Modulation of cyclic AMP-dependent protein kinase isozyme expression associated with activation of a macrophage cell line

We have used the RAW 264.7 macrophage (MO) cell line to study cAMPdPK isozymes during activation by lymphokine (LK) and lipopolysaccharide (LPS). Untreated cells were found to have two isozymes of cAMPdPK in their cytosol. PKI and PKII were differentiated based on the Mr of their regulatory subunits (RI, 45,500; and RII, 52,000, respectively) as determined by photoactivated incorporation of the cAMP analog 8-N3-[32P]cAMP. Loss of the RI subunit of PKI occurred in association with activation of the cell line by suboptimal concentrations of LK and LPS (1/40 dilution, 1 ng/ml) or high concentrations of LPS alone (10 ng/ml to 100 micrograms/ml). No modulation of the RII subunit of PKII was observed under these conditions. The loss of RI was dependent on the addition of a triggering signal to the MO. Treatment of RAW 264.7 cells with LK alone at dilutions from 1/10 to 1/1280 was not sufficient to cause a disappearance of the RI subunit from the cytosol or to induce antitumor activity. The addition of a suboptimal concentration of LPS after LK or a high dose of LPS alone was required for acquisition of cytolytic activity and loss of RI. The kinetics for the disappearance of RI from treated cells were found to be identical after activation with either LK and LPS or high concentrations of LPS alone. RI could no longer be detected in the cytosol 8 hr after the addition of activating agents. The antitumor activity of the RAW 264.7 cell line was transiently expressed after activation. Cells no longer exhibited tumoricidal activity 48 hr after the removal of activating agents. It was observed that the loss of cytolytic function was accompanied by the reexpression of RI in the cytosol. This study provides evidence that modulation of cAMPdPK isozymes occurs during activation, suggesting a potential mechanism for controlling the effects of cAMP on the MO.     

Biosynthesis of the sulfatide/GM1 activator protein (SAP-1) in control and mutant cultured skin fibroblasts

Sphingolipid activator proteins (SAP) are relatively low-molecular-mass proteins that stimulate the hydrolysis of specific sphingolipids by the required lysosomal enzymes. SAP-1 or sulfatide/GM1 ganglioside activator protein has previously been demonstrated to stimulate the enzymatic hydrolysis of sulfatide, GM1 ganglioside and globotriaosylceramide. Using monospecific rabbit antibodies against human liver sulfatide/GM1 activator, the biosynthesis and processing of this activator were studied in cultured skin fibroblasts from controls and patients with GM1 gangliosidosis and a variant form of metachromatic leukodystrophy. When [35S]methionine was presented in the medium to control human fibroblasts for 4 h, the majority of the immunoprecipitable radiolabeling was confined to bands within three regions of apparent molecular mass 65-70, 35-52 and 8-13 kDa. The only immunoprecipitable radiolabeled species excreted into the medium when NH4Cl was present had an apparent molecular mass of 70 kDa. When the excretion products were given to fresh cells followed by incubation for up to 24 h there was production of the mature species. Treatment of the 70 kDa form with endoglycosidase F resulted in production of a 53 kDa molecular mass form. Pulse-chase experiments indicated that the initial immunoprecipitable translation product was 65 kDa which increased to 70 kDa over the next hour. The 65 kDa species must result from co-translational glycosylation of the polypeptide chain. Apparently, intralysosomal processing converts the 13 kDa form to the 8-11 kDa species. The cells from the patient with GM1 gangliosidosis could not process to the smallest species found in controls due to the deficiency of acid beta-galactosidase. Patients who have a variant form of metachromatic leukodystrophy do not make any immunoprecipitable radiolabeled products in the cells or in the media. This indicates a severe mutation in the gene coding for this activator protein. The production of such small mature species from a relatively large precursor form may regulate the production of this interesting protein.     

Type C Niemann-Pick disease. A parallel loss of regulatory responses in both the uptake and esterification of low density lipoprotein-derived cholesterol in cultured fibroblasts

Low density lipoprotein (LDL) internalization by mutant type C Niemann-Pick (NPC) fibroblasts results in uptake of excess total cholesterol. Uptake of excess lipoprotein cholesterol appears to be mediated by the specific LDL receptor pathway. Associated with excessive LDL-cholesterol uptake is a lesion in early intracellular cholesteryl ester synthesis. In vitro acylCoA:cholesterol acyltransferase activity is normal in cell-free extracts of mutant cells. The ability of exogenous sterols to enhance intracellular esterification of [3H]mevalonate-derived [3H]cholesterol was severely limited in mutant cell cultures suggesting that in vivo activation and/or expression of activated acylCoA:cholesterol acyltransferase may be compromised by the primary mutation of type C Niemann-Pick disease. After 2 days of LDL uptake, rates of intracellular cholesteryl ester synthesis in mutant cells paralleled the rates of esterification in normal cells suggesting that specific early in vivo expression of the acyltransferase may be affected in this disorder.     

