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.     

Recognition by peptide mapping of three different structural groups of outer membrane protein YOP-1 of Yersinia enterocolitica and Yersinia pseudotuberculosis

The structural relation of YOP-1 of european and american Yersinia enterocolitica serotypes O3, O9, O5, 27, and O8 and O20, respectively, and Y. pseudotuberculosis serotypes I, II, and III was compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping using Staphylococcus aureus protease V8. Apparent molecular weights of YOP-1 ranged from 206,000 (O3) to approx. 180,000 (O8). According to their respective peptide maps YOP-1 of the european and american Y. enterocolitica serotypes and Y. pseudotuberculosis serotypes could be assigned to three different groups. Evaluation of several isolates of Y. enterocolitica serotypes O3, O9, and O8 by peptide mapping indicated that YOP-1 is conserved within a serotype. However, one serotype O8 isolate differed from the consensus peptide pattern of the other serotype O8 and O20 isolates. The similarity of the peptide patterns of Yersinia serotypes which predominate in certain geographical locations, i. e., european and american Y. enterocolitica serotypes, suggest common evolution of YOP-1 of these serotypes independent of the evolution of the other serotypes.     

Disturbance of delayed match-to-sample in macaques by tetanization of anterior commissure versus limbic system or basal ganglia

Summary Three pig-tailed macaques were trained to select (match) from a pair of colored images that which they had seen (sample) and responded to 5–15 s previously. The anterior commissure (AC) and/or its radiation, various loci in basal ganglia, hippocampal formation and control areas, (splenium of corpus callosum, precentral gyrus, insular cortex), totalling 40 loci, were each tetanized for 4 s during presentation of the sample image, during the delay period, or when the monkey was required to select the matching image. For several loci in the hippocampal formation tetanization at any phase of the task reduced matching to chance levels and gave evidence of electrical after-discharge; but other comparable hippocampal loci had little or no effect. Response to sample or match stimuli were absent during tetanization of basal ganglia or anterior commissure. When finally made, upon cessation of tetanization, responses were equally correct for basal ganglia and control sites, but for AC were at chance levels.     

Effects of sleep deprivation on the activity of selected metabolic enzymes in skeletal muscle

Summary The present study gives the results of a comparison of the recorded and true tibia-calcaneal angles in 17 normal subjects and in 14 patients with abnormally hypoextensible non contracting triceps. 1. For a minimal passive torque, the difference between true and recorded angles varied considerably from one individual to another. The means and ranges for the two groups were respectively: –8 (+7, –21) and –7 (+5, –20). 2. When the passive torque increased as a result of slow passive lengthening of the muscle, the true curve was steeper than the recorded one, owing to differences between the two angle measurements. For each of the two groups the differences in means and ranges were respectively: 6 (0, +13.5) and 8 (3, 12). 3. Subjects made isometric voluntary contractions of the triceps surae at fixed angles which corresponded to step by step muscle lengthening. The resulting true curve was much steeper than the recorded curve. The differences in means and ranges were: 7 (1.5, +15) in children of the two groups and respectively 3 (0, +9) and 12 (10, 14) in adults of the two groups. The present results show that this methodology was the only reliable way of correctly obtaining passive and active torque-angle curves, measuring differences between subjects, appreciating the effects of treatments and these by ascertaining whether or not trophic muscle regulation was defective.     

The Involvement of Dopamine in Strengthening Cortical Signals Activating NMDA Receptors in the Striatum (a hypothetical mechanism)

A possible mechanism is proposed for the enhancement/weakening of those cortical signals in the cortex-basal ganglia-thalamus-cortex neural network which induce/do not induce opening of NMDA channels in the spiny neurons of the striatum and which can be regarded as strong/weak in terms of this measure. The mechanism is based on the modulatory influences of dopamine on changes in the efficiency of corticostriatal inputs. In the absence of dopamine, relative increases in the intensity of strong (weak) cortical signals can lead to the induction of long-term potentiation (depression) of corticostriatal synapses. In this case, because of the differently directed influences on thalamic cells of signals passing via strionigral and striopallidal cells, strong signals at the output of the thalamus are weakened, while weak signals are strengthened. Activation of dopamine D₁ (D₂) receptors on strionigral (striopallidal) neurons may facilitate increases in the extent of long-term potentiation/depression (decreases in the extent of long-term potentiation/depression or induction of long-term potentiation/depression). The consequence of this is that strong signals at the output of the thalamus can be strengthened synergistically, while weak signals cab be weakened synergistically. Background cortical signals evoking tonic release of dopamine in the striatum can decrease strengthening because of weakening of the modulatory influence of dopamine on the modification of corticostriatal synapses.     

