Identification and quantitation of cortisol metabolites in rat liver homogenate and -slices

Summary Cortisol-1, 2-H³ was incubated with rat liver homogenate and/or rat liver slices in the presence of a NADPH-generating system. The following metabolites could be identified in adult male rats: -cortol, allo--cortol, 3-allo--cortol, 20-hydroxy-cortisol, 11, 17, 20, 21-tetrahydroxy-5-pregnan-3-one, 3-allotetrahydrocortisol, tetrahydrocortisol, trace amounts of allotetrahydrocortisol and two highly polar metabolites only partly identified. In female rats only tetrahydrocortisol, allotetrahydrocortisol and allodihydrocortisol could be detected in significant amounts.The radioactive metabolites mentioned above were localized and quantitated on paper chromatograms by a 4-radiochromatogram scanner. A nearly perfect correlation was found between these results so obtained and those given by liquid-scintillation counting of each metabolite after its elution from the paper.Part of this work was supported by grant n° 695 of the National Fonds voor Wetenschappelijk Geneeskundig Onderzoek.Stagiair of the Nationaal Fonds voor Wetenschappelijk Onderzoek.     

Differential expression of β1, β3 and β4 integrins in sarcomas of the small,round, blue cell category

Integrins are a large and complex family of membrane spanning heterodimeric cell surface glycoproteins mediating cell/cell and cell/matrix interactions. Small, round, blue cell sarcomas (SRBCS) are a group of poorly differentiated tumours of various and in part uncertain histogenesis displaying similar cytomorphology. Among them are rhabdomyosarcomas (RMS), ganglioneuroblastomas [(G)NB], primitive peripheral neuroectodermal tumours (pPNET) and Ewing's sarcomas (ES). Thirty-two SRBCS were studied immunohistochemically for the distribution of 1, 3 and 4 integrins in situ. We found complex and to some extent differential patterns of 1, 3 and 4 integrin subunit expression in different types of SRBCS: all of the sarcomas studied were consistently 1⁺, 4^–, 2^–. Four of nine RMS were completely negative for all other integrin subunits studied while one RMS was 5⁺ throughout and three RMS were focally 5⁺. Three RMS expressed the 6 and v chains. In contrast to RMS, pPNET and ES, all of which were 1^–, 3^–, (G)NB were 3⁺ and frequently co-expressed 1. The eight pPNET and seven ES studied showed a similarily restricted integrin profile that was limited to the expression of 1 and 5 in nearly all cases. In summary, RMS were 1⁺, 1^–, 3^– and heterogeneously expressed 5 and 6. (G)NB were generally 1⁺, 1⁺, 3⁺, 5^–, 6^–. pPNET and ES were 1⁺, 1^–, 3^–, 5⁺, 6^–. The data illustrate a complex expression pattern of various integrins in SRBCS, a differential expression pattern of some of the integrin subunits among different types of SRBCS and almost identical integrin profiles in pPNET and ES.This paper is dedicated to Prof. Dr. Dres. h.c. Wilhelm Doerr on the occasion of his 80^th birthday     

Glucocorticoids modulate TGF-beta production

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGB- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFL-1) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TFG-1, -2, and -3. TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TGF-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and -2 production and reduced expression of the upregulated TGF-1 and -2 mRNA induced by exogenous TGF-1, -2 or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Phenotypic characteristics of lewis lung carcinoma cells

We studied adhesive properties and physiological activity in vivo of cells from Lewis lung carcinoma and its metastases. These cells differed in tumorogenic activity and metastatic potential in the syngeneic system. In vivo non-metastasizing cells are characterized by a lower content of surface lectins to tetrasaccharides SiaLe^x [Neu5Ac2-3Gal1-4(Fuc1-3) GlcNAc] and SiaLe^a [Neu5Ac2-3Gal1-3(Fuc1-4)GlcNAc] and trisaccharide HSO₃Le^x [HSO₃2-3Gal1-4(Fuc1-3)GlcNAc] compared to cells forming metastases in the syngeneic system. Metastatic cells with low tumorogenic activity weakly expressed lectins to disaccharide ligands 6-SiaLac [Neu5Ac2-6Gal1-4Glc], 6-HSO₃LacNAc, and A-di [GalNAc 1-3Gal] and trisaccharides H-type 1 [Fuc1-2Gal1-3GlcNAc and Le^x [Fuc1-3(Gal 1-4)GlcNAc] compared to cells that initiated tumor formation in the syngeneic system (similarly to transplanted tumors). We hypothesized that cell receptors to these carbohydrate determinates are involved in the development and growth of primary tumors, while lectins to SiaLe^x, SiaLe^a, and HSO₃Le^x play a role in the progress of tumor process and metastasizing.     

