Cytoplasmic Protein Content Research Articles

Ultrastructure and Some Biologic Properties of Human Oocytes and Granulosa Cells Cultured in Vitro

Of follicular oocytes with germinal vesicles, 79% resumed meiosis within 48 hours in modified Ham's F-10 medium for oocyte culture, and 59% of them reached the second-metaphase stage of meiosis. An increase in cortical granules and tubular aggregates and a decrease in the Golgi apparatus were observed during the maturation process. Scanning electron microscopy of the oocyte surface revealed a decrease in number and length of microvilli. Nuclear DNA contents diminished to approximately one-half, but cytoplasmic protein contents measured by cytofluorometry were unchanged after culture in spite of these structural changes. Insemination with washed spermatozoa resulted in fertilization of 5 of 43 zona-intact cultured oocytes. After removal of the zona, 6 of 17 cultured oocytes showed polyspermic penetration, although immature oocytes were not fertilized even after removal of the zona. Cultured granulosa cells secreted progesterone spontaneously, and its amount was correlated with follicular size. Ultrastructurally, mitochondria with laminar cristae, enlarged nuclei, dispersed Golgi apparatus, smooth endoplasmic reticulum, lipid droplets in the ooplasm, and granular cytoplasmic protrusions were observed on the oocyte surface.

Cytophotometric and morphometric studies of postnatal rat hippocampal neurons and perineuronal gliocytes after electroconvulsive shock

Electroconvulsive shock was evoked by transcorneal electric current (50 Hz, 50 mA; 3 times by 0.2s with 10-min pauses) in 10-day and 20-day old rats. 2 hours after the end of the convulsions, the total cytoplasmic RNA and protein content per cell in CA3 hippocampal neurons has been shown cytospectrophotometrically to increase markedly in 10-day old rats without statistically significant changes in 20-day old animals. No changes in the RNA and protein content were found in perineuronal neuroglia cells in the CA3 hippocampus in both age groups of rats. Morphometric determinations revealed a pronounced augmentation of volumes of the hippocampal neuronal bodies, nuclei, and cytoplasm as well as of nucleo-cytoplasmic ratio in 10-day old rats under effect of electroconvulsive shock. In 20-day old rats, the shock led to less marked increase in volumes of the hippocampal neurons, with no changes in nucleo-cytoplasmic ratio.

SOLUBLE PROTEINS AND ENZYMES IN THE SUBCELLULAR FRACTIONS OF VIRUS-INFECTED TOBACCO LEAF

Leaves of bright tobacco NC 95 infected with tobacco vein mottling virus (TVMV) and tobacco mosaic virus (TMV) were fractionated into subcellular fractions for protein quantitation and enzyme assay. Both viruses increased soluble proteins in the nuclear, chloroplastic and ribosomal fractions, whereas TMV infection lowered cytoplasmic protein content. Leaves infected with TVMV significantly increased total soluble proteins. Virus infection enhanced peroxidase activity in the cytoplasm but decreased activity in the chloroplastic and mitochondrial fractions. Polyphenol-oxidase concentrated in the chloroplast and its activity were not altered by virus infection. Ribosomal fraction was rich in protease in both virus-infected leaves. In the chloroplasts, protease increased in activity in the TMV-infected leaves but maintained a level as low as the control in leaves showing TVMV symptom. The accumulation of soluble proteins in the TVMV-infected leaves is possibly, in part, attributed to a low concentration of protease.

Mean cytoplasmic protein concentration of host erythrocytes and the reticulocyte response inPlasmodium berghei infected mice

The decreasing mean cytoplasmic protein concentration (MPC) of P. berghei host cells is paralled by an increasing parasitemia and percent reticulocyte response. The reticulocyte response parallels the percent parasitemia except during a period of accelerated reticulocytosis noted during the midpoint of the infection at which time the percent reticulocytes increases at a rate more than double the rate of increase of percent parasitemia. Although the reticulocyte population and the host cell population are usually equivalent, the disparity noted suggests the existence of unique characteristics in the reticulocyte response of P. berghei infected mice.

Alterations in Human Endometrial Protein Synthesis During the Menstrual Cycle and in Progestone-Stimulated Organ Culture

The cytoplasmic protein content of endometrium from women at various times in the menstrual cycle has been examined by means of polyacrylamide gel electrophoresis using a double-label isotope technique. Analysis of the gels for altered label ratios suggests an increase in the concentration of a single protein band during the immediate postovulatory period that can be duplicated by the addition of progesterone to organ cultures of preovulatory endometrium. An alteration in the protein content is also observed in the same section of these gels when they are stained and scanned. The protein band of interest migrates on sodium dodecyl sulfate gels at a rate that establishes its approximate molecular weight as 51,500.

