Transcortin in rat kidney tissue: distribution in microsomal fractions

During chromatography of renal tissue cytosolic proteins on DEAE-cellulose the protein specifically binding [3H]corticosterone is eluted within the potassium phosphate concentration range of 0.08-0.10 M. Analysis of kidney slices revealed the synthesis of [3H]transcortin whose electrophoretic mobility was close to that of the blood plasma protein. Using radioimmunochemical methods, it has been found that transcortin-specific [125I]IgG antibodies interact with growing polypeptide chains of membrane-bound polyribosomes. Free polyribosomes do not bind antibodies against transcortin.     

β-lipotropin-like material in human pancreas and pyloric antral mucosa

Evidence for the presence of the endogenous opioid precursor β-lipotropin has been found in human pyloric antral mucosa and pancreas by radioimmunochemical displacement curves, Sephadex chromatography, ion exchange chromatography, and immunocytochemistry. Endogenous opioids are not only present in the pituitary gland and nervous tissue but also in the stomach and pancreas, supporting the concept of a common neuro-endocrine system present in brain and gut.     

Control of protein synthesis in mammalian cells by aminoacylation of transfer ribonucleic acid.

The dependence of protein synthesis on the intracellular content of aminoacylated tRNA has been studied in mouse ascites tumor cells deprived for various amino acids. A remarkable reduction in net protein synthesis has been found only after a drastic decrease in aminoacylation of tRNA. The quantitative correlation of protein synthesis with the degree of aminoacylation suggests that a moderate amino acid starvation primarily influences the rate of elongation at the codon concerned. These results are in contrast to the findings previously reported for HeLa cells. Some crucial steps during the determination of intracellular aminoacyl-tRNA have been investigated. The reliability of the method employed has been discussed on a theoretical basis.     

Free iron in bacteria

Free iron content has been estimated in autotrophic and heterotrophic bacteria. It constituted 40-50 micrograms/g dry weight as compared to 15 micrograms/g dry weight in animal cells. A method for estimation of free iron has been proposed. It is based on formation of paramagnetic dinitrosyl iron complexes by free iron and protein thiol groups or low molecular weight thiol ligands. The reasons for high iron content in bacteria have been discussed.     

125I]-antigammaglobulin antibodies as universal reagents in radioimmunochemical methods of investigation

A new indirect radioimmunochemical method based on the use of [125I]-anti-IgG-antibodies as universal detecting reagents is proposed. Its first step consists in the antibody binding to the antigen to be analyzed; the second step -- in immunoadsorption of the non-bound antibodies by water-insoluble sorbents prepared by chlorocarbonic acid isobutyl ester copolymerization of antigens with serum albumin or by immobilization of the antigen on Sepharose. The third step is the determination of the amount of sorbent-bound antibodies by means of [125I]-anti-IgG-antibodies. The method proposed was used for quantitative estimation of prolactin, somatotropin, lutropin, BB-isoenzyme of human creatine phosphokinase, testosterone and 5 alpha-dihydroxytestosterone. The method does not employ labelled antigens and is highly sensitive and highly specific.     

Scanning microfluorometric measurement of immunofluorescently labelled microtubules in cultured cells

Summary A method for evaluation of microtubule content in cultured cells has been developed. The method is based on scanning microfluorometric measurement of immunofluorescently labelled microtubules. The method has been applied to the comparison of microtubule content in epithelial XTH-2 cells grown in culture at various cell densities. The results have shown that the microtubule content in the cells is not dependent on their proliferative state rather than it depends on cellular contacts.     

Scanning microfluorometric measurement of immunofluorescently labelled microtubules in cultured cells. Dependence of microtubule content on cell density

A method for evaluation of microtubule content in cultured cells has been developed. The method is based on scanning microfluorometric measurement of immunofluorescently labelled microtubules. The method has been applied to the comparison of microtubule content in epithelial XTH-2 cells grown in culture at various cell densities. The results have shown that the microtubule content in the cells is not dependent on their proliferative state rather than it depends on cellular contacts.     

Increased endogenous retroviral gene expression is a consequence of lymphocyte activation

Plasma cells of line 151(5) chickens have been shown to express elevated levels of endogenous retroviral envelope glycoprotein (VEG), measured relative to levels expressed by both immature B cells and resting peripheral B lymphocytes. In this study we analyzed the relationship between peripheral blood lymphocyte (PBL) maturation and the level of VEG expression. A culture system was developed that would support maturation of pokeweed mitogen-activated peripheral B lymphocytes. As analyzed by cytofluorometry, both Ig+ and Ig- lymphoblasts present in the pokeweed mitogen-stimulated cultures expressed detectable levels of VEG in contrast to bursacytes and PBL. Similarly, Ig- blasts, which were present in concanavalin A-stimulated cultures of PBL and presumed to represent activated T cells, were also positive for the expression of VEG. Immature T cells, i.e., thymocytes, although negative by immunofluorescence analysis, expressed VEG at levels that were detectable by radioimmunochemical techniques. These results indicate that T cells as well as B cells constitutively express VEG, and that mitogenic activation of the resting lymphocyte induces an increase in VEG expression.     