The independent expression of HLA andβ 2-microglobulin on human-mouse hybrids

Direct cytotoxicity and absorption analysis were used to detect HLA antigens on man/mouse somatic cell hybrids. Confirmation of the assignment of the Major Histocompatibility Complex to chromosome 6 was obtained. The same series of hybrids were used to study the interaction between HLA and ₂m on the cell surface. Results obtained from a series of clones indicated that there was complete independence of expression of HLA and ₂m. Human ₂m was found to be unnecessary for the normal serological expression of HLA, suggesting that mouse ₂m could possibly replace human ₂m as a subunit of the HLA molecule. Evidence is presented demonstrating that this is in fact the case. Heteroantisera to human ₂m and alloantiserum to HLA-A2 were used, together with complement, to selectively remove the chromosome coding for ₂m and HLA from a hybrid clone. Manipulation of the karyotype in this way further confirmed the independence of HLA and ₂m on somatic cell hybrids.     

The effects of β-2-microglobulin on the infectivity of murine cytomegalovirus

Summary The role of -2-microglobulin (2m) in murine cytomegalovirus (MCMV) infection of susceptible (H-2^d) and resistant (H-2^k) murine embryo fibroblasts (MEF) and peritoneal macrophages was evaluated using serum-free virus (SF-MCMV). The infectivity of SF-MCMV was significantly lower than virus propagated in serum, although the concentrations of virions were similar. Infection of cells with SF-MCMV was assessed by measuring the proportion of cells expressing viral antigens, the sizes of plaques formed in fibroblast monolayers and TCID₅₀ titers. Infection of susceptible fibroblasts was significantly increased 1.6–4.7 fold by the addition of whole FCS, a<20 kDa FCS fraction, or purified human 2m. These supplements also significantly enhanced infection of susceptible macrophages and increased TCID₅₀ titers by 3.5–10 fold in susceptible MEF. In relatively resistant H-2^k cells, the TCID₅₀ titer and the proportion of cells expressing viral antigens after infection with SF-MCMV were not affected by 2m or FCS, but plaque sizes were increased 2.5–3 fold in resistant BALB.K MEF.When human or murine 2m was added to infected cultures, immunogold electron microscopy revealed these proteins to be always associated extracellularly with the tegument material of disrupted multicapsid virions, but rarely with the envelope of intact virions. However, no murine 2m was found in association with the envelope or tegument of SF-MCMV. These relatively modest effects of 2m which were restricted to genetically susceptible cells, may be due to tegument-bound 2m facilitating infection by capsids, or the stabilisation of the conformation of Class 1 molecules by exogenous 2m, promoting MCMV binding to the target cell.     

Thymosin β15 expression in tumor cell lines with varying metastatic potential

We previously isolated thymosin 15 from highly metastatic Dunning rat prostatic carcinoma cells. Immunohistochemical study of human prostate cancer specimens revealed a general correlation between Gleason grade and thymosin 15 expression, with high-grade (more malignant) tumors showing increased staining compared to low-grade tumors. To determine whether thymosin 15 may be differentially expressed in cancer cells with different metastatic potential other than in the prostatic carcinoma cells, we examined thymosin 15 mRNA levels in tumor cell lines from different species. We also examined thymosin 15 protein levels in human breast cancer samples. Thymosin 15 was upregulated in the highly metastatic mouse lung and human breast cancer cell lines in comparison to the nonmetastatic counterparts. Immunohistochemical staining showed the evidence of upregulation of thymosin 15 in malignant human breast carcinomas as compared to benign breast tumors. The expression of thymosin b1 5 was correlated with the metastatic potential of the mouse lung carcinoma and human breast carcinoma cells in addition to the prostatic carcinomas. Thymosin 15 may be a useful marker to predict metastatic potential of certain human cancers. © Rapid Science 1998     

