Morphological changes in the central vacuole ofBlastocystis hominis during in vitro culture

Summary Morphological changes in the central vacuole during the growth in in vitro culture ofBlastocystis hominis were investigated by light and electron microscopy. Most cells in log phase and an early stationary phase showed a positive staining reaction in the central vacuole with PAS or Sudan black B stain, whereas cells in late stationary phase showed few positive reactions. Electron microscopic observations revealed that 95% ofB. hominis cells in log phase and 50% of cells in early stationary phase, had a substantial accumulation of electron-dense material in the central vacuole. In contrast, only 25% of the organisms in late stationary phase had an electron-dense central vacuole, while more than 50% of cells had an electron-lucent central vacuole. These results indicate thatB. hominis accumulated carbohydrates and lipids in the central vacuole during cell growth and that the organism probably consumed these metabolic substances during stationary growth. Therefore, it is strongly suggested that the central vacuole is an important organelle for storage of metabolic substances, such as carbohydrates and lipids, required for cell growth.Abbreviations PBS phosphate-buffered saline - PAS periodic acid-Schiff     

大鼠脊髓内生长抑素mRNA原位杂交的研究

采用地高辛标记生长抑素反意ＲＮＡ探针经原位杂交和显色后,光学显微镜下观察生长抑素ｍＲＮＡ在大鼠脊髓内的定位。结果显示：脊髓内含有大量呈紫蓝色的生长抑素ｍＲＮＡ阳性神经细胞,岍性磷酸酶反应产生     

Effects of exercise on mammary metabolism in the lactating ewe

Mammary metabolism in multiparous lactating ewes fed either lucerne chaff:barley grain (L:B; 70:30) or lucerne chaff:lupin grain (L:Lu; 70:30) diets was measured while at rest, during exercise on a treadmill at 0.7 m s⁻¹ on a 10 ° slope for 60 min, and during 30 min recovery from exercise. The effects of these treatments on plasma glucose, lactate, alpha-amino nitrogen (-amino N), non-esterified fatty acids (NEFA) and acetate were measured. Net mammary uptake of oxygen and metabolites was calculated from mammary blood flow and arteriovenous concentration (A − V) differences.

Mammary blood flow was reduced by 25% during exercise. Arterial concentrations of oxygen, glucose, lactate, -amino N and NEFA increased during exercise, whereas acetate concentration either remained unchanged or declined. Mammary A − V differences were significantly higher for oxygen, glucose, lactate and NEFA, and tended to be higher for -amino N and lower for acetate during exercise. The mammary uptakes of oxygen, glucose, lactate and -amino N were unaffected by exercise, whereas the uptake of NEFA was significantly increased and that of acetate was significantly reduced. The changes in arterial concentrations and mammary uptakes in response to exercise were not significantly affected by the diet. The responses in acetate and NEFA fluxes across the mammary gland might bring a change in the utilization of other metabolites as well as in the fatty acid composition of milk fat.     

Ultra-fastChara myosin: A test case for the swinging lever arm model for force production by myosin

Recent breakthroughs and technological improvements are rapidly generating evidence supporting the “swinging lever arm model” for force production by myosin. Unlike previous models, this model posits that the globular domain of the myosin motor binds to actin with a constant orientation during force generation. Movement of the neck domain of the motor is hypothesized to occur relative to the globular domain much like a lever arm. This intramolecular conformational change drives the movement of the bound actin. The swinging lever arm model is supported by or consistent with a large number of experimental data obtained with skeletal muscle or slime mold myosins, all of which move actin filaments at rates between 1 and 10 μm/sin vitro. Recently myosin was purified, fromChara internodal cells.In vitro the purifiedChara myosin moves actin filaments at rates one order of magnitude faster than the “fast” skeletal muscle myosin. While this ultra fast movement is not necessarily inconsistent with the swinging lever arm model, one or more specific facets of the motor must be altered in theChara motor in order to accommodate such rapid movement. These characteristics are experimentally testable, thus the ultra fast movement byChara myosin represents a powerful and compelling test of the swinging lever arm model.     

Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled,porcine male-specific DNA probe produced by polymerase chain reaction

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5′-AAGTGGTCAGCGTGTCCATA-3′ and 5′-TTTCTCCTGTATCCTCCTGC-3′) for 236 bp fragment of porcine male-specific DNA sequence and 1.25 × 10⁴ template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley-Liss, Inc.     

Bovine cumulus cell expansion does not depend on the presence of an oocyte secreted factor

Communication between the oocyte and its somatic cells has been shown to be important in oocyte development. Here we examined how the oocyte may be involved in bovine cumulus cell expansion. Intact bovine cumulus oocyte complexes (COC) were obtained by puncturing antral follicles. From the intact COC, oocytectomised complexes (OOX) were produced by micro surgical removal of the oocyte. Clumps of cumulus cells (CC) were obtained by micro-dissection. Intact or OOX complexes or CC were matured in the presence of fetal calf serum and hFSH (6 mlU/ml) for 24 hr and the degree of expansion measured. The presence of the oocyte is not essential to allow bovine cumulus expansion to occur as expansion occurred in all groups. Murine OOX complexes from eCG primed 35–40-day-old C57BL6/CBA F₁ hybrids (known to require the presence of an oocyte secreted factor for cumulus expansion) were cultured with or without denuded bovine oocytes (1 oocyte/μl). Murine OOX complexes expanded only in the presence of denuded bovine oocytes. Thus some factor produced by bovine oocytes enabled expansion of murine OOX complexes. To determine whether the factor is secreted by bovine oocytes, murine OOX were cultured with or without media conditioned by bovine oocytes (1 oocyte/μl for 4 hr). Significant expansion of murine OOX occurred in media conditioned by bovine oocytes. This shows that the cumulus expansion enabling effect of bovine oocytes is released into the surrounding media. Media conditioned by bovine oocytes and then frozen for up to 1 month showed that the activity by the factor can withstand freezing. © 1995 wiley-Liss, Inc.     

Mechanism of toxicity of precocene II in rat hepatocyte cultures

Precocene II was more toxic in 24 hour cultures than in 72 hour cultures of rat hepatocytes. In 24 hour cultures, there was no observable toxicity at 75 μM precocene II after exposure for 6 hours, but after 24 hours, 65% of the cells were dead. In contrast, although 794 μM killed 50% of the cells in the 72 hour cultures after a 24 hour exposure, 1 mM killed 96% of the cells within 6 hours. In both 24 and 72 hour cultures, cell death was preceded by a rapid, early loss of mitochondrial membrane potential, followed by decreases in glutathione, reduced pyridine nucleotide status, and plasma membrane Na⁺/K⁺-ATPase activity. There was also a rapid loss of ATP in the 72 hour cultures but not in the 24 hour cultures; therefore, onset of cell death may be closely linked to loss of ATP. Inhibition of cytochrome P-450 prevented the toxicity, and partially protected against the loss of membrane potential and glutathione, in 24 hour cultures but was ineffective in 72 hour cultures. Therefore, in addition to depletion of glutathione, precocene II appears to damage mitochondria and plasma membrane functions and can do so by more than one pathway. © 1996 John Wiley & Sons, Inc.     

Enzyme activity profiles in an overwintering population of freeze-tolerant larvae of the gall fly,Eurosta solidaginis

