Isolierung von 19 S-Antikörpern durch Gelfiltration und Säulenelektrophorese

Zusammenfassung Es wird ein Verfahren zur Isolierung von -M-Globulin beschrieben, das in zwei Trennschritten zu einem weitgehend reinen Präparat führt. Nach der Fraktionierung von Human-Serum durch Gelfiltration an Sephadex G-200 wurde die makroglobulinhaltige erste Fraktion weiter durch Säulenelektrophorese aufgetrennt. Es gelang eine saubere Trennung von ₂-Makroglobulin und -Makroglobulin. In beiden Fraktionen fanden sich immunelektrophoretisch noch Spuren von ₂-Lipoprotein.Das Verfahren wurde zur Isolierung von Antikörpern gegen cytoplasmatische Antigene aus Humanleber verwendet. Die Antikörperaktivität der -M-Globuline wurde durch den Trennvorgang nicht beeinträchtigt.

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Summary A method for the preparation of -M-globulin is described, which gives a nearly pure substance in a two steps operation. After gel-filtration of human serum on Sephadex G-200, the first fraction containing the macroglobulins, was further separated by column electrophoresis. The ₂-macroglobulin was well sparated from the -macroglobulin. Only traces of ₂-lipoprotein were found in both fractions. The method was used for the isolation of antibodies against cytoplasmatic antigens from human liver. The separation procedure did not diminish the activity of the antibodies.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

An improved apparatus for the optical recording of contraction of single heart cells

An optical system for measuring changes in cell length during unloaded contractions of cardiac myocytes is described. A one-dimensional video image of a cell is obtained every 4 ms with a linear photodiode array, which is aligned with the longitudinal axis of the cell. The circuit used to process the image from the photodiode array has a variety of features to aid in the accurate determination of the distance between the ends of the cell, i.e. the cell length. First, the video image of the cell is divided into two windows, one encompassing the front edge of the cell, the other encompassing the rear edge. Other cells or debris beyond the cell edges are excluded. Changes in the general light level, for example as a result of debris floating above the cell, have little effect because within the windows the background light level is subtracted from the signals before they are processed further. To detect the cell edges, the system determines when the signals within the windows exceed (front edge) or drop below (rear edge) chosen threscholds, which are different for the front and rear edges. The system has memory and it identifies the rear edge of the cell as the last time the signal falls below the threshold; because of this bright spots within the cell are not mistaken for the end of the cell. The system has hysteresis, which enables it to ignore small fluctuations in brightness around the threshold. The system is easy to use, accurate, readily calibrated, and it has good spatial and time resolution (about 0.25 m and 4 ms respectively).     

Processing of binaural stimuli by cat superior olivary complex neurons

Summary A method was developed to record stereotactically from the cat Superior Olivary Complex (SOC) using glass micropipettes. Sound stimulation was given through a closed system that permitted independent variation of interaural time (time) and intensity (int) differences. The most common binaural units found (n = 34) were ipsilateral excitatory, contralateral inhibitory (EI¹), cells of the Lateral Superior Olive (LSO). Some Medial Superior Olive (MSO) cells and presumed MSO ascending afferents were found but, as noted by other authors, we found it difficult to obtain single unit recordings from this nucleus. The LSO EI cells were mostly sensitive to higher frequencies and showed Peristimulus Time Histograms (PSTHs) consisting of a sharp On response followed by a plateau when stimulated with Best Frequency (BF) tone bursts or noise bursts. This On response was sensitive to time and int such that ipsilateral time lead or intensity increase resulted in a stronger response. The response reached a minimum around zero time or int. No sharp peaks or dips were seen in the physiological range needed for localization, instead the response increased with increasing ipsilateral lead or intensity to the maximum values tested (2048 s time, 30 dB int). In the physiological range the time and int response were complementary (both increasing response as ipsilaterality was increased). Provided enough sound energy in the unit's sensitive region was present, the same time curves were produced when BF tone bursts, masked tone bursts, sharp onset tone bursts or noise bursts were used. Changing the time of the carrier of the tone burst alone had no effect (except for one cell with a BF of 560 Hz), only the relative time of arrival of the stimulus envelope seemed to be important. In contrast to these LSO EI cells MSO-type units showed EI or EE predominantly low frequency phase-locked responses. When stimulated with interaurally phase shifted (pha) BF tones the unit response was a cyclic function of pha. Some cells (all that were tested, n = 6 including the 560 Hz LSO EI cell) showed these cyclic responses when stimulated with noise bursts or non-BF tones. However, these characteristic delays were not necessarily in the physiological range, i.e. we could find no evidence that these units were responding to time/pha values corresponding to a particular sound source direction. In both LSO and MSO it seems that integration of information higher in the CNS from a population of these cells is necessary for unambiguous coding of sound source direction. The time intensity trading ratios measured in two MSO type cells (11 and 26 /dB) were clearly different to those measured in LSO EI cells (n = 6, 99–550 s/dB). These ratios correspond approximately to those of the psychophysical time and int images measured by Hafter and Jeffress (1968).Supported by the Deutsche Forschungsgemeinschaft (SFB 45)     