Changes in RNA concentration in neurons and perineuronal glia of the caudal nucleus of the vagus nerve after a single maximal overload

By the method of quantitative cytophotometry, the content of ribonucleic acid in neurons and mantle gliocytes of the cat vagus caudal node has been studied after a single maximal overloading of 10 units. It has been stated that a decreased RNA content in the mantle gliocytes is less pronounced; that could be explained by their different nutritional conditions (owing to their various position towards the microvessels of the node), on the one hand, and by different functional loading experienced by the two types of cells under the effect of the extreme factor studied, on the other hand. RNA content in gliocytes is restored more quickly owing to its less decrease, and also owing to a more favourable protein synthesis in glycocytes--"for itself".     

Measurement of the specific lipid content of attached cells in microtiter cultures

A method has been described to quantify intracellular neutral lipid content in attached cells in microtiter cultures. The procedure was based on oil red O staining of neutral lipid and Coomassie brilliant blue G-250 staining of total cellular protein. Results were expressed as the ratio of lipid to protein, the "specific lipid content" index. This measurement was shown to closely correspond to actual lipid per cell measurements under experimental conditions. The procedure was specific for neutral lipids and sensitive (greater than or equal to 50 ng triglyceride/well). Additionally, cell proliferation measurements could be made simultaneously, using protein staining data. Chromatic endpoints were measured using a spectrophotometer capable of reading individual wells of a microtiter plate. The procedure is recommended for applications in which the endpoint is neutral lipid droplet accumulation in attached cultured cells.     

Assessment of skin carbonyl content as a noninvasive measure of biological age.

The oxidative modification of proteins by reactive species, especially reactive oxygen species, is implicated in the etiology or progression of a panoply of disorders and diseases. For the most part, oxidatively modified proteins are not repaired and must be removed by proteolytic degradation. The level of these modified molecules can be quantitated by measurement of the protein carbonyl content, which has been shown to increase in a variety of diseases and processes, most notably during aging. However, these studies have required invasive techniques to obtain cells for analysis. We examined the possibility that desquamating skin cells (corneocytes) would also show an age-related increase in protein carbonyl content, thus providing a noninvasive method for assessing biological age. This was not the case, as we found no age-dependent relationship in the protein carbonyl content of skin cells from volunteers aged 20 to 79 years.     

Gastrin/CCK-like immunoreactivity in the nervous system of coelenterates

Using immunocytochemistry, gastrin/CCK-like immunoreactivity is found in sensory nerve cells in the ectoderm of the mouth region of hydra and in nerve cells in the endoderm of all body regions of the sea anemone tealia. These results are corroborated by radioimmunoassay: One hydra contains at least 5 fmole and one tealia at least 2 nmole gastrin/CCK-like immunoreactivity. Reactivities towards gastrin and CCK antisera with different specificities suggest that the coelenterate gastrin/CCK-like peptide contains the C-terminal amino-acid sequence common to mammalian gastrin and CCK. In addition the radioimmunochemical data indicate that the coelenterate peptide also contains an amino-acid sequence that resembles the sequence 20-30 of porcine CCK-33, but that no other sequences of gastrin are present. Thus, it is probably more CCK-like than gastrin-like.     

Color recovery after photoconversion of H2B::mEosFP allows detection of increased nuclear DNA content in developing plant cells

Many higher plants are polysomatic whereby different cells possess variable amounts of nuclear DNA. The conditional triggering of endocycles results in higher nuclear DNA content (C value) that in some cases has been correlated to increased cell size. While numerous multicolored fluorescent protein (FP) probes have revealed the general behavior of the nucleus and intranuclear components, direct visualization and estimation of changes in nuclear-DNA content in live cells during their development has not been possible. Recently, monomeric Eos fluorescent protein (mEosFP) has emerged as a useful photoconvertible protein whose color changes irreversibly from a green to a red fluorescent form upon exposure to violet-blue light. The stability and irreversibility of red fluorescent mEosFP suggests that detection of green color recovery would be possible as fresh mEosFP is produced after photoconversion. Thus a ratiometric evaluation of the red and green forms of mEosFP following photoconversion could be used to estimate production of a core histone such as H2B during its concomitant synthesis with DNA in the synthesis phase of the cell cycle. Here we present proof of concept observations on transgenic tobacco (Nicotiana tabacum) Bright Yellow 2 cells and Arabidopsis (Arabidopsis thaliana) plants stably expressing H2B::mEosFP. In Arabidopsis seedlings an increase in green fluorescence is observed specifically in cells known to undergo endoreduplication. The detection of changes in nuclear DNA content by correlating color recovery of H2B::mEosFP after photoconversion is a novel approach involving a single FP. The method has potential for facilitating detailed investigations on conditions that favor increased cell size and the development of polysomaty in plants.     