The activity of some enzymes of intermediary metabolism, including enzymes of glycolysis, the hexose monophosphate shunt, and polyol cryoprotectant synthesis, were measured in freeze-tolerant Eurosta solidaginis larvae over a winter season and upon entry into pupation. Flexible metabolic rearrangement was observed concurrently with acclimatization and development. Profiles of enzyme activities related to the metabolism of the cryoprotectant glycerol indicated that fall biosynthesis may occur from two possible pathways: 1. glyceraldehyde-phosphate glyceraldehyde glycerol, using glyceraldehyde phosphatase and NADPH-linked polyol dehydrogenase, or 2. dihydroxyacetonephosphate glycerol-3-phosphate glycerol, using glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Clearance of glycerol in the spring appeared to occur by a novel route through the action of polyol dehydrogenase and glyceraldehyde kinase. Profiles of enzyme activities associated with sorbitol metabolism suggested that this polyol cryoprotectant was synthesized from glucose-6-phosphate through the action of glucose-6-phosphatase and NADPH-linked polyol dehydrogenase. Removal of sorbitol in the spring appeared to occur through the action of sorbitol dehydrogenase and hexokinase. Glycogen phosphorylase activation ensured the required flow of carbon into the synthesis of both glycerol and sorbitol. Little change was seen in the activity of glycolytic or hexose monophosphate shunt enzymes over the winter. Increased activity of the -glycerophosphate shuttle in the spring, indicated by greatly increased glycerol-3-phosphate dehydrogenase activity, may be key to removal and oxidation of reducing equivalents generated from polyol cryoprotectan catabolism.Abbreviations 6PGDH 6-Phosphogluconate dehydrogenase - DHAP dihydroxy acetone phosphate - F6P fructose-6-phosphate - F6Pase fructose-6-phospha-tase - FBPase fructose-bisphosphatase - G3P glycerol-3-phosphate - G3Pase glycerol-3-phosphate phophatase - G3PDH glycerol-3-phosphate dehydrogenase - G6P glucose-6-phosphate - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - GAK glyceraldehyde kinase - GAP glyceraldehyde-3-phosphate - GAPase glyceraldehyde-3-phosphatase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glycerol dehydrogenase - GPase glycogen phosphorylase - HMS hexose monophosphate shunt - LDH lactate dehydrogenase - NADP-IDH NADP⁺-dependent isocitrate dehydrogenase - PDHald polyol dehydrogenase, glyceraldehyde activity - PDHgluc polyol dehydrogenase, glucose activity - PFK phosphofructokinase - PGI phosphoglucoisomerase - PGK phosphoglycerate kinase - PGM phosphoglucomutase - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride - SoDH sorbitol dehydrogenase - V _max maximal enzyme activity - ww wet weight     

Differential expression of Alpha,Mu, and Pi classes of glutathione S-transferases in chemosensory mucosae of rats during development

The expression of three classes of glutathione S-transferases (GSTs), Alpha, Mu, and Pi was investigated in the nasal mucosae of rats during development using immunohistochemical methods. GST Alpha and Mu were first detected in the supranuclear region of sustentacular cells on embryonic days 16. The Bowman's glands expressed differential patterns of immunoreactivity during development, beginning at postnatal day (P) 2 and P6 for Alpha and Mu classes, respectively and being greatest at P11 for both. The acinar cells of vomeronasal glands in the vomeronasal organ expressed Alpha and Mu classes of GSTs from P11 onwards. In the septal organ of Masera, the supranuclear region of sustentacular cells expressed GSTs from P11 with little or no variation during development. In the respiratory mucosa, Alpha and Mu classes of GSTs were detected at the brush borders of ciliated cells and in the acinar cells of posterior septal glands, but not in anterior septal or respiratory glands located on the turbinates. Compared to olfactory mucosa, the changes in immunoreactivity for GSTs were less pronounced in the respiratory mucosa during development. Specific GST Pi immunoreactivity was not detected in the nasal mucosae at any stage of development studied. The occurrence of GSTs in the nasal mucosa, including olfactory, vomeronasal, septal, and respiratory epithelia, suggests that the GSTs are actively involved in the biotransformation of xenobiotics including odorants and pheromones, and may also participate in perireceptor processes such as odorant clearance. In addition, we have developed a working model describing the cellular localization of certain phase I (e.g., cytochrome P-450s) and phase II (e.g., GSTs, -glutamyl transpeptidase) biotransformation enzymes in the olfactory mucosa and their proposed roles in xenobiotic metabolism.