Cellular and Subcellular Localization of Hexokinase, Glutamate Dehydrogenase, and Alanine Aminotransferase in the Honeybee Drone Retina

Abstract: Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[³H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina.     

pH-Dependent Effects of 2,4-Dinitrophenol (DNP) on Proliferation, Endocytosis, Fine Structure and DNP Resistance in Tetrahymena

ABSTRACT. In view of the importance of external pH on cytotoxic effects of ionizable agents, the pH-dependent effects of 2,4-dinitrophenol (DNP) were investigated. As uncoupler of oxidative phosphorylation. DNP interferes with the proton gradient across the inner mitochondrial membrane. DNP was added to proliferating Tetrahymena pyriformis in media of different initial pH. Effects studied were rates of cell proliferation and endocytosis, and fine structure. Findings correlated with the calculated concentration of undissociated DNP, taking into account that pH changes with time and cell density in Tetrahymena cultures. A linear relationship thus emerged between initial concentrations of undissociated DNP and lengths of the lag preceding cell proliferation. Once resumed, the rate of proliferation corresponded to that of control cells, even in different concentrations of undissociated DNP, presumably indicating an adaptation mechanism. Endocytosis was elevated throughout a wide range of undissociated DNP concentrations with a sharp transition towards inhibition at high DNP concentrations causing lethality with time. Changes in fine structure of DNP-treated cells (mitochondria, peroxisomes, nucleoli) also depended on the concentration of undissociated DNP.     

Amino acid concentrations and selected enzyme activities in rat auditory,olfactory, and visual systems

Homogenates of specific brain regions of three sensory systems (auditory, olfactory, and visual) were prepared from pigmented Long-Evans Hooded rats and assayed for amino acid concentrations and activities of glutaminase, aspartate aminotransferase (total, cytosolic, and, by difference, mitochondrial), malate dehydrogenase, lactate dehydrogenase, and choline acetyltransferase. Comparing the quantitative distributions among regions revealed significant correlations between AAT and aspartate, between glutaminase and glutamate, between glutamate and glutamine, and between AAT plus glutaminase, or glutaminase alone, and the sum of aspartate, glutamate, and GABA, suggesting a metabolic pathway involving the synthesis of a glutamate pool as precursor to aspartate and GABA. Of the inhibitory transmitter amino acids, GABA concentrations routinely exceeded those of glycine, but glycine concentrations were relatively high in brainstem auditory structures.     

Isolation and enzymological characterization of infected and uninfected cell protoplasts from root nodules of Glycine max

Infected and uninfected cell protoplasts were isolated from soybean ( Glycine max L. Merr. cv. Akisengoku) root nodules and purified by the use of nylon mesh filters and discontinuous Percoll gradients. Activities of the enzymes involved in carbon and nitrogen metabolism were measured in cytoplasmic fractions of purified protoplasts, as well as in the bacteroids isolated from infected cell protoplasts and in the cortical tissues after enzymatic digestion of the central zone of the nodules.
A high degree of purity of both infected and uninfected cells was demonstrated by microscopic observations and assays of β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and uricase (EC 1.7.3.3) activities and leghemoglobin contents.
As a whole, higher specific activities of enzymes of glycolysis were found in the cortical and uninfected cells than in the infected cells. The activities of glycolytic enzymes were extremely low in the bacteroids. Invertase (EC 3.2.1.26) was highly localized in the cortex. However, activity of sucrose synthase (EC 2.4.1.13) was highest in the cytosol of infected cells. Alcohol dehydrogenase (EC 1.1.1.1) and lactate dehydrogenase (EC 1.1.1.27) activities were much higher in uninfected than in infected cells. Specific activities of enzymes for nitrogen assimilation, that is, glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.14), aspartate (EC 2.6.1.1) and alanine (EC 2.6.1.2) aminotransferase were several-fold higher in uninfected cells than in the infected cells.
The results are discussed in relation to the possible cellular organization of carbon and nitrogen metabolism in soybean root nodules.     

Studies on the stimulation of gluconeogenesis and lipolysis by glucagon and epinephrine in isolated rat Kupffer cells.

Kupffer cells were isolated by collagenease-pronase treatment. Activity and leakage of GOT, GPT, LDH, GlDH and of nucleotide pyrophosphatase were measured and compared to parenchymal cells. In addition, the effects of glucagon and epinephrine on gluconeogenesis and lipolysis were studied. Both glucagon and epinephrine stimulated gluconeogenesis from lactate and alanine. The epinephrine response, however, was far greater than that of glucagon. Additional studies showed a 50% stimulation of lipolysis by epinephrine with triolein and tripalmitin as substrates. No stimulation of lipolysis was observed with glucagon.     

Dietary supplementation of brown seaweed (Sargassum latifolium) alleviates the environmental heat stress-induced toxicity in male Barki sheep (Ovis aries)

Heat stress (HS) is the most potent environmental stressors for livestock in tropical and subtropical regions. HS induced splanchnic tissue hypoxia and intestinal oxidative damage, leading to endotoxemia and systemic inflammation. The present study evaluated and compared the modulatory effects of feeding Barki male sheep (Ovis aries) on a standard concentrated diet containing 2% or 4% of the brown seaweed (Sargassum latifolium) followed by roughage for 40 consecutive days on the toxicity-induced by exposure to severe environmental HS (temperature-humidity index = 28.55 ± 1.62). The present study showed that the diet containing Sargassum latifolium (especially 4%) modulated significantly (P < 0.05–0.001) almost all changes shown in the HS-exposed sheep including the increase in the thermo-respiratory responses (skin and rectal temperatures, and respiration rate) and the resulted dyslipidemia, anemia, and systemic inflammation (blood leukocytosis, the elevation in the erythrocyte sedimentation rate, and the increase in serum proinflammatory cytokines and heat shock protein-70 concentrations). In addition, Sargassum latifolium improved significantly (P < 0.05–0.001) the body-weight gain, kidney functions (especially at the high dose), and blood antioxidant defense system (total antioxidant capacity, and the activities of catalase and superoxide dismutase) in the HS-exposed sheep, as well as protected the animals from oxidative tissue damage and the risk of atherosclerosis. In conclusion, feeding sheep with the diet containing 4% of Sargassum latifolium was safe and suitable for animal nutrition, as well as efficiently alleviated the harmful effects of the environmental HS in Barki sheep through improving the animal antioxidant defense system, and regulating the thermo-respiratory and inflammatory responses.