Effects ofd-penicillamine,dichlofenac sodium and gold sodium thiomalate upon the selective release of lysosomal enzymes from human polymorphonuclear leucocytes to immune complex

The selective release of -glucuronidase (-Gluc) and -N-acetylglucosaminidase (-Glm) from human polymorphonuclear leucocytes (PMN), initiated with bovine serum albumin/anti-bovine serum albumin (BSA/anti-BSA) immune complex (15 g/ml^–1) was significantly reduced by increasing concentrations (10^–7 M, 10^–6 M and 10^–5 M) ofd-penicillamine (d-PEN) in a dose-dependent fashion. These effects upon the exocytosis of the lysosomal enzymes studied are in accordance with the results obtained previously in rats with adjuvant arthritis. In contrast, Dichlofenac Sodium (DICHL), which has been found to exert inhibitory activity upon extracellular release of -Gluc and -Glm in adjuvant arthritic rats in previous studies, had no significantin vitro effect on the exocytosis of these enzymes at the concentrations identical to those ofd-PEN. Also, Gold Sodium Thiomalate (GST), in the same concentrations ranging from 10^–7 M 10^–5 M, failed to inhibit selective release of -Gluc and -Glm in the present investigations. Additionally BSA/anti-BSA,d-PEN, DICHL and GST did not significantly produce the extracellular release of lactate dehydrogenase (LDH) indicating that under experimental conditions described the cell remained intact. Moreover, neitherd-PEN, DICHL, GST or BSA/anti-BSA significantly changed the activities of lysosomal enzyme markers used in these experiments. The possible mechanism(s) of the observed phenomena are discussed.     

Molecular specificity of the tubular resorption of “acidic” amino acids

Single sections of superficial proximal convolutions of rat kidney were microperfused in vivo and in situ. The perfusion fluids contained radioactively labelledl- ord-aspartate,l-glutamate,l-pyroglutamate, or N-methyl-d-aspartate.l--Carboxyglutamate as well as the other amino acids were added in the unlabelled from. Results.l- andd-Aspartate (0.073 mmol·1^–1) are quickly resorbed at about the same rate.d-Aspartate resorption was blocked byl-aspartate (5 mmol·1^–1) but not by -alanine (5 mmol·1^–1).l-Aspartate resorption was inhibited byl-glutamate (2 mmol·1^–1) but not byd-glutamate,l-asparagine,l-phenylalanine or by succinate (2 mmol·1^–1, each). The fast resorption ofl-glutamate (0.073 mmol·1^–1) was blocked byd-aspartate,l-cysteate (2 mmol·1^–1), but not by 3-mercaptopicolinic acid (0.15 mmol·1^–1),l-glutamine, 2-oxoglutarate, taurine, N-methyl-l-glutamate or kainic acid (2 mmol·1^–1, each).l--Carboxyglutamate (0.66 mmol·1^–1) and N-methyl-d-aspartate (2mol·1^–1) were found to be resorbed only at an extremely small rate.l-pyroglutamate (0.076 mmol·1^–1) resorption was not influenced byl-glutamate (1 mmol·1^–1). Fractional excretion of -carboxyglutamate was 7–25% (l-from) or 45–70% (d-form) at an artificially elevated plasma level of 12mol·1^–1.It is concluded thatl- andd-aspartate,l-glutamate,l-cysteate and, to a much smaller extent,l--carboxyglutamate, are accepted by the tubular resorption mechanism highly specific for acidic amino acids. N-Substitution, the amidation of the - or -carboxyl group, or the removal of the -amino moiety almost completely abolish the ability of such compounds to be resorbed via this carrier; N-methylated or -carboxylated derivatives of acidic amino acids are not resorbed at all from the proximal tubule. The resorption of glutamate, but not of aspartate, is highly stereospecific.Parts of this work were presented at meetings of the German Physiological Society in 1978 [28] and of the Gesellschaft für Nephrologie in 1980 [29] as well as at the VIIIth International Congress of Nephrology in Athens in 1981 [26]with technical assistance of Angelika Ascher and Gertaud Vetter     

Regulation of glycoprotein D synthesis of herpes simplex virus 1 by α 4 protein,the major regulatory protein of the virus,in stably transformed cell lines: effect of the relative gene copy numbers