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.     

Der Mechanismus der diabetogenen Wirkung des Pyromekazons

Zusammenfassung Die diabetogene Wirkung des Pyromekazons und des Alloxans wurde verglichen. Beide Verbindungen haben eine diabetogene Aktivität mit dem Unterschied, daß das Alloxan doppelt so stark wirkt. Bei intravenöser Verabfolgung an Ratten zeigen beide Verbindungen die gleiche glykämische Kurve, weshalb auf einen gleichen Mechanismus zu schließen wäre.Die Ascorbinsäure inhibiert die Pyromekazonwirkung, doch läßt sie die Alloxanwirkung unbeeinflußt. Die Frage dieser Behinderung wird in Verbindung mit der diabetogenen Gruppe der Verbindungen, welche bei beiden gleich ist, besprochen.Teilweise mitgeteilt analäßlich des ersten Kongresses der Biologen Jugoslawiens, Zagreb, 12.–15. Juli 1953.Für ihre technische Mitarbeit danken wir Fräulein Danica Paji, Frau Mira Tomaevi, Frau ÐurÐa Urai-Grguri und Herrn Alojz Mikec.     

Inhibitory synaptic channels activated by γ-aminobutyric acid (GABA) in crayfish muscle

Small crayfish muscle fibres were voltage clamped and synaptic current elicited by superfused GABA solutions was measured. Analysis of the fluctuations of synaptic current and of relaxations of the current after voltage steps yielded analogous results. The current has two components. The first component is characterized by the opening of synaptic channels with a single channel conductance =9 pS and an average open time =5 ms, measured at 23°C and-100 mV. depends on the membrane potential, _E = ₀ · e ^E/, and was about +100 mV in the average. The channel open time agrees with the time constant of decay of the inhibitory postsynaptic current (IPSC) elicited by a nerve stimulus. The current is carried by chloride ions. The second current component is much slower, the average channel open time was _s = 33 ms at 23°C and-60 mV. The open time _s of the slow component also was shortened on hyperpolarization. The reversal potential for the current component was more positive than-50 mV. This slow component also seems to be a synaptic one.This investigation was supported by a grant of the Deutsche Forschungsgemeinschaft     

Increase in TCRγδ T lymphocytes in synovia from rheumatoid arthritis patients with active synovitis

To determine the relative presence of TCR+ and TCR+ T cells in synovial tissue from patients with various types of inflammatory synovitis and in tissues from patients with a number of chronic T cell-mediated conditions, we stained frozen tissue sections with monoclonal antibodies in indirect immunofluorescence assays. In tissues obtained from patients with chronic T cell-mediated granulomatous conditions (Wegener's granulomatosis, lymphomatoid granulomatosis, granuloma annulare, Langerhan's cells granulomatosis, pigmented villonodular synovitis, Takayasu's arteritis, and talc granulomatosis), the T cells present were predominantly TCR+, without an increased presence of TCR+ cells. In contrast, 6 of 14 (43%) synovia from patients with rheumatoid arthritis (RA) showed increased TCR+ T cells (3–10 cells/hpf). The RA synovia with increased TCR+ cells present had an increased tissue inflammation score compared to RA synovia with few TCR+ cells [18.6±5.8 versus 11.6±4.2 (mean±SE),P<0.05]. In contrast, synovia from patients with osteoarthritis, systemic lupus erythematosus, and trauma did not show an increased presence of TCR+ T cells. Thus, in cellular inflammatory infiltrates the presence of increased TCR cells is not a component of noninfectious granulomatous inflammation but is found in approximately 40% of RA synovia with high levels of inflammation.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

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.     

