Electrophysiology of Purkinje neurons in the weaver mouse: iontophoresis of neurotransmitters and cyclic nucleotides, and stimulation of the nucleus locus coeruleus. We compared the Purkinje cells of adult normal and weaver mutant (wv/wv) mice by iontophoretic and electrophysiological tests. Although weaver Purkinje cells fire spontaneously at a rate (38 Hz) similar to normal mouse neurons (40 Hz), several abnormalities of firing were seen: high frequency bursts of single (simple) spikes occurred in 5-10-sec episodes in 38% of weaver cells, compared to 8% in normal mice; spontaneous complex spikes (climbing fiber-like burst responses) occurred in several different forms in a given Purkinje cell. As in normal mice and rats, the spontaneous single spike activity is readily depressed by electrical stimulation of the locus coeruleus, the presumed source of a dense noradrenergic plexus in the weaver cerebellar cortex. In a preliminary experiment the adrenergic blocking agent, fluphenazine, antagonized the responses to locus coeruleus stimulation. Iontophoresis of norepinephrine (NE), GABA and serotonin (5-HT) also uniformly depressed Purkinje cell single spike activity in all normal and weaver mice; cyclic AMP depressed 55% of normal and 70% of weaver Purkinje cells. Glutamate was always excitatory. The only qualitative difference was seen with acetylcholine, which was mostly inhibitory in normal mouse, but increased the firing rate in 42% of weaver Purkinje neurons. Cyclic GMP was predominantly excitatory in both types. Thus, despite the absence of parallel fibers, weaver Purkinje neurons grossly resemble normal Purkinje cells electrophysiologically as well as morphologically. Since several sites of indirect presynaptic actions are eliminated in weaver, our results further substantiate the direct post-synaptic inhibitory nature of GABA, 5-HT and NE, and the noradrenergic pathway from locus coeruleus to Purkinje cells. Similarly, consistent inhibitory responses to cyclic AMP in the weaver support the previously hypothesized role of cyclic AMP in the post-synaptic inhibitory response to NE.


Enzymic preparation of neurosecretosome- and pituicyte-enriched fractions from the rat neurohypophysis. A microcellular dispersion procedure for the rat neurohypophysis was developed, comprising tissue softening and dissociation using a special sieving sytringe. In preparatory studies the influence of mesh width, and treatment with trypsin, pronase or collagenase-hyaluronidase was investigated using light and electron microscopy, as well as with microchemistry by means of protein and lactate dehydrogenase activity determinations. Trypsinization gave the best results. In the final adopted procedure, 3 incubated neurohypophyses were sequentially sieved through a 200- and a 50-mum mesh. The resulting 50-mul dispersion was found to contain numerous ultrastructurally well-preserved pinched-off axonal endings (neurosecretosomes), and pituicytes often revealing processes. On the basis of DNA and oxytocin assays 11% of the pituicytes and 28% of the axonal cytoplasm were recovered. Oxytocin immunofluorescence microscopy showed hormone within the neurosecretosomes, but often also in the cytoplasm of pituicytes. Microdensity gradient centrifugation was performed on neurohypophyseal disperions, in order to obtain fractions enriched for neurosecretosomes and pituicytes. Fractions were characterized by means of phase contrast, oxytocin immunofluorescence and electron microscopy, as well as by oxytocin and DNA assays as respective markers. With a 10:14:22% (w/v) Ficoll gradient, fractions were obtained for which the relative purification was by a factor of 4 on the basis of DNA/oxytocin ratios.


Effects of ergosterol on bone mineralisation in chicks given cholecalciferol or ergocalciferol. Groups of chicks were given diets containing cholecalciferol or ergocalciferol supplemented with 0, 0-1, 1 or 10 g ergosterol/kg. 2. Ergosterol had no significant effect on growth, on the plasma concentration of calcium or on the content of bone-ash, indicating that it did not impair the absorption of either form of vitamin D. 3. An explanation is given for the apparent disagreement in the published findings on the relative anti-rachitic potencies of ergocalciferol and cholecalciferol in the chick.