Glucocorticoids modulate TGF-beta production by human fetal lung fibroblasts

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGF- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFLF) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TGF-1, -2, or -3. Post-culture media were collected for ELISA assays of TGF-1, -2, and -3 . TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TFG-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and TGF-2 production and reduced expression of the up-regulated TGF-1 and TGF-2 mRNA induced by exogenous TGF-1, -2, or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Distribution of skeletofusimotor axons in lumbrical muscles of the monkey

Summary The nerve supply to 25 poles of muscle spindles in the monkey was reconstructed by light microscopy of serial 1-m thick transverse sections of lumbrical muscles. Twenty of 60 motor axons that supplied the spindle poles were identified as skeletofusimotor (). Twenty-eight percent of the spindle poles were innervated by axons, in addition to axons. Every -innervated spindle pole transected an endplate zone of extrafusal muscle. Most axons coinnervated extrafusal fibers rich in mitochondria and the nuclear bag₁ or nuclear chain intrafusal fibers. All but two axons innervated one type of intrafusal fiber only. The intramuscular organization of motor system in lumbrical muscles of the monkey was similar to that of the cat tenuissimus muscle. The function of -innervated spindles may be preferentially to monitor mechanical disturbances arising from the activity of extrafusal muscle units with which they share motor innervation.     

In situ expression of β1, β3 and β4 integrin subunits in non-neoplastic endothelium and vascular tumours

Endothelial cells play an important role in adhesive interactions between circulating cells and extracellular matrix proteins. In vitro studies have shown that many of these processes are mediated by a superfamily of heterodimeric transmembrane glycoproteins called integrins. The distribution patterns of 1, 3 and 4 integrin subunits in endothelial cells (EC) in situ were examined immunohistochemically on serial forzen sections of a wide range of non-neoplastic tissues and of vascular tumours, both benign and malignant. Expression of the 1 subunit was a constitutive feature of EC. Among the 1-associated subunits, 5 and 6 were broadly distributed in EC, irrespective of vessel size and microenvironment. The 3 subunit displayed intermediate levels of expression with a slight preference for small vessel EC. Presence of 1 was confined to EC of capillaries and venules/small veins. Expression of 2 in EC was inconsistent. With rare exceptions, the 4 chain was absent in EC. The 3 and v subunits were expressed in most EC, though not always concomitantly. In contrast to the 1 chain, however, these integrin subunits were absent in EC of glomerular capillaries and were expressed variably in sinusoidal EC. The 4 chain was evenly present in the great majority of EC, except for those of large vessels. In vascular tumours, the patterns of 1 and 1 to 6 subunit expression generally corresponded to those found in their non-neoplastic counterparts. Expression of 3, v and 4 chains, however, decreased in neoplasia, especially in angiosarcomas. These data show that EC dispose of broad and at the same time differential repertoires of integrin subunits that presumably reflect vessel-type associated functional differences among these cells. In vascular tumours, the orthologous distribution patterns of 1 and 1 to 6 chains are conserved in most instances while the amounts of 3, v and 4 subunits expressed in EC tend to decrease in the course of malignant transformation.Dedicated to Prof. Dr. med. Dres. h.c. Wilhelm Doerr on the occasion of his 80th birthday     

Die Ausscheidung von β-Aminoisobuttersäure bei Gesunden und Kranken

Zusammenfassung 1. Bei größerer interindividueller Schwankung zeigt die Ausscheidung von -AIB im Urin eine erstaunliche intraindividuelle Konstanz.2. Untersuchungen von Leberkranken ergaben, daß der Funktionszustand der Leber keinen wesentlichen Einfluß auf die Ausscheidung von -AIB im Urin hat.3. Die Hälfte der untersuchten Patienten mit Hämochromatose scheidet größere Mengen dieser Aminosäure aus.4. Der bei der Ahorn-Sirup-Krankheit auftretende Enzymdefekt (Ausfall der -Ketosäureoxydasen) hat keinen Einfluß auf die Menge der eliminierten -AIB.

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Summary 1. -AIB excretion varies greatly from individual to individual, whereas normal day to day differences in -AIB excretion rates for any one person appear to remain constant.2. Impaired liver function does not markedly alter urinary -AIB excretion.3. Half of our patients with hemochromatosis excrete large amounts of -AIB.4. Impaired activities of -ketoacid-oxydases in maple syrup urine disease have no apparent effect on -AIB excretion.