Summary Earlier studies concerning 1 gene regulation by the 4 protein, the major regulatory protein of herpes simplex virus 1 (HSV-1), in stably transformed cell lines, reported conflicting results, i.e., 4 protein positively regulated the ₁ gB gene in 4/gB cells, while it negatively regulated the ₁ gD gene in 4/BJ cells. Both cell lines were derived from a common parental cell line 4/c 113 that contains 1 copy of the 4 gene, and the only apparent difference between them was the relative copy number of the gB and gD sequences (1 and 30–50, respectively) resident in the cell genome. We investigated this disparity by constructing a cell line (BA 4) that contains one copy each of the 4 and ₁ gD sequences, by fusion of 4/c 113 and BJt cells, containing and expressing respectively 1 copy of the 4 and gD genes. BA 4 cells constitutively expressed both the 4, gD genes inherited from the parental cell lines ( 4/c 113 and BJt). In BA 4 cells the 4 protein positively regulates the gD gene as evidenced from (i) higher levels of gD expression than the parental BJt cells lacking the 4 gene, and (ii) significant decrease in gD expression under conditions that render the 4 protein produced in BA 4 cells non-functional. In addition the ₂gG gene contained within the DNA fragment encoding the gD gene, is also expressed in BA 4 cells. On the basis of these data, we propose that gene regulation by the 4 protein is affected by the relative copy number of these genes, resident in the cell genome.     

Spontaneous and agonist-induced openings of an acetylcholine receptor channel composed of bovine muscle α-, β and δ-subunits

During the development of mammalian muscle the -subunit of the nicotinic acetylcholine receptor (AChR) is replaced by the -subunit to produce well-defined alterations in the conductance and gating of the channel. To gain a better unterstanding of the functional role of the and -subunits, we have studied the properties of an AChR channel lacking these subunits. The AChR expressed in Xenopus oocytes injected with the bovine -, and -subunit-specific mRNAs (referred to as -AChR) is unusual in that its channel opens spontaneously at a high frequency in the absence of agonist. From a comparison of the -AChR with complete receptors containing either the or -subunit, we conclude that the and -subunits influence most channel properties, including agonist binding, and are especially important for stabilizing the closed state of the unliganded receptor channel. The -AChR can form when a complete set of four subunit-specific mRNAs is injected. The ease with which it is assembled raises the possibility that the - AChR contributes to some of the variations in receptor properties that occur during development.     

Increased serum N-acetyl-β-d-glucosaminidase and α-ddd-mannosidase activities in obese subjects

Summary We have studied N-acetyl--d-glucosaminidase and -d-mannosidase activities in human sera from 35 control subjects, 47 normo- and hyperinsulinemic obese persons, and 12 diabetic patients after a fasting period of 12 h and at 30, 60, 90, and 120 min after an oral glucose overload. The results show a significantly higher activity of these 2 enzymes in obese subjects and diabetic patients, of similar magnitude, especially in those obese persons with a higher grade of obesity. Moreover, the activity of these glycosidases decreases in a similar way in all these 3 groups after the oral glucose overload.Abbreviations BMI Body mass index - NAG N-acetyl--d-glucosaminidase - -Man -d-mannosidase     

The localization of lectin-binding sites on Schwann cell basal lamina

Summary Basal laminae were separated from Schwann cells of mouse sciatic nerves by Bonification, and the distributions of lectin-binding sites were demonstrated by electron microscopy using ferritin-conjugated lectins. Only three out of the 11 lectins examined were bound to the basal laminae of Schwann cells: they wereRicinus communis agglutinin-I (RCA-I),Canavalia ensiformis agglutinin (ConA) andTriticum vulgaris agglutinin (wheat germ agglutinin, WGA). It was notable that WGA was bound more densely to the cellular side than to the interstitial side, whereas in the case of RCA-I and ConA there were no differences in the binding density on the two sides of the basal lamina.These results indicate that there are sugar residues such as -d-galactose, -d-mannose, -d-glucose and (1–4) linkedN-acetyl-d-glucosamine in the Schwann cell basal laminae. The first three sugar residues are almost equally densely distributed on the cellular and interstitial sides of the basal laminae, whereas (1–4) linkedN-acetyl-d-glucosamine is more densely distributed on the cellular than on the interstitial side. This result suggests that the basal lamina has a polarity in chemical composition between the cellular and interstitial sides. These findings are discussed in the context of the preferential attachment of regenerating axons to the cellular side of the Schwann cell basal laminae.     