[Correlation between permeabiltiy to albumin and levels of cyclic adenosine monophosphate in the incubated rat mesentery]. Histamine, bradykinin and prostaglandin E1 increase the permeability coefficient of albumin; this effect is mimicked by dibutyryl-cAMP and by theophylline. They also cause a significant rise in the cAMP levels of the incubated tissue. Serotonin has lesser activities on these parameters. Presumably, the first three mediators control the transcellular passage of albumin by increasing the cAMP content of the tissue, serotonin being less effective. These findings on the mesentery seem to have a couterpart in vascular tissue as shown by others using different methods.


[Action of lithium salts on hepatocytes in histiotypic culture]. Addition of lithium salts to cultures of hepatocytes increases glycogen in these cells. Glycogenolytic action of glucagone or AMPc becomes slow. Toxic action of paraquat is suppressed.


[Demonstration of specific receptor sites of 3, 5, 3'-triiodo-L-thyronine on isolated rat hepatocytes]. The polarographic respiration studies of isolated liver cells from normal and thyroidectomised rats show that oxygen consumption rate is decreased by 40% after thyroidectomy. The number of specific binding sites estimated with 125I labelled T3 experiments, is about 130 per cell.


[Mast-cell rosettes in the immunized rat]. After stimulation by ovalbumin + Bordetella pertussis, rat peritoneal mast cells can form rosettes with antigen-coated erythrocytes. This phenomenon is inhibited by previous incubation of mast cells with antigen; it is attributed to apparition of membrane-bound anaphylactic antibody. The variations of percentage of rosette forming mast cells during the course of immunization are reported and compared with P.C.A. titers.


[Induction of cytochrome P-450 and b5 of the mouse liver by phenytoin during chronic hypoxia (Fi02 : 0,08)]. During chronic hypoxia at sea level (FiO2 : 0,08) the liver concentrations of cytochrome P 450 and b5 decreased. Phenytoin administration induces an increase in cytochrome P 450 and b5 liver content. The hydroxylation of phenytoin that was decreased at low level of cytochrome P 450 and oxygen, reached again a normal level after induction of cytochrome P 450 in spite of the persistency of a low level of available oxygen. Liver cytochrome P 450 concentration is the main limiting factor of phenytoin hydroxylation.


[Role of various regulation factors of interrenal gland activity in response to stress in the green frog Rana esculenta L]. The effects of an ether stress on the corticosterone levels were studied in variously experimented frogs. The removal of the distal lobe of the pituitary gland, with preservation of the intermediate lobe which contains also ACTH, suppresses, as total hypophysectomy, the increase of corticosterone level observed in normal frogs. The administration of dexamethasone reduces but not suppresses this increase. A second ether stress 20 minutes after the first is effective. The injection of frog plasma treated or not with an corticosteroid adsorbant do not alter the response to stress. These results suggest that modifications of corticosterone level, in some limits, do not disturb the effects of a stress.


[Renal production and excretion of AMPc after intravenous infusion of salmon calcitonin in man]. Intravenous infusion of salmon calcitonin in man produced an increase in the plasma levels and urinary excretion of cyclic AMP. This study demonstrates a net extraction of cyclic AMP from plasma by the kidneys but salmon calcitonin does not act only on the kidney and stimulates the production of cyclic AMP in extra renal tissues. The excess of cyclic AMP formed is catabolized by the kidneys.


[Immunization of the nude mouse against mouse hepatitis virus through the transfer of sensitized lymphocytes]. Nude (nu/nu) mice failed to resist to virulent mouse hepatitis virus (MHV) infection after vaccination with inactivated virus. Resistance was induced in nu/nu mice by the transfer of spleen cells from heterozygous haired (nu/ +) mice concomitantly with the vaccination, and the effect was more remarkable with spleen cells from immunized nu/ + mice. Antibody was demonstrable in nu/nu mice having received nu/ + cells and survived challenge infection.