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Abkürzungen -AIB -Aminoisobuttersäure - DNFB 2,4 Dinitro-1-Fluorobenzol     

Changes in the serum proteins of rats with radiation sickness

Summary Immunoelectrophoretic analysis of serum proteins in rats of the August strain irradiated with -rays in a dose of 630 R showed an increase in the heterogenity of ₂B- and ₂B-globulins with the appearance of electrophoretically faster or slower fractions retaining (₃B) or slightly changing (₂B) their antigenic properties. Beginning with the 7th day after irradiation the serum of irradiated animals is found to contain a new radiation antigen migrating during electrophoresis together with slow ₂-globulins and indicated by ₂x-protein. The protein fails to appear after irradiation of sera in vitro and is absent both in healthy and artificially infected nonirradiated animals.(Presented by Active Member of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 62, No. 10, pp. 57–61, October, 1966.     

Transforming growth factor β upregulates the integrin-mediated adhesion of human prostatic carcinoma cells to type I collagen

Prostate cancer frequently metastasizes to bone, and we propose that this process may be facilitated by the adhesion of metastatic cells to bone-derived type I collagen. We examined collagen receptor function and regulation in osteotropic PC-3 human prostatic carcinoma cells. PC-3 cell adhesion to immobilized human type I collagen was promoted by Mn and Mg ions and was RGD-independent. Antibodies directed against 1 or 2 integrin subunits inhibited adhesion to collagen by 90% and 53%, respectively, suggesting involvement of the 21 receptor. Anti-1 or anti-3 antibodies had no effect on adhesion. Flow cytometry and immunoprecipitation of [S]methionine-labeled cells demonstrated that 21 was the major collagen receptor expressed by PC-3 cells. The pretreatment of PC-3 cells with transforming growth factor-1 (TGF-1), a major bone-derived growth factor, caused a rapid (2 h) 2-fold increase in the de novo synthesis of 2 and 1 integrin subunits, and also increased by 2- to 3-fold the adhesion and spreading of PC-3 cells on collagen. We conclude that 21 is the major collagen receptor employed by PC-3 cells, and that 21 upregulation by TGF- is associated with an increased adhesion and spreading on collagen. The data suggest that exposure of metastatic PC-3 cells to the high levels of TGF- in bone may promote their ability to adhere to bone-derived collagen, which may thereby facilitate the localization of metastatic cells in the skeleton.     

Mapping thyrotropin β subunit gene in man and mouse

Thyrotropin (TSH) is composed of two subunits: and . Previously, we have mapped the TSH gene to human chromosome 6 and mouse chromosome 4. In this study we have located the human TSH gene on chromosome 1 and the mouse TSH gene to chromosome 3. These data suggest that the TSH gene lies in a conserved linkage group with the genes for amylase 1 and 2, nerve growth factor, and the protooncogene Nras.     

Yeast artificial chromosome cloning of the β-catenin locus on human chromosome 3p21–22

-Catenin has emerged as an important component of the adherens junctions between epithelial cells. As a result of studies of its interaction with theAPC gene product, it has been implicated in the development of colorectal cancer. -Catenin, -catenin, E-cadherin and APC appear to mediate contact inhibition in epithelia. As part of the study of the organization of the -catenin gene, we have isolated yeast artificial chromosomes (YACs) to characterize its intron/exon structure. YAC fluorescencein situ hybridization analysis and polymerase chain reaction analysis of somatic cell hybrid DNAs show that -catenin maps in the 3p21–22 region, the location of tumour-suppressor genes deleted in small-cell lung cancer (SCLC) and other disorders. -Catenin YACs will provide a source of microsatellite markers useful in loss of heterozygosity studies to assess the importance of -catenin deletions in SCLC.     

Evidence for a multigenic system controlling methyl-β-carboline-3-carboxylate (β-CCM)-induced seizures

-CCM is a -carboline known to have properties opposite to those of benzodiazepines. Our approach was to analyze, in mice, the genetic mechanisms involved in -CCM-induced myoclonic seizures using recombinant congenic strains and F₁ hybrids issued from these strains. Our aim was to define the extent of the multigenic character of -CCM-induced myoclonic seizures, while also evaluating the distribution of the strength of the genes implicated in this trait. The results show that the control of reactivity to -CCM is multigenic with notable epistatic involvement.     

