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     

Immunohistochemical studies on S-100 cells in the anterior pituitary gland of Sprague Dawley rats and spontaneous dwarf rats

The S-100 cells in the pituitary glands of adult male Sprague Dawley rats (SDs) and spontaneous dwarf rats (SDRs) were immunohistochemically examined using anti-S-100 and anti-S-100 monoclonal antibodies. The immunoreactive cells against S-100 protein were divided into three subtypes on the basis of their immunore-activity against subunits of S-100 protein: S-100 dominant type (the -type cell), S-100 dominant type (the \-type cell) and immunoreactive against both S-100 and S-100 (the -type cell). In the SD, -type cells represented 26% of the total S-100 immunoreactive cells (S-100 cells) and were localized in the peripheral area of the ventral region of the pituitary gland. This type of cell was observed forming clusters, with more abundant cytoplasm than the -type cell. The proportion of -type cells was 53%. They were diffusely distributed throughout the gland, and their processes were thicker than those of the -type cell. In the SDR, the proportion of -type cells was 55%, and they were observed throughout the gland. In contrast, -type cells totalled 12% and were localized in small areas of the central and peripheral region of the gland. The proportion of -type cells was 21% in the SD and 33% in the SDR and they were observed forming small clusters in both animal groups. The proportion of -type cells compared with the total of S-100-immunoreactive cells was significantly higher (P < 0.05) in the SDR than in the SD, while the proportion of -type cells was markedly lower (P < 0.05).     

Harnsteroidprofile bei Cushing Syndrom und Tumoren der Nebennierenrinde

Summary The analysis of 24-h excretion profiles of urinary steroids in 18 patients suffering from Cushing's syndrome or adrenocortical tumors revealed typical patterns when compared to 37 healthy control persons, 24 patients with obesity, and 6 patients with hirsutism. The validation of eight criteria — increased excretion of free cortisol, 6-hydroxycortisol, 20-dihydrocortisol, 11-hydroxyandrosterone, and 3-hydroxy-5-en steroids, decreased ratio of tetrahydrocortisone (THE) to tetrahydrocortisol (THF), and increased ratios of THF to allotetrahydrocortisol (a-THF) and metabolites of androgens (AM) to metabolites of cortisol (CM) — afforded reliable detection of disorders in steroid biosynthesis. The analysis of urinary steroid profiles can therefore be recommended as a screening procedure in patients with clinical symptoms of disorders in steroid production and/or metabolism.

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Abkürzungen An Androsteron - Aet Aetiocholanolon - AM Androgenmetaboliten: An plus Aet - 11-O-An 11-Ketoandrosteron - 11-OH-An 11-Hydroxyandrosteron - 11-OH-Aet 11-Hydroxyaetiocholanolon - DHEA Dehydroepiandrosteron - 16-OH-DHEA 16-Hydroxydehydroepiandrosteron - 16-OH-DHEA 16-Hydroxydehydroepiandrosteron - A⁵T-16 Androsten-3,16,17-triol - P⁵T Pregnentriol-3-hydroxy-5-en Steroide: DHEA plus Androstendiole plus 16-OH-DHEA plus 16-OH-DHEA plus - P⁵D Pregnendiol plus A⁵T-16 plus 16-Hydroxypregnenolon plus P⁵T - THE Tetrahydrocortison - THF Tetrahydrocortisol - a-THF Allotetrahydrocortisol - CM Cortisolmetaboliten: THE plus THF plus a-THF. - -C,-C Cortole - -CL,-CL Cortolone - 6-OH-F 6-Hydroxycortisol - 20-OH-F 20-Dihydrocortisol - NNR Nebennierenrinde - CRF Corticotropin Releasing Hormon - ACTH Adrenocorticotropes Hormon - CPB Competitive Protein Binding - RIA Radioimmunoassay - HPLC High Pressure Liquid Chromatography - DHEAS Dehydroepiandrosteron-Sulfat. Mit Unterstützung der Deutschen Forschungsgemeinschaft, Ho-471/5-1     