Neuromuscular block by neomycin in the cat. The neuromuscular block produced by neomycin is unique. Despite nearly complete blockade of the twitch the tetanus is not blocked and does not fade. The pathognomonic post-tetanic exhaustion has not been reported previously. Anticholinesterases and calcium antagonize the block, but the antagonism may not be complete. Germine antagonizes the block in the cat, but its clinical value has not been tested. These characteristics of neuromuscular block by neomycin strongly suggest respiratory support and sedation to decrease the respiratory drive, rather than attempts at antagonism, as the rational management of patients suffering from toxic paralysis due to neomycin.


Synthesis of diphosphoinositide by a supernatant fraction from Acanthamoeba castellanii. Homogenates of the free-living amoeba Acanthamoeba castellanii incorporate phosphate from [gamma-32P]ATP into a lipid which co-chromatographs with diphosphoinositide on one- and two dimensional chromatography. Incorporation into lipids similar in mobility to triphosphoinositide is not detected. The product co-chromatographs with diphosphoinositide whether exogenous phosphatidylinositol or total amoeba lipid is the substrate. The inositide kinase is almost entirely located in the supernatant fraction after centrifugation at 100 000 g. Incorporation of phosphate from [gamma-32P]ATP is linear for at least 15 min in the presence of 0.5 mM phosphatidylinositol. The enzyme requires Mg2+ of Mn2+ as well as ATP and it is not affected by low concentrations of Ca2+. The apparent Km for phosphatidylinositol in 2 mM. Both ADP and cAMP inhibit the reaction.


Effects of indomethacin and aminoglutethimide phosphate in vivo on luteinizing-hormone-induced alterations of cyclic adenosine monophosphate, prostaglandin F, and steroid levels in preovulatory rat ovaries. Changes in levels of cyclic adenosine monophosphate (cAMP), prostaglandin F (PGF), progesterone, testosterone, and estradiol-17beta, in preovulatory rat ovaries induced by exogenous luteinizing hormone (LH) have been measured. Ovarian cAMP reached maximal levels 15 min and 1h after LH administration by intravenous and intraperitoneal routes, respectively, and then declined to pre-LH levels by 8 h. Progesterone levels in ovaries and serum rose approximately in parallel with cAMP, but remained elevated throughout the 8-h sampling period. Ovarian testosterone increased to maximal levels 1 h after LH injection, followed by a rapid decline to below pre-LH levels. Ovarian estradiol-17beat concentrations declined steadily throughout the sampling period, reaching almost undetectable levels 8 h after LH treatment. Elevated ovarian PGF levels were observed only at the 4- and 8-h sampling times. Indomethacin treatment, 1 h before LH, prevented the LH-induced increase in ovarian PGF levels, depressed PGF values considerably in saline-injected controls but produced no significant inhibition of ovarian cAMP and progesterone levels. Aminoglutethimide phosphate depressed ovarian concentrations of all three steroids (progesterone, testosterone, and estradiol-17beta) to essentially undetectable levels, both in control and LH-injected rats, but did not alter the LH-induced changes in ovarian cAMP and PGF levels. These observations support the concept of cAMP as a mediator of the LH-induced alterations of ovarian steroidogenesis in vivo during the preovulatory period, but argue against an obligatory role of PGF in this process.


