Glycerol Phosphate Dehydrogenase, Glucose-6-Phosphate Dehydrogenase, and Lactate Dehydrogenase: Activities in Oligodendrocytes, Neurons, Astrocytes, and Myelin Isolated from Developing Rat Brains

Abstract: Glycerol phosphate dehydrogenase (GPDH), glucose-6-phosphate dehydrogenase (G6PDH), and lactate dehydrogenase (LDH) activities were determined in Oligodendrocytes, neurons, and astrocytes isolated from the brains of developing rats. The activity of each enzyme was significantly lower in both neurons and astrocytes than in Oligodendrocytes. The GPDH activity in Oligodendrocytes increased more than 4-fold during development, and at 120 days cells of this type had 1.4-fold the specific activity of forebrain homogenates. The G6PDH activities in Oligodendrocytes from 10-day-old rats were 1.4-fold the activities in the forebrain homogenates. The activities of this enzyme in Oligodendrocytes were progressively lower at later ages, such that at 120 days the cells had 0.8 times the specific activities of homogenates. The Oligodendrocytes had 0.6 times the homogenate activities of LDH at 10 days, and this ratio had decreased to 0.2 by 120 days. These enzymes were also measured in myelin isolated from 20-, 60-, and 120-day-old rats. By 120 days the specific activities of G6PDH and LDH in myelin were <8% of the respective activities in homogenates. The GPDH activity in myelin was, however, at least 20% the specific activity in the homogenates, even in the oldest animals. It is proposed that LDH could be used as a marker for oligodendroglial cytoplasm in subfractions of myelin and in myelin-related membrane vesicles.     

Properties of Bovine Oligodendroglia Isolated by a New Procedure Using Physiologic Conditions

A new class of procedures, previously shown to permit the isolation of pure oligodendroglia from whole rat cerebrum, has been applied with equal or greater success for the bulk isolation of this cell type from bovine white matter. Thus, the generality of this approach has been demonstrated. The bovine preparations have a purity of greater than 90% intact, phase-bright oligodendroglia and are obtained in a yield of 8 x 10(6) cells per gram of white matter. Within 1 day it is possible to obtain a preparation containing 60 mg of protein from a single cell type. These cells show a higher degree of ultrastructural preservation of all cytoplasmic constituents than previously obtained. The values for protein (33 pg/cell), DNA (5.4 pg/cell), and lipid (5-6 pg/cell) are very similar to those obtained with an earlier procedure. The cell lipids are rich in galactolipid, which comprises 20% of the total. The activity of the "myelin-specific" enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37), is 4.7 mumol/min/mg protein, similar to that obtained previously for isolated oligodendroglia and about 25-40% of that found in myelin. The activity of 5'-nucleotidase (EC 3.1.3.5) in the cells is about 10% of that in myelin or white matter.     

Differentiation of embryogenic pollen in cold-treated buds ofNicotiana tabacum var. Badischer burley and nutritional requirements of the isolated pollen to form embryos

Summary Embryogenic pollen were selectively isolated from buds after cold treatment at 10 °C for 10 days; it was immaterial whether the buds were taken from short day and low temperature (SD and LT; 8 hours light, 18 °C) or long day and high temperature (LD and HT; 16 hours light, 24 °C) plants. However, in buds from SD and LT plants the differentiation of embryogenic pollen could be detected as early as 7 days after the cold treatment, and pollen from these plants formed embryos at higher frequency (up to 4% of cultured pollen) than those from LD and HT plants (up to 1% only).The embryogenic pollen, in isolated buds, differentiated by way of pollen dimorphism. During cold treatment a fraction of pollen remained small, retained clear cytoplasm and was capable of embryogenesis in comparison to gametophytic pollen which enlarged and acquired granular cytoplasm. In our experiments cold treatment was a key factor in the induction of pollen dimorphism. This aspect of cold treatment in pollen embryogenesis is reported for the first time and was possible on the basis of selection of embryogenic pollen by density gradient centrifugation. The ratio of embryogenic pollen was about one fifth of the total population.The nutritional requirements of isolated pollen for embryogenesis were rather simple. These pollen formed embryos which readily developed into plantlets on a mineral medium supplemented with sucrose provided the pH was 6.8.     

Sodium chloride resistant cell line from haploidDatura innoxia mill

Summary A cell line resistant to sodium chloride was selected from callus cultures of haploidDatura innoxia by cloning under selective pressure. Cells of the resistant cell line retained their resistance even after subculture in absence of NaCl. Plantlets could be regenerated from resistant cells in the presence as well as absence of NaCl. In contrast, regeneration of plantlets was not possible from normal cells in the presence of NaCl, although regeneration readily occurred in the absence of NaCl.To examine the stability of the resistance in the long-term, callus cultures were initiated in presence of NaCl from stem expiants of the differentiated plantlets. All expiants of plantlets derived from resistant cells showed callus formation. This callus, derived from resistant explants, retained the trait of resistance upon subculture.     

