Phospholipid modulation of low-Km cyclic AMP phosphodiesterase activity on rat adipocyte microsomes

The ability of nine phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase was examined in microsomal fractions of rat adipocytes. The enzyme was activated by phosphatidylserine (21% at 300 microM) and phosphatidylglycerol (36% at 300 microM). The activation was concentration dependent over the range 1-1000 microM. Six other phospholipids were without effect. Phosphatidylinositol 4-phosphate inhibited the activity of the enzyme over the same range of concentrations (26% at 300 microM). Phosphatidylserine also activated a partially purified preparation of the enzyme, whereas phosphatidylinositol 4-phosphate was ineffective. The mechanism of the activation of the enzyme by phosphatidylserine and phosphatidylglycerol involved an increase in the apparent Vmax of the enzyme, while the inhibition by phosphatidylinositol 4-phosphate was associated with an increase in the Km of the enzyme for substrate. The phospholipid modulators of low-Km cyclic AMP phosphodiesterase activity did not alter the activity of high-Km cyclic AMP phosphodiesterase. The ability of phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase in native membranes suggests a possible role for phospholipids in metabolic regulation.     

Communication of gender from human breath odors: Relationship to perceived intensity and pleasantness

During five consecutive daily test sessions, 10 men and women rated the relative intensity and pleasantness of breath odors from 14 males and 19 females on a no-oral-hygiene regimen. In addition, the likely gender of the donor of each odor was also estimated. The breath odors of males were rated, on the average, as more intense and less pleasant than the breath odors of females. Women consistently gave lower pleasantness ratings to the odors than did men. Both the male and female judges assigned the breath odors to the correct gender classes at a frequency unlikely due to chance, although the females were more accurate in this regard. An inverse relation between breath odor intensity and pleasantness was noted. Systematic changes in the rated intensity and pleasantness of the odors were present across the 5 days of the study period. These data suggest that differences exist between the breath odors of men and women, and that humans, like many other mammals, may be capable of assessing gender from oral odors. However, such assignments conceivably reflect the strategy of assigning stronger and less pleasant odors to the male category, and weaker and less unpleasant odors to the female category, regardless of the true sex of the odor donor.     

Partial purification from hepatoma cells of an intracellular substance which mediates the effects of insulin on pyruvate dehydrogenase and low Km cyclic AMP phosphodiesterase

A substance capable of stimulating the activities of pyruvate dehydrogenase and low K_m cyclic AMP phosphodiesterase was prepared from H4-II-EC3′ hepatoma cells by acid extraction and partially purified by molecular exclusion chromatography. The material thus prepared by gel chromatography was found to stimulate the activities of these enzymes in a concentration-dependent manner. The amount or activity of the pyruvate dehydrogenase stimulating factor was increased in cells which had been treated with physiological concentrations of insulin (0.2 mU/ml). Increasing the concentration of insulin increased the amount or activity of the factor generated. High concentrations of insulin did not cause a reversal of the effects of insulin. The stimulation of pyruvate dehydrogenase activity by the factor was eliminated when sodium fluoride (75 mm) was present in the enzyme assay, implying that activation was mediated by the pyruvate dehydrogenase phosphatase. The enzyme-stimulating factor isolated from hepatoma cells shares a number of important characteristics with the putative second messenger of insulin prepared from other cell types: (1) it is heat and acid stable, (2) it has a similar apparent molecular weight, (3) it is generated in an insulin-dependent manner, (4) it stimulates the activity of pyruvate dehydrogenase by a fluoride-sensitive mechanism, and (5) it elutes from the anion-exchange resin AG 1-X8 at an ionic strength of 0.4 m. These findings suggest that the stimulator of pyruvate dehydrogenase and of low K_m cyclic AMP phosphodiesterase isolated from hepatoma cells has chemical properties identical with those of the putative second messenger of insulin action isolated from a number of other insulin-sensitive tissues.     