Isolation and carbohydrate composition of glycopeptides of human apo low-density lipoprotein from normal and type II hyperlipoproteinemic subjects. Glycopeptides were prepared from the delipidized protein of low-density lipoprotein (LDL, d=1.019-1.063) of three normal and three familial heterozygous type II hyperlipoproteinemic (HLP) subjects. The glycopeptides of all subjects were resolved into three groups by gel filtration on Bio-Gel P6 following papain (EC 3.4.22.2) digestion and initial purification on Bio-Gel P2. In normal individuals the component of largest molecular weight (F-1) contained mannose (Man), N-acetyl glucosamine (GlcNAc) galactose (Gal), and N-acetyl neuraminic acid (NANA) in the respective amounts of 45.9 +/- 6.7, 37.3 +/- 5.9, 28.6 +/- 3.4, and 27.0 +/- 3.9 nmol/mg original apoprotein. The group of smallest molecular weight (F-3) contained essentially only Man (25.8 +/- 1.5 nmol/mg protein) and GlcNac (3.0 +/- 0.4 nmol/mg protein) with traces of Gal and NANA. A group of intermediate molecular weight (F-2) exhibited considerable heterogeneity and contained Man, GlcNAc, Gal, and NANA in the amounts of 45.9 +/- 5.1, 18.3 +/- 1.7, 11.0 %/- 1.7, and 7.7 %/- 1.2 nmol/mg protein. While the major portion of NANA (78%), Gal (71%), and GlcNAc (64%) was present in F-1, approximately 22% of the total Man was in F-3. No major differences were detected in the carbohydrate composition of the three glycopeptide fractions of LDL apoptotein from normal and Type II subjects.


Regulation by zinc and adenosine 3',5'-cyclic monophosphate of growth and citric acid accumulation in Aspergillus niger. The citric acid fermentation by Aspergillus niger is divided into two consecutive phases, growth phase when the cells proliferate but do not accumulate citrate, followed by an accumulating phase when they excrete citrate but do not proliferate. The phase alternative was controlled by the concentration of zinc: high zinc (2 muM) maintained growth phase, and a zinc 'deficiency' apparently signaled the ransition to the accumulating phase. Cyclic AMP affected the rates of growth and acidogenesis when added to cultures growing at low but not at high zinc: that is, cAMP did not induce the phase transition, zinc deficiency did, Cyclic AMP enhanced growth early in the fermentation, but at later stages the response of the mycelia to cAMP changed, and then cAMP inhibited growth. When citrate eventually began to accumulate cAMP augmented its sypthesis. The growth and acidogenic responses were quite specific to cAMP, and were sensitive to concentrations of about 1 muM. Cyclic AMP also either promoted or retarded the appearance of an unidentified yellow pigment. It is proposed that the growth and accumulating phases are distinct differentiated states, at least with respect to cAMP metabolism.


A general survey of the proton spin-lattice relaxation-times of some oligo- and poly-saccharide derivatives. A Fourier-transform method has been used to measure the spin-lattice relaxation-times (T1 values) of the anomeric protons of a selection of oligo- and poly-saccharide derivatives. Although systematic variations are found for the substances of lower molecular weight, these variations are essentially non-existent at higher molecular weights. Data for the disaccharides cellobiose, maltose, lactose, gentiobiose, and melibiose demonstrate that proton T1-values may provide a powerful method for evaluating conformations of oligosaccharides.


Concerning the independence of the basis of hypertension due to ACTH or renovascular constriction. Administration of ACTH to four sheep with chronic renovascular hypertension resulted in an increase in blood pressure which was at least as high as that described in normotensive sheep and could be completely accounted for by an increase in cardiac output.


Adenohypophysial cell-types in the pituitary gland of the soft-shelled turtle, Trionyx sinensis. I. Seasonal cycles. By using various staining techniques, two acidophilic and three basophilic cell-types have been distinguished in the pars distalis of Trionyx sinensis. Seasonal fluctuations in their cytoplasmic granulation, cell and nuclear size have been compared with seasonal patterns of activity in the gonads, adrenals and thyroid. On the basis of the results, specific functions have been ascribed to each cell-type. Basophils 1 activity closely parallels the seasonal thyroid cycle. Basophils 2 are probably FSH-secreting gonadotropes and their activity correlates with the seasonal cycle of spermatogenesis in the seminiferous tubules. The functional activity of the testicular interstitial cells correlates with the secretory pattern of the basophils 3. That these are LH-secreting cells is also suggested by the epididymidal cycle, which indicates a high level of androgen secretion coincident with the rapid degranulation of these cells, at a time of no spermatogenetic activity. Adrenal activity shows no relationship with the basophils 3 cycle, and chromophobic cells may be the locus of ACTH secretion. Acidophils 2 remain stable throughout the year and are probably somatotropes, but acidophils 1 increase their activity in autumn and are likely to be lactotropes.