Cytophotometric determination of nuclear non-histone protein in the pre-replicative phase of rat liver regeneration

Nuclear non-histone protein (NHP) and cytoplasmic protein was determined cytophotometrically in Feulgen-Naphthol Yellow S (NYS) stained, isolated parenchymal liver cells during the first 16 h following partial hepatectomy in rats. Nuclear NYS binding was also measured in histological sections, and the RNA polymerase activity of parenchymal nuclei was assayed autoradiographically. The following results were obtained: 1. 1. From 4 h on, the mean nuclear NHP in regenerating livers is significantly higher than in sham-operated controls; at 16 h the difference is 33%. 2. 2. The frequency distributions of nuclear NHP suggest that the increase in mean nuclear NHP is due to the gradual emergence of cells that have higher nuclear NHP values than any cells in the sham-operated controls. The number of such cells amount to a third of the population at 12 and 16 h after the partial hepatectomy. 3. 3. The measurements of nuclear NYS binding on histological sections show that the cells with high nuclear NHP values at 12 and 16 h after partial hepatectomy are predominantly located to the periportal area. 4. 4. The mean RNA polymerase activity and the mean cytoplasmic protein content also increase after partial hepatectomy, and the highest values of both parameters were found in cells with high nuclear NHP values.

Pharmacokinetic aspects of sulfobromophthalein transport in chronically carbon tetrachlorideintoxicated rats

In order to elucidate the factors affecting the hepatic transport of sulfobromophthalein (BSP) in a pathological condition, the kinetics of the disappearance of BSP from the blood and appearance in the bile were studied in rats chronically intoxicated with carbon tetrachlorde, and the importance of factors that might affect the kinetics, i.e. binding activites of plasma and hepatic cytoplasmic proteins, and glutathione S-transferase activity of Y-fraction, was examined. During chronic intoxication, although plasma protein albumin concentration decreased, no qualitative difference was shown in the high affinity binding site ( K 1). A significant decrease was shown in the cytoplasmic protein concentration for the Y- and Z-fractions in intoxicated rats; no significant difference was observed in the number of binding sites for both fractions, but their binding constants decreased. Glutathione S-transferase activity and glutathione (GSH) content were not altered, although a decrease in conjugating activity per rat was expected. The time course for the plasma disappearance and biliary excretion of BSP showed a remarkable delay after CCl 4 intoxication, while no difference was shown in the bile flow rate. It was concluded that hepatic blood flow might play a primary ro)e in the initial plasma disappearance of BSP and that the decrease in the Y-fraetion binding activity might explain the decrease in BSP uptake rate into the liver which was observed in intoxicated rats.

Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. Effects of castration and androgen replacement.

Four intrinsic soluble secretory proteins are synthesized in vitro by isolated seminal-vesicle mucosa from sexually mature guinea pigs. Newly synthesized specific proteins labelled with [(14)C]glycine and [(14)C]lysine were precipitated by using double-antibody immunoprecipitation techniques and their radioactivity was assessed. Rates of synthesis were determined on each of 5 days after castration. By 5 days after castration the wet weight of the epithelium decreased to 42% of intact control values; the absolute amount of specific protein synthesized in vitro after 60min incubation decreased to 28% and the 27500g cytoplasmic protein content decreased to 31%. Thus androgen deprivation leads to a decrease in general protein synthesis in vivo, as well as to a decrease in specific protein synthesis in vitro. Specific protein synthesis comprised 76% of the total protein formed in isolated tissue from animals 5 days after castration as compared with 99-100% in tissue from intact animals. At 72h after an injection of testosterone or dihydrotestosterone, seminal-vesicle epithelium wet weight, cytoplasmic protein content and capability for synthesizing specific proteins in vitro were restored to approx. 70% of normal values. At 72h after onset of therapy with 3alpha-androstanediol, both epithelium wet weight and cytoplasmic protein content had increased significantly, but without a corresponding increase in the capability of the isolated tissue to synthesize specific proteins. The soluble labelled proteins synthesized in vitro by isolated epithelium from intact animals during 60 or 120min incubation were essentially entirely immunoprecipitable, i.e. specific. In contrast, approx. 29% of all soluble protein newly synthesized by isolated epithelium from animals 5 days after castration was acid-precipitable, but not immunoprecipitable, i.e. ;non-specific'. The injection of testosterone into castrated animals inhibited the synthesis of the non-specific fraction by isolated tissue. The effects of castration on the ultrastructure of guinea-pig seminal-vesicle epithelium are also presented.