Ongoing DNA synthesis continues in young human diploid cells (HDC) fused to senescent HDC, but entry into S phase is inhibited

Previous experiments have shown that when young human diploid cells (HDC) were fused to senescent HDC, neither nucleus synthesized DNA. This paper demonstrates that when young HDC are in S phase at the time of fusion to senescent HDC, they do make DNA in heterodikaryons; therefore, ongoing DNA synthesis is not inhibited by senescent cells. On the other hand, entry into S phase is inhibited: young HDC nuclei in G1 phase do not make DNA in heterodikaryons with senescent HDC.     

Isolation of myelin basic protein-reactive T-cell lines from normal human blood

T-Cell lines which responded by proliferation to the autoantigen, myelin basic protein (MBP), were isolated from the blood of six of nine normal humans. These T-cell lines could be maintained in in vitro culture for up to 2 months through the use of Interleukin 2 and repeated MBP stimulation. Optimal antigen-induced proliferation required both antigen and antigen-presenting cells found in the adherent cell population of autologous peripheral blood mononuclear cells (PBM). The T-cell lines were predominantly of the helper phenotype (OKT3+, OKT4+, OKT8-) and responded to both human and guinea pig myelin basic protein.     

Azodipyrroles of unconjugated and conjugated bilirubin using diazotized ethyl anthranilate in dimethyl sulfoxide

We have developed a diazotization technique in which both conjugated and unconjugated bilirubin react completely. The method represents a crucial modification of the ethyl anthranilate diazo reaction originally described by K. P. M. Heirwegh, J. Fevery, J. A. T. P. Meuwissen, and J. de Groote (1974, Methods Biochem. Anal.22, 205–250). In the presence of dimethyl sulfoxide (2 ml/ml of sample and diazo reagent), conjugated and unconjugated bilirubin in human serum and human, rat, and mouse bile reacted rapidly and completely. The azopigments were stable for at least 4 h. Addition of human serum to unconjugated bilirubin, bilirubin monoglucuronide, and human bile did not influence azopigment formation. Because the reaction solution was optically clear, total azopigments could be measured by spectrophotometry or separated and quantitated by high-performance liquid chromatography without prior extraction into nonpolar solvents. Alternatively, the pigments could also be extracted into 2-pentanone for analysis by thin-layer or high-performance liquid chromatography. This method allows the quantitation of total bilirubin and analysis of individual ethyl anthranilate azopigments after a single diazotization step.     

Structural characterization of proteoheparan sulfate isolated from plasma membranes of an ascites hepatoma,AH 66

A proteoglycan isolated from plasma membranes of an ascites hepatoma, AH 66, was characterized structurally. The glycosaminoglycan was obtained by alkali treatment and was identified as heparan sulfate. It was essentially the only type of carbohydrate chain attached to the core protein. The identification was based on chemical analysis, electrophoresis, and digestibility with heparitinase from Flavobacterium heparinum. Analysis of neutral sugars of the proteoglycan by mass fragmentography indicated the presence of xylose and galactose which should be involved in the linkage region between a heparan sulfate chain and the core protein. The weight-average molecular weights of the proteoglycan and its heparan sulfate chain were determined to be 71,000 and 21,000, respectively, by meniscus depletion equilibrium centrifugation. The latter value was in good agreement with those obtained by chemical analysis and by gel filtration. From these values for molecular weight and the protein content of the proteoglycan (10.6%), the molecular weight of the core protein was estimated to be 7500. On the basis of these molecular parameters, it was proposed that three heparan sulfate chains on average are linked to the core protein.     