Beitrag zur Anatomie,Statik und Mechanik der Wirbelsäule des Pferdes mit besonderer Berücksichtigung der Zwischenwirbelscheiben

Ohne ZusammenfassungDamit bin ich am Schlusse meiner Arbeit angelangt. Mit Freude erfülle ich meine Dankespflicht gegen alle, die mich in meinen Untersuchungen förderten und unterstützten: Herrn Prof.Rubeli für die freundliche überlassung dieses Themas und für das allzeit rege Interesse, das er meiner Arbeit angedeihen ließ. Herrn Prof.Strasser sei der herzliche Dank für manche wertvolle Ratschläge und Belehrungen betreffend mechanische Fragen, wie auch für die mir zur Verfügung gestellte Wirbelmeßlade wiederholt. Herrn Prof.Noyer, Herrn Prof.Studer, wie auch Herrn Prof.Rubeli für das mir in zuvorkommender Weise überlassene Material. Herzlichen Dank schulde ich ferner Herrn P.-D. Dr.Ries für die mikrophotographische und die kinematographischen Aufnahmen. Ebenso danke ich der Firma A. G.Fritz Marti in Bern, Fabrik landwirtschaftlicher Maschinen und Geräte, für den mir bereitwillig zur Verfügung gestelltenSackschen Kraftmesser.     

Human platelet basic protein/connective tissue activating peptide-III maps in a gene cluster on chromosome 4q12–q13 along with other genes of the β-thromboglobulin superfamily

Summary Pro-platelet basic protein (pro-PBP) is the precursor of the two platelet -granule proteins, PBP and connective tissue activating peptide-III. Upon platelet activations they are released and further processed in plasma to -thromboglobulin and neutrophil-activating peptide-2. The gene encoding pro-PBP is mapped in this study to chromosome 4q12–q13. At least four other members of this family of small inducible cytokines, including NAP-1/Il-8 and platelet factor 4, reside within the same locus, indicating a gene cluster for the -thromboglobulin family.     

DNA containing non-nucleosidic phenanthrene building blocks with asymmetrical linkers

The synthesis and hybridization properties of oligonucleotides containing phenanthrene building blocks with non-nucleosidic linkers of different length are described. It was found that the length of the linkers, as well as the combination of unequal linkers can have a substantial influence on the thermal stability of the modified DNA.     

Indoor intake fraction considering surface sorption of air organic compounds for life cycle assessment

Purpose

Life cycle assessment (LCA) has largely focused on characterizing the impact of outdoor emissions. However, the intake fraction (iF) of indoor air emissions could be more important. The present paper aims to determine the long-term intake fractions of indoor emissions, including multiple indoor removal pathways such as sorption on indoor surfaces, and to compare it to the outdoor intake fraction.

Method

The developed model accounts for the different removal pathways in buildings, including air exchange, degradation in the gas phase, degradation on surfaces, and finally partitioning between air, walls, and furniture assuming a kinetically limited material transfer between gas phase and a near-surface film. The indoor intake fraction is presented as a function of the adsorption and degradation rate on surfaces.

Results and discussion

The intake fraction of volatile substances is only affected by the ventilation rate, with a constant intake fraction of 1?×?10^?2. For ozone-sensitive substances, indoor gas phase reactions can significantly reduce the intake fraction. Semi-volatile substances are affected by the adsorption and degradation on room surfaces. For highly adsorbing substances, the decrease in intake fraction is limited to a minimum value of 2.5?×?10^?4 by the mass transfer rate between air and room surfaces for a typical office or residence room in developed countries with temperate climate. Indoor intake fraction is compared to outdoor intake fraction calculated using the Impact 2002 multimedia model. Typical calculated indoor intake fraction values are in a significantly higher range (2.5?×?10^?4 to 1?×?10^?2) than inhalation outdoor values (1?×?10^?9 to 1?×?10^?6).

Conclusions

This paper opens new possibilities to assess the health impact of indoor and outdoor air emissions in a consistent way, including surface sorption??a major removal pathway for semi-volatile compounds. By combining the newly calculated intake fractions with effect factors and with indoor and outdoor emissions per functional unit, it becomes possible to consistently account for indoor exposure in methods such as LCA