Effect of molsidomine on ex vivo platelet aggregation and plasma guanosine 3′∶5′-cyclic monophosphate levels in healthy volunteers

Summary To find out whether 3-morpholino-sydnonimine (SIN 1), the active metabolite of molsidomine, exerts its antiaggregatory effects not only in vitro but also in vivo, we tested ex vivo aggregation before and after intravenous application of molsidomine in healthy volunteers. We also measured plasma levels of guanosine 35-cyclic monophosphate (cyclic GMP) as SIN 1, the bioactive metabolite of molsidomine, becomes effective via activation of soluble guanylate cyclase. In eight out of ten subjects molsidomine had an inhibitory effect on platelet aggregation and a higher threshold concentration of platelet-activating factor was required after molsidomine application to induce irreversible aggregation. Despite the effect on platelets, plasma cyclic GMP levels did not increase. These results suggest that the nitric oxide-containing SIN 1 inhibits platelet aggregation not only in vitro but also in vivo and that this property can be a beneficial effect in antianginal therapy.Abbreviations Cyclic GMP guanosine 35-cyclic monophosphate - NO nitric oxide - PAF platelet-activating factor - PRP platelet-rich plasma - SIN 1 3-morpholino-sydnonimine     

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.     

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).     

Myelin gene expression in glia treated with oligodendroglial trophic factor

Summary Oligodendroglia synthesize myelin in the CNS.in vitro, oligodendroglia may be identified by the binding of monoclonal antibodies against galactocerebroside, a myelin-specific galactolipid. Oligodendroglial trophic factor is a protein mitogen for cells of the oligodendroglial lineage. When oligodendroglia in cerebral white matter cultures are treated with oligodendroglial trophic factor, galactocerebroside-positive cells undergo mitosis but fail to express the myelin structural proteins, myelin basic protein and proteolipid protein. Oligodendroglia treated with oligodendroglial trophic factor, however, do express 2, 3-cyclic nucleotide 3-phosphodiesterase and myelin-associated glycoprotein in a manner similar to oligodendroglia treated with platelet-derived growth factor. Oligodendroglial trophic factor, therefore, generates a population of somewhat immature oligodendroglia, which are galactocerebroside, myelin-associated glycoprotein and 2, 3-cyclic nucleotide 3 phosphodiesterase positive but myelin basic protein and proteolipid protein negative.     

Poly ADP Ribose-Polymerase Inhibitors Prevent the Upregulation of ICAM-1 and E-selectin in Response to Th1 Cytokine Stimulation

We studied the role of poly-ADP-ribose polymerase (PARP) in the mobilization of ICAM-1, VCAM-1, and E-selectin by TNF- and IL-1 in cultured human endothelial cells. Enzyme linked immunosorbent analysis (ELISA) was used to assess if ICAM-1, VCAM-1, and E-selectin were expressed at the cell surface, and if PARP inhibition (using the selective PARP inhibitor GPI 6150) blocked the induced expression. Endothelial cell adhesion molecule expression was evaluated at 4 and at 24 h after cytokine stimulation. At 4 h ICAM-1 and E-selectin, but not VACM-1, were stimulated by both IL-1 and TNF-. Blocking PARP via GPI 6150 only affected TNF- induced E-selectin expression at 4 hours. ICAM-1, VCAM-1, and E-selectin expression were all stimulated by both IL-1 and TNF- in the 24 h assays. PARP inhibition with GPI 6150 blocked the IL-1 mediated stimulation of both ICAM-1 and E-selectin expression, and blocked TNF- stimulation of ICAM-1 expression at 24 h. These experiments suggest that specific PARP inhibition may provide a novel method of controlling leukocyte dependent inflammation through the reduction of ICAM-1 and E-selectin expression in endothelial cells in response to cytokines.     

Induction of interferon and host resistance in vivo by double-stranded complexes of copolyribonucleotide of inosinic and guanylic acids with polyribocytidylic acid

Summary Several double-stranded complexes of copolyribonucleotide of inosinic and guanylic acids with polyribocytidylic acid (poly IGC) were found to possess interferon inducing activity stronger than poly ICin vivo, Their activity increased in parallel with increase in the ratio of guanine base to hypoxanthine base in these copolymers as far as double-strand formation was observed with polyribocytidylic acid. Many other combinations of copolyribonucleotide with homopolyribonucleotide were also investigated, and several of them were found to induce interferon. However, the interferon inducing effects of these combinations including complementary base-pairings of hypoxanthine and cytosine increased in parallel with the length of the base-pairings, thus approaching to that of poly IC. It is, therefore, supposed that the activity of poly IG C is somewhat different from poly IC and that those of other combinations owe to the essential structure of poly IC. Furthermore, kinetics of interferon induction, cross tolerance to reinduction, and antiviral effectsin vivo of poly IGC and poly IC were studied.     