Assorbimento dopo colorazione al Fast-green di cellule della caliptra di Allium cepa localizzate in differenti regioni istologiche

Abstract Fast-green absorption of cells localized in different regions of Allium cepa root cap.—The nuclear and cytoplasmic protein content of two cell population (C2 = columella cells; P2 = peripheral cells) has been determined by Barr and Stroud cytophotometer after Fast-green staining (after DNA extraction and without DNA extraction). The collected data seem indicate that: 1) histone content is higher in nuclei of C2 cells than in nuclei of P2 ones; 2) acid proteins content is higher in nuclei of P2 cells than in those of C2 ones; 3) the cytoplasmic proteins of the two cell populations are quite different in their Fast-green reactivity.

Enzymes of carbohydrate and lipid metabolism in the placenta and liver of pregnant rats

Changes in the activity of enzymes involved in glycosis, gluconeogenesis and lipogenesis during the last 7 days of gestation were determined in the placenta and liver of pregnant rats and expressed in relation to the cytoplasmic protein content of these tissues. On Day 15 of gestation the activity of pyruvate kinase (EC 2.7.1.40) in the placenta was considerably higher than in the liver, whereas the activity of placental enzymes associated with gluconeogenesis was much lower than in the liver. This indicated that the placental capacity of glucose utilization is preponderant over gluconeogenesis. The activity of enzymes associated with lipid synthesis was lower in the placenta in comparison to the liver. However, the activity of enzymes of NADPH generation and of ATP citrate lyase (EC 4.1.3.8) in relation to that of acetyl-CoA carboxylase (EC 6.4.1.2) was markedly higher in the placenta than in the liver. This was suggestive of the importance of synthesis of lipids other than fatty acids in the placenta. With the progress of gestation a decrease in the activity of most placental enzymes was observed, particularly in that of pyruvate kinase. In the liver the activity of enzymes of NADPH generation and of ATP citrate lyase also decreased. The possible background of these changes in enzyme activity in the liver and placenta is discussed.

Metabolic changes in diseased insects: IV. Radioautographic studies on protein changes in nuclear polyhedrosis, densonucleosis, and Tipula iridescent virus infections

Changes in glutamic acid, leucine, arginine, and tyrosine in Lepidoptera and Hymenoptera tissues infected with nuclear polyhedrosis (NPV), densonucleosis (DNV), or Tipula iridescent (TIV) viruses were studied by radioautography with a view to determining the effect of the viruses on protein metabolism. During NPV and DNV infection cycles, enlargement of the nucleus was accompanied by an increase in the nucleus and a simultaneous decrease in the cytoplasm of the amino acids. Total cell protein increased at the intermediate stage of infection and then decreased. The rate of change in nuclear protein content was inversely proportional to the rate of change in cytoplasmic protein content. The spindle-shaped cytoplasmic inclusions which characterize the NPV of Lambdina fiscellaria lugubrosa incorporate tritiated glutamic acid, leucine, and tyrosine and stain deeply with Naphthol Yellow S indicating the presence of lysine, arginine, and histidine. The inclusions are produced in L. f. lugubrosa infected by the NPV of Caripeta divisata and Orgyia pseudotsugata but not in the latter two insect species infected by homologous virus. Changes in amino acids of tissues infected with TIV differed from those described above in that an increase in total cell arginine was maintained up to the advanced stage of viral pathogenesis. There was very little change in rate of nuclear or cytoplasmic protein content during the infection cycle.

Morphological and biochemical changes in rat liver during the early stages of ethyl chlorophenoxyisobutyrate administration.