Bioactive 5-Reduced 16α,17α-Cyclohexanoprogesterone Derivatives Weakly Interact with Proteins of the Soluble Uterine Fraction

We studied competitive activities of 16,17-cyclohexano-5- and 5-dihydroprogesterone in replacing ₃H-progesterone and ₃H-16,17-cyclohexano-6-methylprogesterone from protein complexes. Direct binding of ₃H-5-reduced derivatives with proteins of soluble fractions from rat and rabbit uteri was also assayed. Cd values for 5-reduced derivatives were in the micro- or submicromolar range. The data suggest that biological effects of these analogues are not mediated via soluble uterine receptors.     

Mechanism of modulation of single sodium channels from skeletal muscle by the β 1-subunit from rat brain

We studied the molecular mechanism of the rat skeletal muscle -subunit (_I) gating kinetics modulation by the brain ₁-subunit by heterologous expression of single sodium channels from _I and ₁ in Xenopus laevis oocytes. Coexpression of ₁ reduced mean open time at –10 mV to 21% when compared to channels expressed by _I alone. Channels formed by _I exerted multiple openings per depolarization, which occurred in bursts, in contrast to the channels formed by the _I/₁ complex that opened in average only once per depolarizing voltage pulse. Macroscopic current decay (mcd), as evidenced by reconstructed open probability vs. time , was greatly accelerated by ₁, closely resembling mcd of sodium currents from native skeletal muscle. Generally was larger for channels expressed from the pure _I subunit.From our single channel data we conclude that ₁ accelerates the inactivation process of the sodium channel complex.     

Secretion of proinflammatory cytokines by villous chorion tissue in spontaneous abortion

Secretion of proinflammatory cytokines IL-1, TNF-, IL-6, IFN-, and IFN- by the chorion tissue (8-12 weeks) was studied in normal gestation and spontaneous abortion. The production of IL-1, TNF-, and IFN- virtually did not change in spontaneous abortion, while IFN- was not secreted in all experimental groups. The production of IL-6 increased more than 2-fold in patients with spontaneous abortion during the first trimester. These data confirm the involvement of this cytokine in the reproductive processes and in the pathogenesis of miscarriage.     

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.     

Comparative evaluation of integrin α- and β-chain expression in colorectal carcinoma cell lines and in their tumours of origin

The integrin family consists of broadly expressed cell surface adhesion receptors, each member of which is composed of a non-covalently linked / heterodimer. Integrin receptors are involved in the interaction with matrix proteins and may contribute to invasion and metastasis of carcinomas. To examine the biological role integrins play in colorectal carcinoma we compared the expression of integrin - and -subunits in situ and in vitro. Eight newly established cell lines derived from immunohistochemically characterized colorectal carcinomas together with two sublines obtained after nude mouse passage and the commonly used colon carcinoma lines HT-29, SW480, SW620, and COLO 205 were investigated by immunocytochemistry and flow cytometry. The carcinomas in situ expressed 1-, 2-, 3-, 6-, v-and 1-subunits in variable amounts while being devoid of 4, 5 and 3. The individual integrin profile of the tumour in tissue was essentially maintained in vitro. However, a neo-expression of the 5 chain was found, together with an induction or increase in 1, 2, 3, v and 1 levels. No decrease in integrin subunit expression was observed. Standard-serum and serum-free medium revealed no striking differences in - and -chain expression in the cell lines HT-29 and COLO 205. In serum-free medium, SW480 showed a slight increase of 1 and 5 and a decrease of 3 and v while SW620 expressed more 1. We conclude that the great variability of adhesion receptor expression of the integrin family in colorectal carcinomas in situ is essentially maintained in vitro, although culture conditions which are only marginally influenced by serum factors unpredictably lead to some increase in expression or even induction of several integrin subunits.This work is dedicated to Prof. Wilhelm Doerr on the occasion of this 80th birthday     

Nachweis von 6 neuen Steroiden im Plasma von menschlichem Placentablut (Nabelsehnurblut)

Zusammenfassung 16-Hydroxyprogesteron, 17-Hydroxyprogesteron, ⁴-Pregnen-20-ol-3-on, ⁴-Pregnen-17, 20-diol-3-on, Adrenosteron und 11-Hydroxyandrostendion wurden in Extrakten von Plasma des menschlichen Placentablutes (Nabelschnurblut) nachgewiesen.     