Regional location of α1-antichymotrypsin and α1-antitrypsin genes on human chromosome 14

The human protease inhibitor genes ₁ antitrypsin (₁-PI) and ₁-antichymotrypsin (₁-ACT) are acute-phase proteins which are induced in response to inflammation. These inhibitors function to limit the activity of serine proteases in vivo. ₁-PI acts as an inhibitor of neutrophil elastase to protect the elastin fibers of the lung. Genetic deficiencies of ₁-PI result in development of chronic pulmonary emphysema. The physiologic role of ₁-ACT has not been clearly defined, but it also appears to function in the maintenance of protease-protease inhibitor equilibrium in the lung. Nucleic acid and protein sequence homologies detected between ₁-PI and ₁ t-ACT suggested an evolutionary relationship. Gene mapping experiments were performed to determine if these protease inhibitor genes reside at the same chromosomal locus in man. In situ hybridization data demonstrate that both ₁-PI and ₁-ACT map to the same region, q31–q32.3, on chromosome 14.     

Immunohistochemical localization of αl-antitrypsin and αl-antichymotrypsin in salivary pleomorphic adenomas

Summary Immunohistochemical identification of l-antitrypsin (l-AT) and l-antichymotrypsin (l-ACh) in pleomorphic adenomas of salivary glands is reported in order to compare their distribution profiles with those of lysozyme and lactoferrin, already described elsewhere.Normal salivary glands indicated positive l-AT staining in ductal segments and had no l-ACh in any glandular cell. Pleomorphic adenomas displayed moderate positivity to l-AT staining in duct-like, tubular and glandular epithelia which was particularly intense in luminal cells. The limited number of tumour cells which showed duct-like structures with a single cellular layer arrangement, displayed the highest staining to l-ACh. Strongly l-AT positive tumour cells located on the inner side of luminal cavities were also markedly positive to l-ACh. Spindle shaped tumour cells existed outside tubular and ductal structures and were negative to l-AT and l-ACh.Distribution of l-AT in salivary glands was similar to that of lysozyme as is usual in ductal segments or their transformed cells, and occurrence of l-ACh localization rather resembled that of lactoferrin, with occurrence in acinar compartments and changed epithelia within acini.The biological role of a specific immunohistochemical distribution of l-AT and l-ACh in pleomorphic adenomas may be associated with a self regulating mechanism which inhibits degradation by tissue proteinases.     

Differential expression of basement membrane type-IV collagen α1, α2, α5 and α6 chains among the histological subtypes of adenoid cystic carcinoma

Six distinct (IV) chains in the basement membrane (BM) of adenoid cystic carcinoma (ACC) of the salivary gland were immunohistochemically examined by anti-(IV) chain-specific antibodies, and their expressions were compared with the histological subtypes and the expressions of cytokeratin 19 (CK19), cytokeratin 14 (CK14) and -smooth muscle actin (-SMA) and Ki-67. In the BM of normal salivary ducts, 1(IV), 2(IV), 5(IV) and 6(IV) chains were continuously stained, but 3(IV) and 4(IV) chains were negative. In the tubular and cribriform subtypes of ACC, tubules with continuous staining of 5(IV) and 6(IV) chains showed the biphasic-staining pattern among the expressions of CK19, CK14 and -SMA. However, in cancer-cell nests with discontinuous or negative staining of 5(IV) and 6(IV) chains, the biphasic pattern was ambiguous. In the solid subtype, the staining of 1(IV) and 2(IV) chains was discontinuous, the staining of 5(IV) and 6(IV) chains was negative and the biphasic-staining pattern was unclear. The mitotic activity of cancer cells analyzed by the Ki-67 labeling index was significantly related to the expression of 5(IV) and 6(IV) chains in the cribriform subtype. These results suggest that BM irregularity with the differential expression of (IV) chains in ACC closely relates to cell proliferation, cell differentiation and histological structure.     

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.