Expression ofβ1 integrins in non-neoplastic mammary epithelium,fibroadenoma and carcinoma of the breast

1 Integrins were examined immunohisto-chemically in normal and mastopathic mammary glands, 12 benign tumours and 90 carcinomas of the breast using monoclonal antibodies against1 and1 to6 subunits. When compared with epithelial cells of non-neoplastic mammary glands and of benign tumours, carcinoma cells showed considerable quantitative changes in the pattern of2,3 and6 subunit expression. In contrast, the distribution pattern of1,1,4 and5 antigens corresponded to the situation observed in non-neoplastic mammary gland epithelium in most instances. An abnormal expression of2 was found in 71.0% of the carcinomas ranging from a remarkably low number of2-positive tumour cells in 27.5% of the cases to a complete absence of the2 molecule in 43.5% of the carcinomas. Of the carcinomas 39.9% exhibited quantitative changes in3 expression with an abnormally low content of3-positive neoplastic cells in 15.4% and a complete absence of this molecule in 24.5% of the cases. Expression of6 was abnormal in 73.2% of the carcinomas, consisting in a greater number of6-negative tumour cells in 31.9% and in a complete absence of6 in 41.3% of the tumours. The abnormally low expression/absence of2 and3 subunits correlated with oestrogen receptor negativity (P<0.033 andP<0.04, respectively). In addition, abnormally low expression/absence of2 correlated with poor differentiation of the tumours (P< 0.014). The quantitative changes in the expression pattern of1-associated subunits in breast carcinomas may cause a disturbed cell-cell and/or cell-matrix interaction that increases the invasive and migratory property of the tumour cells.     

The effectiveness of the control of pH in the extracellular fluid of the brain by the respiratory control system

Summary The effectiveness of the respiratory control system as a regulator of the pH in the extracellular fluid of the brain is defined by pH_{ECF op}/pH_{ECF cl} where pH_{ECF op} means the primary or open loop shift and pH_{ECF cl} the final or closed loop shift of brain extracellular fluid pH. The analysis of a steady state model described in a preceding paper (Loeschcke, 1973) allows, in the limits of the suppositions and simplifications, to calculate the effectiveness of the feedback regulator in the cases of increased metabolism, metabolic acidosis-alkalosis and inhalation of CO₂. The effectiveness is diminished if CO₂ production is increased, it drops in metabolic acidosis and rises in metabolic alkalosis and it drops steeply if CO₂ is inhaled. The effectiveness of this control system depends on the controlling action of the controlling element (the ventilation) rather than on varying sensitivity of the sensing element. The controlling effect is defined asC=–d P _CO2/d V _A orC=d pH_ECF/d V _A.Im Rahmen des Programms des Sonderforschungsbereiches 114 (Bionach) der Deutschen Forschungsgemeinschaft.     

Expression of the L-type calcium channel with two different β subunits and its modulation by Ro 40-5967

The smooth muscle 1_Cb subunit of the L-type calcium channel was expressed alone (CHO ₁ cell) or together with the skeletal 1 (CHO1 cell) subunit or smooth muscle 3 (CHO ₁ 3 cell) subunit in Chinese hamster ovary (CHO) cells. The interaction of the expressed calcium channels with the non-dihydropyridine calcium channel blocker Ro 40-5967 was studied. Ro 40-5967 decreased isradipine binding by an apparent allosteric interaction and blocked the barium inward currents (I _Ba) in a voltage- and use-dependent manner in all cells. The steady-state inactivation curves were shifted to hyperpolarizing potentials in the presence of Ro 40-5967. The rate of channel inactivation was increased in CHO ₁ and CHO ₁ 3 cells. The shift in the steadystate inactivation curve and the increase in channel inactivation were less pronounced in CHO ₁ 1 cells than in the other cell lines. Low concentrations of Ro 40-5967 increased I _Ba by up to 198% in 33% of the CHO ₁ 1 cells. In addition, higher concentrations of Ro 40-5967 were required to inhibit I _Ba in 60% of the CHO ₁ 3 cells. These results suggest that the subunits modify the interaction of the non-dihydropyridine Ro 40-5967 with the expressed calcium channel ₁ subunit.Dedicated to the late Professor Dr. W. Osterrieder     

d-glucose balance in the enterocyte of rat jejunum “in vitro”