Transient expression of myosin heavy chain MHCIα in rabbit muscle during fast-to-slow transition

The expression of an -cardiac-like myosin heavy chain, MHCI, was investigated at both the mRNA and protein levels in rabbit tibialis anterior muscle undergoing fast-to-slow transition by continuous chronic low-frequency stimulation (CLFS). According to sequence analyses of the PCR product, the MHCI isoform was found to be identical to the -cardiac MHC expressed in rabbit atrium. In muscles at different degrees of transformation, the upregulation of MHCI mRNA preceded that of the MHCI mRNA. At more advanced stages of the transformation, MHCI mRNA decayed while MHCI mRNA persisted at high levels. The expression of MHCI, therefore, was transitory. Studies at the protein level were based on immunoblotting using a monoclonal antibody (F88 12F8,1), characterized to be specific to MHCI in rabbit muscle. These studies revealed a similar relationship between initial increase and successive decline of the MHCI protein as seen at themRNA level. Immunohistochemistry of 30-day stimulated muscle revealed that up to 65% of the fibres expressed the MHCI isoform in combination with other adult MHC isoforms. The most frequent patterns of coexistence were MHCIIa+MHCI + MHCI (28%), MHCI+MHCI (18%), and MHCIIa + MHCI (11%). According to these combinations, the upregulation of MHCI may be assigned as an intermediate step in the transformation of existing fibres during theMHCIIa MHCI transition. A small fraction of fibres contained, in addition to the MHCI + MHCI and MHCIIa + MHCI combinations, developmental myosin, suggesting that MHCI was also expressed in regenerating fibres originating from satellite cell-derived myotubes.     

Synchronous appearance of fibronectin,integrin α5β1, vinculin and actin in epithelial cells and fibroblasts during rat tracheal wound healing

The distribution of integrin 51 (51) and associated components during wound healing was investigated in the rat trachea following mechanical injury. Under anesthesia, the ventral surface of the trachea was scratched, and tissue specimens were obtained from 6 h to 3 weeks after injury and studied using light and electron microscopy and immunohistochemistry. 51, vinculin and actin in regenerating epithelial cells and extracellular fibronectin appear virtually simultaneously after injury (from 12 h to 7 days) as do 51, vinculin and -smooth muscle actin in fibroblasts and cellular fibronectin in granulation tissue (from 3 to 10 days). Immunoelectron microscopy 2 days after injury showed that 51 and vinculin were localized on the basal and lateral surfaces of regenerating epithelial cells and fibroblast surfaces, and fibronectin was localized just under the regenerating epithelial cells, around collagen fibrils and sporadically around fibroblasts. Bromodeoxyuridine labeling showed that the appearance of these components was associated with the period of cell proliferation. The appearances of fibronectin, 51, vinculin and actin in regenerating epithelial cells and fibroblasts during tracheal wound healing are well coordinated. During the initial cell migration phase, plasma fibronectin may stimulate cell migration before cellular fibronectin is produced in situ, and regenerating epithelial cells appear to begin to migrate into the wound before cell proliferation starts.     