Radioimmunochemical determination of carbamoyl-phosphate synthase (ammonia) content of adult rat liver

1. Rabbit antiserum was raised against purified carbamoyl-phosphate synthase (ammonia) from rat liver. 2. The antiserum proved to be specific in double-diffusion test and reacted in an in situ immunohistochemical test on rat liver proteins fractionated on a sodium dodecyl sulphate polyacrylamide gel only in the region where carbamoyl-phosphate synthase (ammonia) migrated. 3. This antiserum was used for setting up a radioimmunochemical determination of carbamoyl-phosphate synthase (ammonia) in cetyltrimethylammonium bromide extracts of rat liver. To obtain reproducible results in this assay it was necessary to treat the unlabelled ligand with sodium dodecyl sulphate and dithiothreitol. This treatment led to a large increase in the percentage of labelled ligand displaceable by added unlabelled ligand. 4. Radioimmunochemical determination showed that adult rat liver (3-month old) contains 5.5 mg carbamoyl-phosphate synthase (ammonia) protein per gram wet weight.     

Characteristics of bacterial lipopolysaccharides depending on extraction method

Carbohydrate-containing biopolymers have been isolated from Erwinia carotovora subsp. atroseptica 549 by two methods--the aqueous-phenol and with using physiological solution--with addition and without addition of ethylenediaminetetraacetate (EDTA). The biopolymers yield from the cells of bacteria are shown to depend on the extraction method. Lipopolysaccharide-protein complex have been isolated by the sparing method. The purest lipopolysaccharide have been isolated by the aqueousphenol method. The preliminary treatment of cells by EDTA increased the biopolymers output. Carbohydrate containing biopolymers isolated from the cells of bacteria by different methods possess the similar qualitative composition of monosaccharides but they differ in the quantitative content of monosaccharides, spectrum of fatty acids of lipid components as well as in the protein content.     

Gastrin/CCK-like immunoreactivity in the nervous system of coelenterates

Summary Using immunocytochemistry, gastrin/CCK-like immunoreactivity is found in sensory nerve cells in the ectoderm of the mouth region of hydra and in nerve cells in the endoderm of all body regions of the sea anemone tealia. These results are corroborated by radioimmunoassay: One hydra contains at least 5 fmole and one tealia at least 2 nmole gastrin/CCK-like immunoreactivity. Reactivities towards gastrin and CCK antisera with different specificities suggest that the coelenterate gastrin/CCK-like peptide contains the C-terminal amino-acid sequence common to mammalian gastrin and CCK. In addition the radioimmunochemical data indicate that the coelenterate peptide also contains an amino-acid sequence that resembles the sequence 20–30 of porcine CCK-33, but that no other sequences of gastrin are present. Thus, it is probably more CCK-like than gastrin-like.     

The effect of epidermal growth factor on protein phosphorylation in normal and transformed hepatocytes

Qualitative differences in the content of tyrosine-phosphorylated proteins in normal and transformed hepatocytes have been found using the method of two-dimensional electrophoresis. Epidermal growth factor (EGF) has induced quantitative changes in the spectra of phosphotyrosine-containing proteins in normal cells and qualitative changes in the transformed ones. Results of immunoprecipitation with antibodies against phosphotyrosine permit revealing a protein with Mm 50 kDa which is subjected to EGF-dependent tyrosine-phosphorylation in normal hepatocytes. The intensity of protein phosphorylation is 10 times higher in the transformed cells but the dependence of this process on the EGF is not exhibited. The role of protein-tyrosine-kinases in the transmission of a mitogenic signal and in liver carcinogenesis is discussed.     

A rapid and simple method for staining of the crystal protein ofBacillus thuringiensis

Summary A rapid and simple method of staining for the crystal protein (-endotoxin or parasporal body) ofBacillus thuringiensis has been developed. Changes in colonial morphology were observed when cells lost their ability to form crystal protein or both crystal protein and spore.     

Simplified method for DNA and protein staining of human hematopoietic cell samples

A rapid reproducible method yielding high resolution analysis of DNA and protein in human hematopoietic cell samples has been developed by modification of the propidium iodide and fluorescein isothiocyanate procedure. Cell staining involves sequential addition of each reagent (RNase, fluorescein isothiocyanate and propidium iodide) to ethanol-fixed cells and requires no centrifugation steps. Stained cells are analyzed in the reagent solutions. Analysis of bone marrow samples from multiple myeloma patients showed mixed normal and aneuploid populations with a major portion of the aneuploid cells having a significantly higher protein content. This approach permitted differential cell cycle analysis of normal and the aneuploid populations.