An everted sac of male albino rat jejunum (Wistar strain) incubated in vitro is used. Netd-glucose and Na⁺ transport together withd-glucose concentration in the emerging fluid [5], or in the serosal fluid, and in the enterocytes are determined. Celld-glucose concentration does not change significantly in a range between 20–200 moles or between 50–500 moles of netd-glucose transepithelial transport, depending on the experimental conditions.As far as cellulard-glucose and Na⁺ concentration is concerned, the enterocyte behaves as an homeostatic system.The mechanism involved ind-glucose extrusion is extensively discussed. Two hypotheses seem to be possible. First, the mechanism is an active metabolically dependent one, just as it is for sodium transport. Second, the metabolic activity favoursd-glucose facilitated permeability through the basolateral membrane in such a way as to maintain a constant relationship betweend-glucose and Na-extrusion, notwithstanding the fact thatd-glucose concentration gradient across the basolateral membrane lowers by increasing Na and glucose extrusion rate.     

Übererregbarkeit der γ-Motoneurone beim „Stiff-man“-Syndrom

Zusammenfassung Es wurde eine 40jährige Frau mit sog. Stiff-man-Syndrom untersucht. In Narkose und Lumbalanaesthesie verschwand die abnorme Muskelverspannung. Ebenso normalisierte sich unter Differentialblockade des N. femoralis durch Procainumspritzung der Tonus im Quadriceps vorübergehend ohne Lähmungserscheinungen. Daraus kann gefolgert werden, daß die-Motoneurone wesentlich am Zustandekommen der Muskelverspannung beteiligt sind. Histologisch ergab sich kein krankhafter Muskelbefund. Elektromyographisch bestand eine langanhaltende Aktivität in den befallenen Muskeln, die sich von Willkürentladungen nicht unterscheiden ließ. Bei passiven Bewegungen wurde eine ungewöhnliche Entdehnungsaktivität registriert. Unter Willkürinnervation bestand ein normal dichtes Muster. Bei Entspannung nach aktiver Innervation trat nach kurzer Entlastungsreaktion Überdauerungsaktivität auf. Einzelpotentiale, Nervenleitgeschwindigkeit, mechanisch und elektrisch (H-Reflex) ausgelöste Muskeleigenreflexe waren normal, die silent period nicht verändert. Der H-Reflex zeigte unter subparalytischer Dosis von Succinylcholin einen Abfall wie beim Gesunden.Unter Behandlung mit Diazepam und einem Amino-Buttersäurederivat verschwand die abnorme Muskelspannung fast vollständig. Es wird die Annahme vertreten, daß es sich bei dieser Form des Stiff-man-Syndroms, die dem Syndrom vonMoersch undWoltman entspricht, um ein Parabiosephänomen der-Motoneurone handelt (Spindelmyotonie). Analog dazu liegt beim Isaacs-Syndrom eine Überaktivität der peripheren -Motoneurone vor (Neuromyotonie). Bei anderen in der Literatur beschriebenen Formen des Stiff-man-Syndroms handelt es sich offensichtlich um primäre Muskel- oder Bindegewebserkrankungen.