Sublocalization on chromosome 21 of human interferon-alpha receptor gene and the gene for an interferon-gamma response protein

The cellular responses to alpha and beta interferons (IFN- and -) are mediated through the IFN-/ (type I) receptor, while the response to IFN- is mediated through the IFN- (type II) receptor. The receptors for IFN-/ and IFN- are encoded by genes on human chromosomes 21 and 6q, respectively. The presence of chromosome 21q confers both ligand binding and responsiveness to human IFN-/, whereas chromosome 6q confers binding of Hu-IFN-, but not cellular responsiveness on somatic cell hybrids. Chromosome 6q (i.e., the Hu-IFN- receptor gene) and chromosome 21q are both necessary for the cellular response of somatic cell hybrids (from fibroblasts) to Hu-IFN-. It is conceivable that the factor mediating activity through the IFN- receptor is, in fact, the IFN- receptor, or that the two genes are distinct but part of an interferon response region. Here we more precisely localize on human chromosome 21 the genes for the IFN- receptor and for the factor(s) mediating the action of IFN- through the chromosome 6-encoded receptor. Hamster-human somatic cell hybrids containing various fragments of human chromosome 21 were used. The presence of the human IFN-/ receptor was determined by binding³²P-labeled human IFN- to cells, covalently cross-linking the [³²P]IFN--receptor complex, and analyzing it by SDS-polyacrylamide gel electrophoresis. The presence of the IFN- receptor-related factor mediating cellular responsiveness was determined by HLA induction in hybrid cells containing the IFN- receptor (chromosome 6q), a transfected copy of the human HLA-B7 gene, and various portions of chromosome 21. In all hybrids examined, the two genes cosegregate. Specifically, both genes are localized to the region of chromosome 21 containing the markers D21S58, D21S65, and GART and appear to be proximal to D21S58. The implications for IFN action are discussed.     

Specific alterations in the expression of α3β1 and α6β4 integrins in highly invasive and metastatic variants of human prostate carcinoma cells selected by in vitro invasion through reconstituted basement membrane

Highly invasive cell subpopulations from a human prostate carcinoma cell line, PC-3, were selected for by allowing the parental PC-3 cells to invade through reconstituted basement membrane, Matrigel. These cells were collected, cultured and then selected further by repeated invasion through the in vitro invasion chamber. The invasive subpopulations (I-PC3 (2) and (3)) were found to be approximately 15-fold more invasive in vitro than the parental cells, had a distinct rounded morphology in culture, and proliferated more rapidly than the parental cells. When injected either subcutaneously or intraperitoneally into immunocompromised SCID mice, the I-PC3 cells were found to form tumors at the primary sites and to be highly invasive and metastatic. In contrast, the parental PC-3 cells formed tumors at the site of inoculation in these mice but failed to invade or metastasize. The I-PC3 cells attached equally as well as PC-3 cells to fibronectin, laminin, collagen type IV and vitronectin, but unlike the parental PC-3 cells these invasive variants failed to spread on any of these substrates. On Matrigel, the PC-3 cells became highly organized, whereas the I-PC3 cells remained rounded, clumped together and penetrated into the Matrigel. Biochemical analysis of the expression of adhesion proteins and integrins demonstrated that whereas the parental cells synthesized and secreted substantial amounts of fibronectin, the I-PC3 cell variants did not secrete any fibronectin. Although both PC-3 and I-PC3 cells expressed equivalent levels of cell surface v 3, 21 and 51 integrins, the expression of the 31 integrin, which is expressed at very high levels on the parental PC-3 cells, was drastically reduced on the invasive I-PC3 cells. This decrease in expression of 3 occurred also at the level of mRNA expression. Finally, whereas the PC-3 cells express 61, in the invasive I-PC3 cells the a6 subunit was associated mostly with the 4 subunit. Since the 64 integrin is analogous to the A9 tumor antigen which is associated with aggressive human squamous cell carcinomas, the apparent overexpression of 64 may also participate in the aggressive behavior of these variant prostate carcinoma cells. Alterations in the expression of the 31 and 64 integrins may thus allow these cells to become more invasive, and lead to an increased propensity for metastasis.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Differential effect of Fc gamma receptor ligation on LPS-stimulated TNF-alpha secretion by hepatic,splenic, and peritoneal macrophages