Humanβ:α but not γ interferon binding site is a product of the chromosome 21 interferon action gene

The binding of human interferons to their binding site(s) was measured by the amount of radioiodinated human beta Interferon (HuIFN) displaceable by unlabeled human beta, alpha, and gamma Interferon (HuIFN, , and ). By this approach, HuIFN and HuIFN were found to interact with specific binding sites in cell membranes derived from human cells and mouse-human cell hybrids containing chromosome 21 as their only human chromosome. Specific binding was not observed with cell membranes derived from parental mouse cells or from mouse-human cell hybrids in subsequent generations that have lost human chromosome 21. Although the chromosome 21-positive mouse-human cell hybrids are sensitive to the antiviral effects of HuIFN and HuIFN, they are found to be insensitive to the antiviral effect of HuIFN and to lack specific HuIFN binding sites. These results suggest that the HuIFN and HuIFN but not HuIFN binding sites are coded for by genes located on chromosome 21. The lack of a chromosome 21 gene dosage effect on the inducibility of the antiviral state by HuIFN is consistent with this hypothesis.     

Loss of beta1-integrin-deficient cells during the development of endoderm-derived epithelia

1-Integrins (1) represent cell surface receptors which mediate cell–matrix and cell–cell interactions. Fässler and Meyer described chimeric mice containing transgenic cells that express the LacZ gene instead of the 1 gene. They observed 1-negative cells in all germ layers at embryonic day E 8.5. Later in development, using a glucose phosphate isomerase assay of homogenized tissue samples, high levels of transgenic cells were found in skeletal muscle and gut, low levels in lung, heart, and kidney and none in the liver and spleen (Fässler and Meyer 1995). In order to study which cell types require 1 during development of the primitive gut including its derivatives, chimeric fetuses containing 15 to 25% transgenic cells were obtained at days E 14.5 and E 15.5. They were LacZ (-galactosidase) stained en bloc and cross-sectioned head to tail. In esophagus, trachea, lung, stomach, hindgut, and the future urinary bladder, we observed various mesoderm-derived 1-negative cells (e.g. fibroblasts, chondrocytes, endothelial cells, and smooth muscle cells) but no 1-negative epithelial cells. Since the epithelia of lung, esophagus, trachea, stomach, hindgut, and urinary bladder are derived from the endodermal gut tube, we hypothesize that 1 is essential for the development and/or survival of the epithelia of the fore- and hindgut and its derivatives.     

Effect of recombinant mouse interferon-β on acute and latent herpes simplex infection in mice

Summary The antiviral effect of recombinant mouse interferon-(rMuIFN-) on herpes simplex virus type 1 (HSV-1) in experimentally infected mice was examined at several stages of infection as a model for the treatment of human HSV infection. Recombinant MuIFN- protected mice from lethal intraperitoneal challenge with virulent HSV-1 strains. The in vitro reactivation of HSV from latently infected trigeminal ganglia was also suppressed by treatment with rMuIFN-. Thus, rMuIFN- was effective against HSV-1 during acute infection and during in vitro reactivation of latent HSV. However, rMuIFN- was not effective in preventing the establishment of latent infection, or in eliminating a previously established latent infection.     

Histochemischer Nachweis der β-Glucuronidase (EC 3.2.1.31) und β-Acetylglucosaminidase (EC 3.2.1.30) in der Rattenleber nach Gabe von 1-Naphthyl-Isothiocyanat und D-Galaktosamin-Hydrochlorid

Zusammenfassung In der vorliegenden Arbeit konnte gezeigt werden, daß die histochemisch nachweisbaren Reaktionsprodukte der-Glucuronidase und-Acetylglucosaminidase der Rattenleber nach Gabe von 1-Naphthyl-isothiocyanat und D-Galaktosamin-HCl zunehmen. Die-Glucuronidase ist vorwiegend in den Kupfferschen Sternzellen nachweisbar.

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Summary In this study the authors, for the first time, could demonstrate by histochemical methods that the activities of-glucuronidase and-acetyl glucosaminidase in rat liver increase in animals which are pretreated with 1-naphthyl-isothiocyanate and galactosamine. Best reaction of -glucuronidase was found in Kupffer cells.