Summation and adaptation in stimulation of the motor cortex in adult and newborn animals

Summary In rabbits and their progeny an investigation was made of summation and adaptation of the neurones of the motor cortex; unipolar stimulation was used, and the muscular response recorded. Two types of summation curves were found: exponential and Y-shaped,, and were related to the direct and indirect summation of stimulation. In adults, Y-shaped summation curves were more frequent, whereas in rabbits aged 24 h exponential curves preponderted. We found very little capacity for adaptation at any age.(Presented by Active Member AMN SSSR A. F. Turom) Translated from Byulleten Éksperimental'noi Biologii i Medisiny, Vol. 55, No. 6, pp. 3–7, June, 1963     

Eine neue Bestimmung der Neutralfette im Blutserum und Gewebe

Zusammenfassung Die Neutralfette im Blutserum und Gewebe lassen sich über Glycerin exakt ermitteln. Freies und nach alkoholischer Verseifung nachweisbares Gesamt-Glycerin werden mit Glycerokinase in einer dreistufigen enzymatischen Nachweisreaktion im Mikroverfahren bestimmt. Die Differenz wird als Glycerid-Glycerin angesprochen. Reaktionsprinzip, Durchführung der Methode und Berechungsweise werden ausführlich besprochen.Für freies Glycerin werden 0,2 ml Serum, für Gesamtglycerin etwa 0,007–0,015 ml Serum äquivalente Hydrolysatmengen in den Test eingesetzt. Bei einer Empfindlichkeit von 0,0008 µMol (wenn bei 340 nm, bzw. 0,0015 µMol Glycerin, wenn bei 366 nm gemessen wird), können noch 1/20 bis 1/10 des im Normalserum vorhandenen freien bzw. Gesamtglycerins mit den Standardansätzen erfaßt werden. Die obere Nachweisgrenze reicht dann bis 0,15 bzw. 0,3 µMol Glycerin, das entspricht der 8- bzw. 15fachen Normalmenge für freies und Gesamtglycerin. In den angegebenen Grenzen liegen die über Glyceridglycerin nachweisbaren Triglyceridkonzentrationen. Empfindlichkeit und obere Nachweisgrenze können durch andere Serum- bzw. Hydrolysatmengen in weiten Grenzen variiert und der jeweiligen Fragestellung angepaßt werden.

---

Summary Triglycerides in blood serum and tissue can be identified exactly by means of glyceride-glycerol. Free and total glycerol, measurable after alcoholic saponification, are determined by glycerolkinase in a three-step enzymatic proof-reaction by means of micro-methods. The difference between total and free glycerol is called glycerideglycerol. Reaction principles, working instructions and the calculation will be discussed in detail.The sensitivity of the standard test runs to 0,0008 respeccively 0,0015 µMol glycerol or 1/20 resp. 1/10 of the normal contentrations. The upper proof limit is at 0,15 respectively 0,3 µMol glycerol per test. This is equal to 8–15 times more than the normal of free and total glycerol. Sensitivity and upper proof limit can easily by varied and adapted to the formulation of the question.

     

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.     

Further observations on the morphogenesis of human rotavirus in MA 104 cells

Summary Human rotavirus KUN strain was cultivated in a fetal rhesus monkey kidney cell line, MA 104 cells. Four types of virus particles in cells infected with KUN strain were clearly identified: nucleoid cores, single-shelled particles, double-shelled particles, and membrane band, enveloped particles. Enveloped particles were found only in the thin sections of infected cells. When first visible, the virus precursors appeared at the ribosome free membrane of rough endoplasmic reticulum (RER), increasing in size while simultaneously being coated with nucleocapsid, inner shell. Single-shelled particles were also synthesized within bundles of filaments of viroplasm in the cytoplasma. During subsequent virus maturation two types of budding processes were observed. Double-shelled particles arising at the RER membrane entered the cisternae of the RER through an exocytosis-like process. In contrast, the enveloped particles developed in the cisternae by being completely enclosed with RER membrane, and later during cytolysis released the single-shelled particles. These enveloped virus particles appeared to be the result of inefficient virus maturation at the last stage of outer capsid formation.With 11 Figures     

Farmerlunge

Ohne ZusammenfassungAus den Laboratorien der Marshfield Clinic und Marshfield Clinic Foundation for Medical Research and Education, Marshfield, Wisconsin, USA. Die Arbeit wurde durchgeführt mit Unterstützung des US Public Health Service, Bewilligungsnummer UI-00398.Die Autoren sind Dr.Dieter M. Voss für seine wertvolle Hilfe bei der Vorbereitung des Manuskriptes zu Dank verpflichtet.Deutsche Übersetzung: Dr.M. v. Bubnoff, München.     