Lipopolysaccharide (LPS) increases serum TNF- levels due to TNF- secretion by macrophages. The serum TNF- response to LPS was augmented 10× when FcR ligation was induced by the intravenous injection of Gig-coated erythrocytes (IgG) prior to the administration of LPS. The macrophage population responsible for the augmented TNF- secretion was determined by isolating Kupffer cells, splenic macrophages and peritoneal macrophages from mice that had been given ElgG prior to LPS and determining TNF- secretion ex vivo. The intravenous injection of ElgG augmented LPS-stimulate TNF- secretion by Kupffer cells and splenic macrophages. In contrast, LPS-stimulated TNF- secretion by peritoneal macrophages was not altered by either the intravenous or intraperitoneal injection of ElgG. In vitro phagocytosis of ElgG by isolated peritoneal macrophages also did not augment LPS-stimulated TNF- secretion. These results show that FcR ligation augments LPS-stimulated TNF- secretion by Kupffer cells and splenic macrophages but not by peritoneal macrophages. Thus, the ability of FcR ligation to influence TNF- secretion may be specific to the tissue source of the macrophages.     

An improved method for the quantitation of cellular migration: Role of αvβ3 integrin in endothelial and smooth muscle cell migration

The present study was undertaken to develop a simple and improvedmethod for the accurate quantitation of cellular migration and to examinethe role of v3 integrins in different cellular migration. Usingour newly developed micro-volume chemotaxis assay, we developed an improvedquantitative method to measure in vitro chemotaxis of smooth muscle orendothelial cells toward different extracellular matrix proteins. Theconvenience in setup and counting of migrated cells using this methodallows for large capacity screening and for various research applicationswith other cells as well. The signal. to noise ratios were in the range of10/1, along with about 10–20% intra- or inter-assayvariabilities. Using this method, we have determined that eithervitronectin at 0.4 µg/well or osteopontin at 0.4 µg/well areselective v3 chemoattractants for endothelial or smooth musclecells (0.5 × 105 cells/well). Additionally, a selective v3small molecule peptiddomimetic, monoclonal antibody LM609, or an anti-3 (v3/II3) anti-body, c7E3 demonstratedmaximal inhibition of cellular migration toward vitronectin or osteopontin.These data suggest the potential utility of this method in assessing therole of various mechanisms in cellular migration and also suggests the potential implication of an v3 antagonist in blocking pathologicalprocesses involving endothelial or smooth muscle cell adhesion/migration.     

Integrins and extracellular matrix-proteins in the different components of the Wilms' tumour

The Wilms' tumour (WT) is composed of blastema, epithelium and mesenchyme; the epithelium and possibly also the mesenchyme develop from the blastema, parallel to embryonal development. Since interactions between cell adhesion receptors and extracellular matrix (ECM) proteins play an important role in tissue maturation, we examined the expression of the integrin subunits 1–6, 1 and 4, and of the ECM proteins fibronectin, laminin and collagen I and IV, in 20 frozen WT samples and in 5 fetal and 2 adult kidneys. The integrin and ECM protein distribution in tumour epithelium and mesenchyme showed strong similarities to that in their fetal counterparts, whereas the tumour blastema differed strongly from the fetal blastema. In the WT blastema different components were recognized. Undifferentiated blastema, characterized by expression of 3 and 6 and the virtual absence of ECM proteins. Blastema with epithelial commitment, showing increased expression of 3 and 6 and the appearance of 2 and, as a very early phenomenon, production of laminin. Blastema with mesenchymal commitment, with loss of 3 and 6 and expression of 1, 4 and 5 and presence of ECM proteins. It is speculated that the inability of the (undifferentiated) blastema to produce ECM proteins is related to its relatively high metastatic potential when compared with epithelium and mesenchyme.     