Dark requirement for cell regeneration and colony formation by mesophyll protoplasts ofNicotiana plumbaginifolia Viv.

Summary The protoplasts ofNicotiana plumbaginifolia required darkness for cell regeneration and colony formation. Maximal plating efficiency of the protoplasts could be achieved by keeping the cultures in dark instead of light or dark/light sequence. Only two days of darkness prior to the illumination at 400 or 3,000 lux resulted in appreciable plating efficiency, than those of light from the beginning, but these values could not match the high plating efficiency in total darkness.     

Glycogenosis in the Dog

Four cases of a generalized form of glycogenosis occurring in German Shepherd dogs, all females, are described. Symptoms could be noticed as early as the age of two months and progressed slowly for months. They appeared as dizziness, muscular weakness, and in two of the cases as poor nutritional state. The abdomen became gradually distended. The main lesion seen at postmortem was a greatly increased liver size with some moderate liver fibrosis. Heavy deposits of a granular substance behaving as glycogen in histochemical tests and at electron microscopy were found in the hepatic cells, muscle fibres of the heart, skeletal and smooth muscles, and in nerve and glial cells of the central nervous system. The substance was lying freely dispersed in the cell cytoplasm without any indication of lysosomal storage. The disease of dogs does not seem to be fully comparable with any of the types observed in man, but is probably much related to Type III or Cori's Disease. Structure analysis of the deposits and enzyme investigations have been done and are published (Čeh et al. 1976).     

T-rosettes in hemodialysis patients and renal allograft recipients

Subcellular fractions, isolated from the lymphoid cell line IM-1, are capable of stimulating a weak proliferative response in allogeneic lymphocytes. They also stimulate the generation of cytotoxic effector lymphocytes. The proliferative response to subcellular fractions, as measured by ³H-thymidine incorporation, is only one-fourth to one-sixth as great as that to intact IM-1 cells, suggesting that a component(s) synthesized during the mixed lymphocyte reaction (MLR), or a short-lived cellular constituent, may be responsible for the ability of intact cells to stimulate a lymphocyte proliferative response. This component appears to be lacking or in limiting quantity in subcellular fractions, including the soluble fractions. In contrast to the decreased proliferative response to subcellular fractions, the cytotoxic capacity of the stimulated lymphocytes is comparable to that after stimulation by intact IM-1 cells. The data demonstrate that, in this system, cytotoxic effector lymphocytes can be generated in the absence of the extensive proliferative response normally observed in the MLR. The antigenic stimulus responsible for the generation of cytotoxic effector cells appears to reside on intracellular components as well as on plasma membrane. In these reactions, specificity is shown by the failure of the cytotoxic cells to release ⁵¹Cr from autologous target cells. In fact, reactivity of lymphocytes stimulated by subcellular fractions is more specific than the reactivity of cells stimulated by intact IM-1 as judged by their lytic capacity for another target cell, RPMI 4265.     

A natural fusion of flavodiiron,rubredoxin, and rubredoxin oxidoreductase domains is a self-sufficient water-forming oxidase of Trichomonas vaginalis

Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. This oxygen consumption results from H₂O-forming NADH oxidase (NOX) activity of two distinct flavin-containing systems: H₂O-forming NOXes and multicomponent flavodiiron proteins (FDPs). Neither system is membrane bound, and both recycle NADH into oxidized NAD⁺ while simultaneously removing O₂ from the local environment. However, little is known about the specific contributions of these systems in T. vaginalis. In this study, we use bioinformatics and biochemical analyses to show that T. vaginalis lacks a NOX–like enzyme and instead harbors three paralogous genes (FDPF1–3), each encoding a natural fusion product between the N-terminal FDP, central rubredoxin (Rb), and C-terminal NADH:Rb oxidoreductase domains. Unlike a “stand-alone” FDP that lacks Rb and oxidoreductase domains, this natural fusion protein with fully populated flavin redox centers directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of oxygen to water within a single polypeptide with an extremely high turnover. Furthermore, using single-particle cryo-EM, we present structural insights into the spatial organization of the FDP core within this multidomain fusion protein. Together, these results contribute to our understanding of systems that allow protozoan parasites to maintain optimal redox balance and survive transient exposure to oxic conditions.