Ultrastructural and cytochemical features of SV 40 transformed hypothalamic cell lines. A continuous cell line was previously obtained by Simian Virus (SV) 40 transformation of primary cultures of dissociated mouse fetal hypothalami. One clone from this cell line has been previously shown to possess some of the ultrastructural features, immunological properties and synthesizing capacities of magnocellular hypothalamic neurons which secrete vasopressin and neurophysins. The present paper reports on the morphological characterization of 14 other clones or subclones of the original cell line, using the following criteria: phase contrast microscopy, electron microscopy, Gomori's aldehyde fuchsin staining, cytochemical detection of beta-glucuronidase, immunochemical staining with antisera against bovine neurophysin I, bovine neurophysin II, lys-vasopressin, oxytocin, LH-RH and TRH. The results allowed the conclusion that the clones as well as the subclones can be distributed into two groups: 1) neurosecretory neurons which all possess several of the ultrastructural and cytochemical features of the neurophysin-vasopressin synthesizing clone, and 2) primitive nerve cells which are devoid of such features but display numerous bundles of filaments. In addition some clones were found to display intermediate features between groups 1 and 2. A similar diversity was observed within clones of the original strain and subclones of a neurosecretory clone. It is suggested that the primitive clones could represent precursors of the neurosecretory clones.


A unique RNA species involved in initiation of vesicular stomatitis virus RNA transcription in vitro. Purified virions of vesicular stomatitis virus (VSV) are capable of synthesizing two distinct types of virus-specific RNA in vitro. The first consists of several viral mRNAs which have been previously shown to contain the blocked 5' terminal sequence GpppApApCpApGp and 3' terminal poly(A). The second type of RNA has an unblocked 5' terminus and does not contain poly(A) stretches long enough to bind to oligo (dT)-cellulose columns. It migrates in 20% polyacrylamide gels as a single homogeneous peak with an estimated chain length of 68 nucleotides. Base analysis demonstrated that this small RNA molecule is composed of 48% AMP, 20% CMP, 11% GMP, and 21% UMP. The 5' terminal sequence of the small RNA is ppApCpGp, which appears to be complementary to the 3' terminal sequence of the VSV genome RNA (...PypGpU). These results indicate that this small RNA molecule probably represents the intitiated lead-in RNA segment which is removed during formation of VSV mRNAs by a possible processing mechanism.


The number of superhelical turns in native virion SV40 DNA and minicol DNA determined by the band counting method. By a method of overlapping the results obtained after agarose gel electrophoresis under two different sets of conditions, it has become possible to determine the number of superhelical turns in a given DNA by counting the bands present after partially relaxing the DNA (Keller and Wendel, 1974) with highly purified nicking-closing (N-C) enzyme from LA9 mouse cell nuclei. Because native supercoiled DNA is heterogeneous with respect to superhelix density, an average number of superhelical turns was determined. Virion SV40 DNA contains 26 +/- 0.5 superhelical turns, and native Minicol DNA contains 19 +/- 0.5 superhelical turns. The above are values at 0.2 M NaCl and at 37 degrees C, the condition under which the enzymatic relaxations were performed. The superhelix densities determined by the band counting method have been compared with superhelix densities determined by buoyant equilibrium in PDl-CsCl gradients. The Gray, Upholt, and Vinograd (1971) calculation procedure has been used for evaluating the superhelix densities by the latter method with the new statement, however, that relaxed DNA has zero superhelical turns. Comparison of the superhelix densities obtained by both methods permits a calculation of an unwinding angle for ethidium. The mean value from experiments with SV40 DNA is 23 +/- 3 degree. The average number of superhelical turns in SV40, 26, combined with the value, 21, obtained by both Griffith (1975) and Germond et al. (1975) for the average number of nucleosomes per SV40 genome, yields an average of 1.25 superhelical turns per 1/21 of the SV40 genome. If the regions of internucleosomal DNA are fully relaxed, 1.25 correesponds to the average number of superhelical turns with a nucleosome. When analyzed under identical conditions, the limit product generated by ligating a nicked circular substrate in the presence of 0.001 M Mg2+ at 37 degrees C (ligation conditions) is slightly more positively supercoiled than the limit product obtained when the N-C reaction is performed in 0.2 M NaCl at 37 degrees C. The difference in superhelix density as measured in gels between the two sets of limit products for both Minicol and SV40 DNAs is 0.0059 +/- 0.0005. This result indicates that the DNA duplex is overwound in the ligation solvent relative to its state in 0.2 M NaCl.


