the stimulation of glycogen synthesis and the reverse was true during hyper-osmotic exposure. These observations were supported by changes in glycogen phosphorylase a (GPase a) and glycogen synthase a (GSase a) activities. During hypo-osmotic exposure (−80 mOsmol l⁻¹), the GPase a activity decreased by 1.93 fold and GSase a activity increased by 1.63 fold, while during hyper-osmotic exposure (+80 mOsmol l⁻¹), the GPase a activity increased by 1.58 fold and GSase a activity decreased by 1.95 fold. The total activity of both the enzymes were not effected by a short term exposure to aniso-osmotic conditions, suggesting that the alterations in GPase a and GSase a activity were mainly due to changes of their phosphorylation status during aniso-osmotic exposure. A direct correlation exists between glucose efflux and the hydration status of the perfused liver. These alterations of glucose metabolism are probably necessary by this walking catfish to meet the different energy demand, and also for maintenance of glucose homeostasis under osmotic stress.     

A comparison of time-lapse cinemicrography and flow cytometry for the study of accelerated cell-cycle transit

Abstract. Two methods for the study of cell-cycle progression, time-lapse cinemicrography (TLCM) and flow cytometry (FCM), were compared for their ability to measure the shortening of cell-cycle transit time induced by temporary inhibition of DNA synthesis. DNA synthesis was reversibly inhibited by aphidicolin (APH) in synchronized HeLa cells obtained by mitotic collection. TLCM directly measured intermitotic time intervals and thereby directly obtained the cell-cycle transit time distribution. In contrast, FCM measured time dependent changes in the fractions of cells in the cell-cycle phases from which the distribution of cells traversing a cell-cycle boundary, such as that between G₁ and S phase, was determined. Nevertheless, both methods provided equivalent measures of the cell-cycle transit time and its dispersion. However, TLCM apeared to provide a better measure of skewness of the transit time distribution than did FCM. Further, both methods were able to detect changes in the cell cycle transit on the order of 1 h or less. The TLCM data showed a greater precision (due to a larger number of data points) than that from FCM. However, FCM was able to directly measure changes in the transit of G₁ phase whereas TLCM would require two different experiments to make a similar determination. The results obtained in this study show that FCM can replace TLCM to study most aspects of cell-cycle progression.     

Polyphenol Oxidase Activity and Phenolic Content Pattern During Shoot Development of Grape (Vitis vinifera L.) in Different Growing Seasons

The PPO activity and content of phenolic compounds were estimated during the different growing seasons and developmental age of shoots in seven grape genotypes. Both, the total phenol content and catecholase activity were found significantly affected by the growing season, developmental age of shoot and the genotype. But, season as well as age of shoot could not influence cresolase activity. Spring as season; 30 days of sprouting as age; Gold and Anab-e-Shahi as genotypes were found better in comparison to summer as season and 15 days of sprouting as age in respect to level of phenols and PPO activity.