A kinetic study comparing the light-reversal properties of carbon monoxide inhibition of ethylmorphine, benzphetamine, and 7-ethoxycoumarin dealkylation with those of hydroxylation of 17-hydroxyprogesterone and testosterone

The light-reversal properties of carbon monoxide (CO) inhibition of the dealkylation of benzphetamine, ethylmorphine, and 7-ethoxycoumarin by microsomes from phenobarbital (PB)-induced rat livers were compared with those of the 6 beta-, 7 alpha-, and 16 alpha-hydroxylations of testosterone by the same rat hepatic microsomes and C-21 hydroxylation of 17-OH progesterone by steer adrenal microsomes. CO inhibited all reactions studied to essentially the same degree. The significant finding was that the dealkylations were reversed most effectively by light of wavelengths between 440 and 445 nm, rather than around 450 nm, the optimal wavelength for steroid hydroxylations. Moreover, the dealkylations required several-fold higher light intensities for equivalent light reversal. These studies suggest that the heme protein-CO complex responsible for dealkylations has a spectrum corresponding to the shape of the pass band of the 445-nm filter, whereas that of the steroid hydroxylations has its light-reversal maximum at 450 nm and appears to be broader. The measurable differences in the light-reversal properties between the monooxygenations of two groups of substrates, (i) dealkylations and (ii) hydroxylations of lipid substrates, furnish biophysical properties that allow a better characterization of microsomal monooxygenases which should be of value in forwarding progress in the study of these systems.     

Analysis of microtubule polymerization inhibitors in sea urchin egg extracts: Evidence for a protease

Inhibitors of microtubule polymerization have been found in extracts of unfertilized sea urchin eggs using neural tubulin polymerization assays without glycerol. The inhibitory activity is partially destroyed by boiling or by reduction and carboxymethylation and is nondialyzable. When chromatographed on DEAE-cellulose, the inhibitory activity is eluted over a broad NaCl gradient and is in association with several peaks. This partially purified inhibitor is not destroyed by incubation with RNase A. When the partially purified inhibitor is incubated with brain microtubule protein under conditions which support microtubule polymerization, both high molecular weight-microtubule associated proteins and tubulin appear to be digested when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteolytic digestion as well as inhibition of microtubule polymerization depend upon similar concentrations of partially purified inhibitor present in the polymerization reaction. It appears as though at least part of the microtubule polymerization inhibitory activity present in unfertilized sea urchin eggs is due to this protease.     

Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis

Mouse oocytes are reversibly inhibited from resuming meiotic maturation in vitro by cAMP phosphodiesterase inhibitors such as 3-isobutyl-1-methyl xanthine (IBMX) and cAMP analogs such as dibutyryl cAMP (dbcAMP). Oocytes cultured in IBMX-containing medium were transferred to and cultured in IBMX-free medium for various periods of time prior to their return to either IBMX- or dbcAMP-containing medium. Results from these experiments defined a period of time in which oocytes became committed to resuming meiosis. Forskolin, which elevated the intracellular oocyte cAMP concentration, transiently inhibited oocytes from resuming meiosis. Levels of cAMP were determined in oocytes incubated in medium that allows resumption of meiosis. The level of oocyte cAMP decreased significantly during the time in which oocytes become committed to resuming meiosis. This decrease in oocyte cAMP was not observed in oocytes inhibited from resuming meiosis by IBMX. In addition, cAMP levels were determined in preovulatory antral follicles, cumulus cell-oocyte complexes, and oocytes during gonadotropin-induced resumption of meiosis in vivo. A decrease in oocyte cAMP preceded resumption of meiosis as manifested by germinal vesicle breakdown (GVBD). This decrease apparently occurred before or during a period of time in which follicle and cumulus cell cAMP were increasing. Associated with commitment to resume meiosis was a characteristic set of changes in oocyte phosphoprotein metabolism that preceded GVBD. These changes are, to date, some of the first reported biochemical changes that precede GVBD. Results from these experiments are discussed in terms of a possible role cAMP may play in regulation of resumption of meiosis in mammals.     