The early changes in the rat liver cells after the administration of ethyl chlorophenoxyisobutyrate (CPIB) were studied. At first, mitochondrial changes appeared 6 to 12 hours after the first dose of CPIB; the mitochondria were deformed and the succinic oxidase activity decreased; these changes disappeared in 24 hours. At 12 hours after administration an increase in smooth endoplasmic reticulum was observed and this proliferation reached the maximum on the 4th day accompanying increase of activity of drug metabolizing enzyme system. After 24 hours increased formation of microbodies without nucleoids followed the appearance of smooth ER and it was parallel to the increase of enzyme activities associated with microbodies, catalase and D-amino acid oxidase. These augmentations attained the plateau after 4 days. On the other hand, CPIB caused less remarkable change in lysosomes. A consistent increase in the liver weight of rats receiving CPIB was found and this rise was not accompanied by any histological evidence of liver damages. easurements of protein, RNA and DNA show that the increase in liver weight is not due to the increase in the number of cells, but to the increase of the protein content in cytoplasm. The relationship among intracellular organelles on the effect of CPIB was discussed.

Histochemical changes in Melolontha melolontha (Linnaeus) (Coleoptera) infected by a new virus.

Changes in DNA, RNA, protein, and glycogen in the fat body of a beetle, Melolontha melolontha (Linnaeus), were studied histochemically at intervals of 15, 23, 75, and 195 days after intrahemocoelic injection of a cytoplasmic polyhedrosis virus, Vagoiavirus melolonthae. As the disease progressed, the average nuclear diameter increased considerably without any apparent change in DNA content. Cytoplasmic RNA increased progressively for about 75 days and then decreased slightly. Nuclear protein content increased during the first 23 days after virus inoculation. Cytoplasmic protein content progressively increased throughout the disease process. There was no significant change in glycogen reserves 23 days after inoculation; thereafter, glycogen was drastically reduced.Three morphologically different types of cytoplasmic inclusions all staining intensely with naphthol yellow S (thus establishing their proteinaceous nature) were observed. The pyroninophilic "round" type is considered to be "mature" inclusions containing the virus particles demonstrated by electron microscopy. The resemblances between this virus and some viruses of higher animals are discussed.

Chromatolysis in the superior cervical ganglion of the cat

Chromatolysis was studied morphologically and histochemically following axon section of superior cervical ganglia of 80 cats over a 110-day interval. Three distinct cell types, considered representative stages in chromatolysis, were defined, and the relative chronologic distribution of each cell type was presented. Microspectrophotometric determinations of cytoplasmic concentrations of RNA and protein-bound SH and SS groups in each cell type was correlated with area measurements of respective cell bodies, nuclei, and nucleoli. Autonomic ganglion cells respond promptly to axon section, as do spinal sensory ganglia (Hyden, 1943) and anterior horn cells ( Bodian and Mellors, 1945), by a marked decrease in cytoplasmic RNA concentration but with a modest increase in cytoplasmic protein concentration. The increase in cell size noted at this stage appears to result from active protein synthesis rather than osmotic uptake of water as suggested by Gersh and Bodian (1943). Cytophotometric evidence for the reappearance of cytoplasmic RNA first in the perinuclear area of the ganglion cell may be related to a concomitant enlargement of the nucleolus. This lends suggestive support to steadily increasing data noting the importance of the nucleolus in the synthesis of cytoplasmic RNA.

Refractometry of living cells part I. basic principles

ABSTRACT The principles underlying a new method of refractometry of living cells are discussed. The method was evolved from the chance observation that the amoebocytes of the blood of the earthworm, examined in their own blood, appeared bright instead of dark by positive phase-contrast microscopy. This was shown to be due to the presence of dissolved haemoglobin which raised the refractive index of the medium above that of the cytoplasm. In order to determine the refractive index of the latter it was only necessary to dilute the blood until the cytoplasm became virtually invisible. Non-pigmented proteins and other high molecular weight substances have now been substituted for haemoglobin. The nature of the initial observations suggested that if the cell could be regarded to a first approximation as being composed entirely of proteins, the cytoplasmic protein concentration could be equated to the protein concentration of the immersion medium which made the cell appear with minimum contrast. This would only be true if equal concentrations of different proteins in solution had the same refractive index. The nature of refractive index and its relationship to density are discussed and it is shown that for nearly all unconjugated proteins so far investigated the specific refraction increments (i.e. the increase in refractive index per 1 per cent, increase in concentration) have almost the same values (00185±2 per cent.). The effects of many factors such as pH, salts, temperature, wavelength, concentration, and nature of the solvent are discussed. Since living cells contain substances other than proteins the specific refraction increments of protein derivatives, lipides, carbohydrates, and salts are considered and it is shown that the presence of moderate amounts of such substances is unlikely to affect the refractive index of cells to any great extent. It is suggested that the mean specific refraction increment of protoplasm should be taken as 0018 and that this value can be used in order to calculate the solid and water content of protoplasm from values of refractive index.