5' terminal nucleotide sequences of the messenger RNA's of Dictyostelium discoideum. As in the mRNA from all other eucaryotic cells examined, the 5' nucleotide in messenger RNA from Dictyostelium discoideum is linked by a 5'-5' triphosphate bridge to the unusual nucleoside 7-methyl guanosine. In mammalian cellular mRNA, the 5' terminal sequences have the general formula m7GpppXmpYp(m), where X and Y can be either purine or pyrimidine nucleotides and Y, as well as X, may contain a 2'0-methylated ribose. Although at least 32 5' terminal sequences are possible in cellular mRNA, only four are present in Dictyostelum mRNA. They are (I) m7GppppAp (65%); (II) m7GpppGp (10%); (III) m7GpppAmpAp (10%); (IV m7GpppAp (65%); (II) m7gpppGp (10%); (III) m7GpppAmpAp (10%); (IV) m7GpppAmpUp (10%). Sequences I and II are simpler than those previously reported for mammalian cells because they lack 2'0-methylated nucleosides. Another difference is that in all Dictyostelium mRNAs. the nucleoside X is a purine. The nucleoside 6-methyl adenosine which is found internal to the 5' end of the mRNA of mammalian cells is not detectable in Dictyostelium mRNA. Thus neither 2'0-methylated nucleotides nor 6-methyl adenosine can represent sites for processing of a primary nuclear transript to yield mRNA.


The effect of SV40 transformation on the chromosomal proteins of 3T3 mouse embryo fibroblasts. The composition and metabolism of chromosomal proteins-histones and nonhistones chromosomal proteins-were examined in normal and SV40 transformed 3T3 mouse cells. Variations were observed, many of which were similar to those previously reported for normal and SV40 transformed W138 human diploid fibroblasts. The possible implications of these viral induced changes in the protein component of the genome for the phenotypic modifications which occur in transformed cells are discussed.


Evidence for superoxide-dependent reduction of Fe3+ and its role in enzyme-generated hydroxyl radical formation. This report describes studies yielding additional evidence that superoxide anion (O2) production by some biological oxidoreductase systems is a potential source of hydroxyl radical production. The phenomenon appears to be an intrinsic property of certain enzyme systems which produce superoxide and H2O2, and can result in extensive oxidative degradation of membrane lipids. Earlier studies had suggested that iron (chelated to maintain solubility) augmented production of the hydroxyl radical in such systems according to the following reaction sequence: O2 + Fe3+ leads to O2 + Fe2+ Fe2+ + H2O2 leads to Fe3+ + HO-+OH-. The data reported below provide additional support for the occurrence of these reactions, especially the reduction of Fe3+ by superoxide. Because the conditions for such reactions appear to exist in animal tissues, the results indicate a mechanism for the initiation and promotion of peroxidative attacks on membrane lipids and also suggest that the role of antioxidants in intracellular metabolism may be to inhibit initiation of degradative reactions by the highly reactive radicals formed extraneously during metabolic activity. This report presents the following new information: (1) Fe3+ is reduced to Fe2+ during xanthine oxidase activity and a significant part of the reduction was oxygen dependent. (2) Mn2+ appears to function as an efficient superoxide anion scavenger, and this function can be inhibited by EDTA. (3) The O2-dependent reduction of Fe3+ to Fe2+ by xanthine oxidase activity is inhibited by Mn2+, which, in view of statement 2 above, is a further indication that the reduction of the iron involves superoxide anion. (4) Free radical scavengers prevent or reverse the Fe3+ inhibiton of cytochrome c3+ reduction by xanthine oxidase. (5) The inhibition of xanthine oxidase-catalyzed reduction of cyt c3+ by Fe3+ does not affect uric acid production by the xanthine oxidase system. (6) The reoxidation of reduced cyt c in the xanthine oxidase system is markedly enhanced by Fe3+ and is apparently due to enhanced HO-RADICAL formation since the Fe3+-stimulated reoxidation is inhibited by free radical scavengers, including those with specificity for the hydroxyl radical.