Phytohemagglutinin-lnduced acquisition of T-cell surface markers by rat bone marrow precursor cells in the absence of the thymic microenvironment

Two monoclonal antibodies specific for different rat T-cell subpopulations, the anti-helper-T-cell antibody, W3/25, and the OX8 suppressor cell antibody were used to investigate lectin-stimulated T-lymphocyte differentiation of F-344 rat bone marrow cells in culture. Cytofluorometric analysis of freshly isolated lymphocytes from thymus and spleen revealed that these tissues contained both W3/25? and OX8-positive populations but differed with respect to the number of cells and receptor density distribution. By contrast, bone marrow-derived lymphocytes exhibited negligible W3/25? or OX8-associated fluorescence. However, several days after stimulation of bone marrow lymphocytes with phytohemagglutinin (PHA), cells appeared bearing these markers. Two-parameter histogram analysis of light scatter measurements with cell surface immunoflu-orescence indicated that this phenomenon represented the appearance of a new population of cells, presumably mature T cells, bearing an increased density of marker. These findings suggest an induction of differentiation of bone marrow T precursor cells by nonthymic factors (PHA) since lymphocytes lacking mature T-cell marker expression developed this characteristic after several days in culture.     

Subcellular fractionation of isolated rat hepatocytes a comparison with liver homogenate

An improved method for the homogenization and the subsequent subcellular fractionation of hepatocytes isolated from adult rat liver is described.The homogenization procedure developed in the present study allows the preservation of the integrity of subcellular structures, as demonstrated by measurement of the activities of representative enzymes as well as by determination of their latency.The activities of representative marker enzymes, as calculated on subcellular fractions obtained by differential centrifugation of the homogenate, are identical whether the homogenate arises from isolated hepatocytes or from the whole liver.Moreover, there is a close similitude between the kinetic parameters (K_{m and} V) of two microsomal cytochrome P₄₅₀-dependent mixed-function oxidases, namely aniline hydroxylase and aminopyrine demethylase determined on microsomal preparations obtained either from isolated cells or from the whole liver.     

Efficient extraction and quantitative determination of nanogram amounts of cellular RNA

An assay for quantitating nanogram amounts of cellular RNA is described. RNA is efficiently extracted from cells, using RNA-free DNA as carrier, by conventional chloroform: phenol procedures and the nucleic acids are precipitated with ethanol. Isolated RNA is hydrolyzed by RNase T2 to ribonucleoside 3′-monophosphates which in turn are converted to 5′-³²P-labeled ribonucleoside 3′,5′-diphosphates in the presence of T4 polynucleotide kinase and [γ-³²P]ATP. Radiolabeled products are separated from remaining [γ-³²P]ATP by chromatography on polyethyleneimine-cellulose, located by autoradiography, excised from the chromatogram, and subjected to liquid scintillation counting to quantitate the amount of RNA. Using mouse liver ribosomal RNA as a standard, the assay is linear over a range of 0 to 64 ng of RNA. The assay has been used to determine the amount of RNA in fully grown mouse oocytes arrested at the dietyate stage of first meiotic prophase. Each oocyte contains 0.61 ± 0.05 ng of RNA and only 25 oocytes have been used for such assays.     

Isolation and chemical characterization of a melanoma-associated proteoglycan antigen

Many melanoma-associated antigens have been identified by monoclonal antibodies. One of these monoclonal antibodies, O₁-94-45, binds only to melanomas, nevus cells, some astrocytomas, and fetal epitheloid cells. There are approximately 100,000 cell surface antigens per melanoma cell with an association constant of 3 × 10⁸m^?1. The antigen is efficiently extracted from the membrane only in the presence of detergent and is, therefore, bound by hydrophobic forces. However, it is also shed into the culture supernatant during normal cell growth. The two components of the O₁-95-45 antigen are a chondroitin sulfate proteoglycan (CSP, >500,000 Da) and a glycoprotein gp260 (260,000 Da, pI 6.9). CSP contains chondroitin sulfate and N-linked and O-linked oligosaccharides. Only N-linked saccharides were associated with gp260. The antigenic site is expressed on both components and is heat-sensitive. Since the CSP was converted to gp260 by chondroitinase, the protein cores of the two molecules are the same or similar. For more detailed study the O₁-95-45 antigen was purified by immunoaffinity chromatography. The amino acid composition of the purified antigen was relatively polar with an unusually high Leu content and low Lys content. Initial attempts to sequence the antigen were unsuccessful probably due to a blocked N-terminus. CSP and gp260 were partially separated by gel filtration chromatography, and both were found to carry the O₁-95-45 antigenic determinant. Three other monoclonal antibodies were found to bind the purified antigen at a site or sites different from the O₁-95-45 epitope and one other monoclonal antibody may bind at the same site. Two of these antibodies were used for a double determinant immunoassay.     