RGD-mutants of B-lymphotropic polyomavirus capsids specifically bind to αvβ3 integrin

Summary. Integrins are a family of cell surface proteins that function as receptors for extracellular matrix ligands and for some viruses. A subset of integrins recognises peptide sequences containing arginine-glycine-aspartic acid (RGD) motifs as ligands. The B-lymphotropic polyomavirus (LPV) has a non-enveloped capsid that recognises a sialylated cell surface receptor. To change the receptor binding specificity we have replaced sets of three amino acids in three predicted surface loops of the major capsid protein VP1 of the B-lymphotropic polyomavirus LPV by RGD.Ten mutants gave rise to the expected 40kDa VP1 protein upon expression from a baculovirus vector in insect cells. Five of the VP1 mutants representing all three surface loops have retained the ability to spontaneously assemble to capsids in the nuclei of the insect cells. Structural changes of the mutant capsid surface were shown by differential reactivity with a set of 7 neutralising monoclonal antibodies that recognise conformational surface epitopes of wildtype LPV virions. In addition all mutant capsids had lost specific binding to the LPV receptor.Three mutant capsids of one loop (BC) showed specific binding to v3 integrin but not to integrins v5, v6, or to IIb3 known also to recognise RGD containing peptide sequences. This selective binding of the mutant capsids could be inhibited by synthetic peptides that specifically bind to v3 integrin with IC₅₀ values between 10 and 40nM.     

In vitro Corticosteroidbiosynthese in menschlichen Nebennieren

Zusammenfassung Die Bildung von Pregnenolon, Progesteron, 11-Desoxycorticosteron, Corticosteron, Aldosteron, 11-Desoxycortisol und Cortisol aus endogenen Vorstufen und der Radioaktivitätseinbau in diese Steroide aus zugesetztem 4-¹⁴C-Pregnenolon während der in vitro Inkubation normalen menschlichen Nebennierengewebes wurden untersucht.Ergebnisse aus Experimenten mit Schnitten frischen Gewebes zeigten gute Korrelation zum Corticosteroidsekretionsmuster und zur Sekretionskapazität bei Normalpersonen. ACTH (Synacthen, Ciba) stimulierte die Corticosteroidsynthese aus endogenen Vorstufen und beeinflußte den Radioaktivitätseinbau nur geringfügig.Die spezifischen Radioaktivitäten von 11-Desoxycorticosteron waren 3–4 (Schnitte frischen Gewebes) und 12–14 (Schnitte gelagerten Gewebes) mal höher als die des Progesterons. Daraus ist die mögliche Existenz eines alternativen Syntheseweges abzuleiten, in dem Progesteron als Zwischenstufe nicht vorkommt.In Schnitten aus Gewebe, das 4–5 Monate bei –20°C gelagert worden war, zeigte ACTH keine Wirkung auf die Steroidbildung, und im Vergleich zu frischen Gewebeschnitten konnte ein signifikanter Kapazitätsverlust der 11- und 17-Hydroxylierung nachgewiesen werden.Das aus der Inkubation von Homogenaten frischen Gewebes resultierende Steroidmuster unterschied sich signifikant von dem aus entsprechenden Experimenten mit Schnitten. Homogenisieren hatte offensichtlich zu Schädigungen im Bereich der 11- und 17-Hydroxylierung geführt. Im Gegensatz zur Wirkung des ACTH auf die Steroidsynthese in Gewebeschnitten stimulierte NADPH (NADP⁺+G-6-P) in Homogenaten den Radioaktivitätseinbau stärker als die endogene Steroidbildung. In Homogenat frischen Gewebes bewirkte NADPH eine leichte Reaktivierung der 11-Hydroxylierung, die für Cortisol stärker ausgeprägt war als für Corticosteron.Homogenate aus gelagertem Gewebe zeigten zusätzliche Kapazitätsverluste der 11- und 17-Hydroxylierung. Unter der Einwirkung von NADPH ergab sich eine leichte Reaktivierung der 17-Hydroxylierung, jedoch keine Reaktivierung der 11-Hydroxylierung.Auszug aus der Dissertation von Peter Herzog, Fachbereich 08 Konservative Medizin der Johannes Gutenberg-Universität Mainz, 1974.     

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