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Summary A 40 years old woman suffering from the so-called stiff-man syndrome is described. The abnormal muscle-tonus disappeared in narcosis and in lumbar anaesthesia. When procaine was infiltrated close to the nervus femoralis, the tonus in the quadriceps muscle was normalised temporarily without the muscle being paralysed. Consequently the-motoneurones may be considered as an essential factor in bringing about the pathological muscle stiffening. Histologycally there was no evidence of muscle disease. The EMG showed a long-lasting activity in the stiffened muscles which did not differ from a normal pattern. During passive shortening of the muscle abnormal activity was registered. An interference pattern was seen with maximum voluntary contraction. Active innervation was followed by anew electrical activity in the relaxing muscle, only interrupted by a short silent interval. Single units, nerve conduction velocity, the potentials of the muscle stretch reflex an the H-reflex were normal. The silent period was not altered. Under subparalytical dosage of succinylcholine the amplitude of the H-reflex showed a decrease in a physiological manner. The muscle stiffness disappeared almost completely when the patient was treated with diazepam (Valium) and a derivate of amino-butyric acid (CIBA 34-647 Ba).There is some reason to believe that this variety of stiff-man syndrome, being identical with the syndrome described byMoersch andWoltman, is caused by the parabiotically diseased-motoneurones (spindlemyotonia). Likewise a hyperactivity of peripheral -motoneurones is known to bring aboutIsaacs syndrome (neuromyotonia). Other varieties of the stiff-man syndrome described by literature are obviously primary diseases of the muscle or connective tissue.

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Herrn ProfessorG. Schaltenbrand zum 70. Geburtstag gewidmet.     

Intracellular lectin-binding sites in symbiont-bearingCrithidia species

Crithidia oncopelti, C. deanei, andC. desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Direct and indirect lectin-gold labeling techniques were used at the electron microscopic level in Lowicryl K4M-embedded cells to demonstrate the presence of intracellular lectin-binding sites. We used the lectinsUlex europaeus I, Griffonia simplicifolia II, Ricinus communis I, Arachis hypogaea, G. simplicifolia I, Wistaria floribunda, Limulus polyphemus, andCanavalia ensiformis, which recognize -l-fucose, - and -N-acetylglucosamine, -galactose and -N-acetylgalactosamine, -galactose, -galactose, -N-acetylgalactosamine, sialic acid and -d-mannose, and -d-glucose residues, respectively. The nucleus was the cellular structure most frequently labeled by the lectins. The Golgi complex was seldom labeled, whereas the endoplasmic reticulum and the flagellar pocket presented a large number of binding sites. Symbionts had their two unit membranes weakly labeled by the different lectins but displayed no labeling of the space between the membranes.     

Expression of an antigenic polypeptide of the human parvovirus B19

The DNA fragment of the human parvovirus B19, with 715 nucleotides between nucleotide positions 3141–3856 was expressed in Escherichia coli as a -galactosidase fusion protein. The plasmid vector pSS20d used for this purpose permits cleavage of the viral gene product from the -galactosidase moiety by collagenase. After purification by p-aminophenyl--d-thiogalactoside-sepharose and superose, a soluble protein with a molecular mass of 28 kDa was isolated. It represents a common part of the viral capsid proteins VP1 and VP2. This bacterially derived parvoviral gene product can be used for detection of anti-B19 antibodies in human sera.     

Enhanced stability of YEp plasmids in lager brewing yeasts is related to lager brewing yeast 2-μm DNA

Summary YEp plasmid stability in the presence of either Saccharomyces cerevisiae laboratory strain 2-m DNA, or lager brewing yeast 2-m DNA in the same genetic background, was compared under non-selective culture conditions. It was found that YEp plasmids were more stably maintained in the presence of lager 2-m DNA under these conditions. By construction of laboratory-lager 2-m DNA hybrid plasmids, an 867 bp StuI fragment of lager 2-m DNA was shown to be responsible for the enhanced stability of the YEp plasmid. Nucleotide substitutions at two sites were found by sequencing this region. It was also confirmed that increasing cell ploidy enhanced YEp stability under non-selective conditions.     

Concomitant behavioral and neural inhibition and disinhibition in response to subcortical stimulation

Summary Unit and EEG activity were recorded simultaneously from the same electrode in both acute and chronic cats. In chronic cats, parallels between behavioral and electrophysiological activity were observed. Stimulation of a number of brain sites, including the caudate nucleus and parts of the thalamus, induced an inhibition of unit activity with a concomitant positive (extracellularly recorded) slow wave. The inhibition, which lasted from 150–250 msec, was abbreviated when afferent inputs (thalamocortical or peripheral) were paired with the inhibitory stimuli. When many consecutive pairings were made at a rate of 1/3 to 3 pulses per sec, the abbreviation of inhibition tended to disappear, and, in addition, an inhibitory response to the afferent input alone was often seen. In chronic cats, these electrophysiological observations were paralleled by inhibition and disinhibition of behavioral responses.The authors gratefully acknowledge the technical assistance of: Cora Rucker, David Weber and Marion Smith Baldwin.This work was supported by USPHS Grant MH-07097 and N.A. Buchwald's USPHS Career Development Award 5-K3-GM-3509.     