Correlation of biochemical and morphological changes induced by chemical injury to the lung

Comparison has been made of injury to the rat pulmonary alveolar parenchyma evoked by intravenous injection of N-nitrosomethylurethane, intratracheal instillation of 3-methylcholanthrene or repeated inhalation for up to 15 days of carbon tetrachloride, trichloroethylene or gasoline vapour. Biochemical analyses, including assessment of rates of RNA and DNA synthesis and secretion of pulmonary surfactant, were correlated with morphological changes determined by electron microscopy. Single doses of N-nitrosomethylurethane or 3-methylcholanthrene inhibited incorporation of [14C] orotate into lung RNA 1--3 days after treatment. Daily exposure for 30 min to carbon tetrachloride or trichloroethylene vapour caused less marked reduction in orotate incorporation. Ultrastructural examination revealed that 3-methylcholanthrene toxicity was characterised by cytoplasmic change including disruption of surfactant lamellaie of Type 2 pneumocytes and variable degenerative changes Type 1 pneumocytes. Eight to ten days after treatment, the morphological evidence of hypertrophy/hyperplasia and transformation of Type 2 pneumocytes correlated well with biochemical evidence of stimulated incorporation of [3H]thymidine. Inhalation of carbon tetrachloride or trichloroethylene vapour produced milder responses including occasional degenerative changes in Type 1 pneumocytes, reduced numbers of surfactant lamellae in Type 2 pneumocytes and no change in [3H]thymidine incorporation. In contrast to the gradation of injury produced by the various chemicals, all procedures caused a marked and reproducible reduction in secretion of pulmonary surfactant as determined by endobronchial lavage. Following solvent inhalation, reduced recovery of surfactant was detected within 5 days of repeated exposure and thereafter no further change in this depressed level resulted from continued exposure for a further 10 days. The data are discussed in terms of a generalised pattern of response by pulmonary alveolar tissue to chemical injury and the apparent sensitivity of surfactant secretion as an indicator of damage to the lung.     

Migration of recently divided B and T lymphocytes to peritoneum and lung

It is recognized that a population of newly divided (or young) cells migrate preferentially to inflamed foci. It has been shown that a large proportion of lymphocytes residing in the bronchoalveolar airspaces of rat are recently divided cells and that blood may be an important source of these cells. To further delineate how blood may contribute to lymphocyte subpopulations in inflamed peritoneum and lung, a comparison of the capacity of recently divided T and B cells to migrate from blood to inflamed peritoneum and lung was made. To label young lymphocytes, DA strain donor rats were given Initiated thymidine by vein in vivo for 7 days. After thoracic duct drainage, the following labeled cell populations were adoptively transferred by vein into syngeneic recipients: (i) unseparated thoracic duct lymphocytes (TDL), (ii) enriched T cells (>90%) or B cells (>80%) recovered after passage of TDL through nylon columns, and (iii) thoracic duct lymphocytes (> 99% B cells) obtained from “B rats” that were prepared by X irradiation, thymectomy, and bone marrow reconstitution. T and B cells were identified by specific heterologous antisera. The percentage recovery of labeled lymphocytes in the recipients with inflamed peritoneum or lung aspirates was determined from cell counts and autoradiographs. The studies indicated that (a) both labeled T and B cells migrated to inflamed peritoneum and lung; (b) labeled B cells migrated to peritoneum and lung better than did labeled TDL or T cells; and (c) labeled lymphocytes did not migrate to unstimulated peritoneum. The enhanced migration of newly divided B lymphocytes to inflamed peritoneum and normal lung (a site that is likely under chronic antigenic stimulation) was unexpected, but may provide additional information on the relative contribution of these subpopulations in the immune inflammatory response.     