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.

---

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.

---

---

Herrn ProfessorG. Schaltenbrand zum 70. Geburtstag gewidmet.     

Tetrodotoxin exerts a large frequency-dependent depression of the maximum rate of rise of action potentials in guinea pig ventricular myocytes

The action potential configuration in guinea pig ventricular myocytes was unaffected by low concentrations (0.3–1 M) of tetrodotoxin (TTX); high concentrations (10–30 M) depressed both the overshoot (5–10 mV) and duration (5–10%). Although the control was unaffected by stimulation rate (0.1–5 Hz), the depression of by TTX was greatly potentiated at rates above 1 Hz: on dose-response curves, 50% control occurred at 4.3 M (5 Hz) versus 22 M ( 1 Hz). The frequency dependent component of the depression reported here is much larger than the extra block of Na channels observed by others in voltage clamp studies on Purkinje strands. This is not a discrepancy; rather it is a consequence of a non-linear relation between and available Na conductance.     

Ablation of primary afferent terminals reduces nicotinic receptor expression and the nociceptive responses to nicotinic agonists in the spinal cord

A variety of studies indicate that spinal nicotinic acetylcholine receptors modulate the behavioral and autonomic responses elicited by afferent stimuli. To examine the location of and role played by particular subtypes of nicotinic receptors in mediating cardiovascular and nociceptive responses, we treated neonatal and adult rats with capsaicin to destroy C-fibers in primary afferent terminals. Reduction of C-fiber terminals was ascertained by the loss of isolectin B4, CGRP and vanilloid receptors as monitored by immunofluorescence. Receptor autoradiography shows a reduction in number of epibatidine binding sites following capsaicin treatment. The reduction is particularly marked in the dorsal horn and primarily affects the class of high affinity epibatidine binding sites thought to modulate nociceptive responses. Accompanying the loss of terminals and nicotinic binding sites were significant reductions in the expression of 3, 4, 5, 2 and 4 nicotinic receptor subunits in the superficial layers of the spinal cord as determined by antibody staining and confocal microscopy. The loss of nicotinic receptors that follows capsaicin treatment results in attenuation of the nociceptive responses to both spinal cytisine and epibatidine. Capsaicin treatment also diminishes the capacity of cytisine to desensitize nicotinic receptors mediating nociception, but it shows little effect on intrathecal nicotinic agonist elicited pressor and heart rate responses. Hence, our data suggest that 3, 4, 5, 2 and 4 subunits of nicotinic receptors are localized in the spinal cord on primary afferent terminals that mediate nociceptive input. A variety of convergent data based on functional studies and subunit expression suggest that 3 and 4, in combination with 2 and 5 subunits, form the majority of functional nicotinic receptors on C-fiber primary afferent terminals. Conversely, spinal nicotinic receptors not located on C-fibers play a primary role in the spinal pathways evoking spinally coordinated autonomic cardiovascular responses.     

Effect of Individual and Combination Treatment with Cytokines on Expression of Sialoadhesin by Bone Marrow Macrophages

We studied the effects of lipopolysaccharide, interferon-, interleukin-1, or tumor necrosis factor- and combination treatment with tumor necrosis factor- and interleukin-1 or interferon- on the expression of sialoadhesin receptors on the membrane of bone marrow macrophages, macrophage adherent ability, and production of nitric oxide by these cells. Sialoadhesin expression was evaluated by binding of macrophages to sheep erythrocytes labeled with radioactive ⁵¹Cr. Treatment of bone marrow cells with interferon- improved adhesive properties of macrophages, but did not modulate expression of sialoadhesin. Interleukin-1 and tumor necrosis factor- had no effect on the test parameters. Combination treatment with these cytokines enhanced binding of sheep erythrocytes to macrophages. Administration of lipopolysaccharide or combination treatment with interferon- and tumor necrosis factor- increased the count of macrophages adhering to plastic and stimulated expression of sialoadhesin. Combination treatment with interferon- and tumor necrosis factor- stimulated production of nitric oxide by bone marrow macrophages. Blockade of nitric oxide synthesis had no effect on adhesive properties of macrophages and expression of sialoadhesin.     