”Reflexes of purpose and freedom” in the comparative physiology of higher nervous activity

The most complex unconditioned reflexes of aim and freedom, discovered by I. P. Pavlov, are compared with the competence drive and the motivation of the resistance to coercion, respectively, described by contemporary ethologists. On the basis of the unconditioned reflex of purpose, conditioned reflexes were developed in which positive emotions arising in connection with the perfection of a skill, irrespective of its pragmatic significance at a given moment, serve as the reinforcement. The unconditioned reflex of freedom is regarded as a phylogenetic precursor of the will, and its acute extinction as the physiological mechanism of hypnosis. It was demonstrated experimentally that the appearance of the state of animal hypnosis (immobilization catatonia) in rabbits is accompanied by the predominance of electrical activity and heat production in the right hemisphere, i. e., by symptoms which are found in hypnosis in man.Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I. P. Pavlova, Vol. 39, No. 3, pp. 415–420, May–June, 1989.     

Contribution of glial cells to histamine inactivation

dl--aminoadipic acid (dl-AA), a selective gliotoxic agent produced significant reductions in histamine-N-methyl-transferase (HNMT) and monoamine oxidase-B (MAO-B) activities and an enhancement in histamine (HA) level in the hypothalamus of rats 2 and 4 h after single intracerebroventricular (i.c.v.) or subcutaneous (s.c.) injections of the compound. Histidine decarboxylase (HD) and monoamine oxidase-A (MAO-A) were unaffected after these treatments. Following a single i.c.v. injection ofdl-AA of 200 g/rat, or a single s.c. injection of 5 mg/rat, marked diminutions in the astrocytic marker glutamine synthetase (GS) activity occured suggesting marked glial damage in the hypothalamus. In total, these studies indicate an important role for glial cells in HA metabolism (inactivation).     

Isoenzymes of γ-glutamyl transpeptidase in liver diseases

Summary A new method for the separation of isoenzymes of-glutamyl-transpeptidase is described, using electrophoresis on acetate cellulose gel and a developing solution composed by-glutamyl-naphthylamide, and a colored diazonium compound.The method permits the separation of up to four different isoenzymes, which we called-GT₁,-GT₂,-GT₃,-GT₄, the first two showing an electrophoretic migration similar to that of ₁- and ₂-globulins and the other two to that of-globulins.The present technique has proved its usefulness in detecting isoenzymes in serum with values of total-glutamyl-transpeptidase higher than 80 U/L.The application of this method in 52 patients with different types of biliary obstruction and hepatocellular damage has shown that it provides new possibilities in differential diagnosis.     

„Leptospira Mini“ ein neuer Serotyp pathogener Leptospiren

Zusammenfassung Auf Grund ihres immunbiologischen Verhaltens gehören die Leptospirenstämme Sari, Ghidorsi und Szwajizak zu demselben serologischen Leptospirentyp, für den der Name Leptospira Mini vorgeschlagen wird.Der Stamm Sari wurde 1942 vonMino sowieVercelli in Italien isoliert. Der Stamm Ghidorsi wurde von uns im Zuge unserer Leptospirenforschungen bei einer Reisfeldarbeiterin der Po-Ebene nachgewiesen. Der Stamm Szwajizak, der vonSmith, Brown, Tonge u. Mitarb. im Jahre 1954 beschrieben wurde, ist in Nord-Qeensland gefunden worden. Der Stamm Sari und Ghidorsi gehören dem kompletten Biotyp (AB), der Stamm Szwajizak dem inkompletten (A) an.Leptospira Mini gehört zur Serogruppe hebdomadis. Ihre Virulenz ist schwach und ihre Bedeutung als Erreger menschlicher Leptospiren-infektionen scheint gering zu sein.