Morphological and biochemical characterization of light and heavy myelin isolated from developing rat brain

Myelin from developing rat brains was separated on a discontinuous sucrose gradient into subfractions of two different densities, i.e. light and heavy myelin. Electron photomicrographs showed that heavy myelin consisted primarily of large compacted multilamellar structures with a distinct intraperiod line characteristic of myelin in situ. Light myelin, on the other hand, was composed of small vesicles having a unilamellar structure. Similar to whole myelin, both membrane subfractions were highly enriched in 2′,3′-cyclic nucleotide-3′-phosphohydrolase. The specific activity of the enzyme, however, showed no developmental trend. Both subfractions contained all of the four major proteins characteristic of the whole myelin membrane. There were, however, quantitative differences in the relative distribution of these proteins between light and heavy myelin. Basic protein accounted for 55 % and proteolipid protein for 46 % of the total myelin proteins of light and heavy myelin, respectively. DM-20 (Agrawal, H. C., Burton, R. M., Fishman, M. A., Mitchell, R. F. and Prensky, A. L. (1972) J. Neurochem. 19, 2083–2089) exhibited a developmental “switch” between light and heavy myelin. Light myelin appeared to contain more DM-20 in 15- to 20-day-old rat brain, whereas the concentration of this protein was higher in heavy myelin at subsequent ages studied.     

Biochemical and morphological characterization of a plasma membrane-enriched fraction from bovine parathyroid cells

A fraction of enriched plasma membranes from bovine parathyroid cells has been prepared by differential centrifugation. Biochemical characterization shows that this fraction has a specific activity enrichment of 7.2-fold in ouabain-sensitive Na+-K+ ATPase, and 3.5-fold in 5'-nucleotidase. Less than 4% of the total mitochondria and lysosomes are present within the plasma membranes, while microsomal contamination accounts for 14% of total specific activity. Parathyroid hormone radioimmunoassay also reveals the presence of some secretory granules within the plasma membrane fraction. The characteristic morphological aspect of the unusual surface membrane is shown by freeze-fracture electron microscopy. In the enriched pellets, vesicles identified as having a plasma membrane origin have variable sizes, and 50% show an inside-out conformation. Even though the plasma membrane fraction described herein is not absolutely free from contamination by other subcellular components, this protocol represents the first attempt to purify surface membrane from parathyroid tissue and provide the starting material for understanding, at a molecular level, the properties of extracellular Ca2+ regulation and its coupling with secretion of parathyroid hormone.     

Specific binding and pharmacological interactions of apamin,the neurotoxin from bee venom,with guinea pig colon

This paper describes the interaction of apamin, the bee venom neurotoxin, with its receptor in the guinea pig colon. The pharmacological activity of the toxin was assayed by measuring its contracting effect on guinea pig colon preparations that had been previously relaxed by neurotensin. The IC₅₀ value of apamin in this $\text{in vitro}$ bioassay is 7 nM. These pharmacological data are compared to the binding properties of apamin to smooth muscle membranes prepared from guinea pig colon. The highly radiolabeled monoiododerivative of apamin binds to its colon receptor with a dissociation constant $\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}^1\text{= 36}\text{pM}$. The maximal binding capacity of colonic membranes is 30^dfmol/mg of protein. The dissociation constant of the unmodified toxin is 23 pM. The difference between the toxin concentrations that produce half-maximal effects in the binding and pharmacological studies arises from the different experimental conditions used for the two assays.