Endothelial cell dynamics under pulsating flows: significance of high versus low shear stress slew rates (d(tau)/dt)

Shear stress modulates endothelial cell (EC) remodeling via realignment and elongation. We provide the first evidence that the upstroke slopes of pulsatile flow, defined as shear stress slew rates (positive / affect significantly the rates at which ECs remodel. We designed a novel flow system to isolate various shear stress slew rates by precisely controlling the frequency, amplitude, and time-averaged shear stress _ave of pulsatile flow. Bovine aortic endothelial cell (BAEC) monolayers were exposed to three conditions: (1) pulsatile flow (1 Hz) at high slew rate (293 dyn/cm² s), (2) pulsatile flow (1 Hz) at low slew rate (71 dyn/cm² s), and (3) steady laminar flow at /t=0. All of the three conditions were operated at _ave=50{dyn/cm}². BAEC elongation and alignment were measured over 17 h. We were able to demonstrate the effects of shear stress slew rates /t on EC remodeling at a fixed spatial shear stress gradient (/x). We found that pulsatile flow significantly increased the rates at which EC elongated and realigned, compared to steady flow at /t=0. Furthermore, EC remodeling was faster in response to high than to low slew rates at a given tau _ave © 2002 Biomedical Engineering Society. PAC2002: 8719Tt, 8719Rr     

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     

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.     

Zur Ätiologie der Fibrosis mammae virilis

Zusammenfassung Wenn wir das Ergebnis unserer eigenen Untersuchungen mit den oben besprochenen Mitteilungen aus dem Schrifttum zusammenfassen, so ergeben sich zur Ätiologie der Fibrosis mammae virilis nach unserer Auffassung folgende Tatsachen:Grundsätzlich erscheint es notwendig, die Fibrosis mammae virilis, d. h. die umschriebenen knotenförmigen Bildungen in den männlichen Brustdrüsen von der diffusen Brustdrüsenvergrößerung bei Männern, der Gynäkomastie zu trennen. Die Benennung Gynäkomastie sollte nach unserer Auffassung jenen Fällen vorbehalten werden, die meist als Folge klinisch nachweisbarer innersekretorischer Störungen auftreten und auch mit der Neubildung echter Acini einhergehen können, wie es bei Chorionepitheliomen nachgewiesen wurde.Es muß angenommen werden, daß auch für die Entstehung der Fibrosis mammae virilis innersekretorische Störungen in Frage kommen, die zum Teil ernährungsmäßig bedingt sein dürften. Wir sehen in dem Krankheitsbild keine echte Geschwulstbildung sondern eine sehr wahrscheinlich innersekretorisch bedingte Hypertrophie und Hyperplasie des männlichen Brustdrüsenparenchyms, die knotenförmig, einseitig oder auch doppelseitig, oft nacheinander auftreten kann. Die ersten Veränderungen entstehen offenbar im Bereich der Drüsenausführungsgänge bzw. des Drüsenepithels, von dem aus sekundär das Wachstum und die Anordnung des Bindegewebes induziert wird.Herrn Professor Dr.Huebschmann zum 70. Geburtstag.     

Analysis of cytosolic and lysosomal pH in apoptotic cells by flow cytometry

Several reports indicate that the cytosol is acidified during apoptosis although the mechanism is not yet fully elucidated. The most acidic organelle found in the cell is the lysosome, raising the possibility that lysosomal proton release may contribute to the cytosolic acidification. We here describe methods for measurement of the cytosolic and lysosomal pH in U937 cells by a dual-emission ratiometric technique suitable for flow cytometry. Cytosolic pH was analysed in cells loaded with the fluorescent probe BCECF, while lysosomal pH was determined after endocytosis of FITC-dextran. Standard curves were obtained by incubating cells in buffers with different pH in the presence of the proton ionophore nigericin. Apoptosis was induced by exposure of cells to 10ng/ml TNF- for 4h, and apoptotic cells were identified using a fluorescent marker for active caspases. By gating of control and apoptotic cells, the cytosolic and lysosomal pH were calculated in each population. The cytosolic pH was found to decrease from 7.2 ± 0.1 to 5.8s±0.1 and the lysosomal increased from 4.3±0.4 to 5.2±0.3. These methods will be useful in future attempts to evaluate the involvement of lysosomes in the acidification of the cytosol during apoptosis.     

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.