[Rare non-functioning insular tumor of the pancreas]. A case of cystic neoplasia with a cephalopancreatic localization, arising in a young woman aged 26 and derived from the beta-insular elements, is presented. The tumour had invaded the porto-mesenteric tract, and hence duodenopancreatic demolition had to be followed by venous repair by prosthesis. On the basis of the literature the biological complexity of this exceptional tumour is disscussed (especially in relation to prognosis), as well as the surgical treatment adopted in this patient, who is in excellent conditions more than three years later.


Susceptibility of some rodent species to monkeypox virus, and course of the infection. The authors studied the susceptibility of five species of rodent to monkeypox virus inoculated by various routes and the course of the infection. Reactions varied from complete resistance to lethal generalized infection with rash. Rabbits and white mice appeared to be the most susceptible species and young animals were more susceptible than adults. Monkeypox virus was found to infect young animals by natural routes, i.e., per os and intranasally. Transmission by contact occurred among 10-day-old rabbits. Since antibodies to monkeypox virus may persist for over a year in the sera of convalescent animals, serological examination of animals is recommended for studying the ecology of this virus.


Two levels of resting potential in canine cardiac Purkinje fibers exposed to sodium-free solutions. Canine cardiac Purkinje fibers exposed to sodium-free solutions containing 16 mM CaCl2, 20 mM tetraethylammonium chloride, 108 mM tetramethylammonium chloride, and 2.7 mM KCl may be quiescent at a resting potential of either -50 mV or -90 mV. The membrane potential of these fibers can be switched from -50 mV to -90 mV by a hyperpolarizing current pulse and from -90 mV to -50 mV by a depolarizing current pulse. The transition from -50 mV to -90 mV depends on a voltage-dependent increase in potassium conductance, that conductance being low at -50 mV and high at -90 mV. A reduction in potassium conductance causes the fiber to depolarize from -90 mV to -50 mV because of the presence of an inward current which apparently is carried mainly by Ca. Fibers that show a high resting potential cannot be excited except by depolarizing stimuli strong enough to move the membrane from -90 mV to a threshold potential of about -40 mV. Fibers that show a low resting potential are more easily excited and may show rhythmic activity sustained by afterpotentials that appear only if the low membrane potential is accompanied by a low potassium conductance. Slow changes in membrane potential also are seen; these changes may result from movements of chloride.


Origin of epicardial ST-T wave potentials in the intact dog. Ventricular repolarization was analyzed by measuring epicardial potential distributions in intact dogs with single or multiple ectopic foci and a minimum at the terminal site(s) of excitation. During the latter half of the T wave the distributions became more complex, and two maxima evolved from the initial one at each ectopic site. The measured epicardial potentials were simulated by means of a model of ST-T wave events that is suitable for study of single and multiple ectopic beats with fusion, a a model we call 'SI model.' Intracellular potentials around the ventricles during repolarization were calculated from measured excitation sequences and known action potential shapes. The extracellular potentials around the ventricles were computed from the intracellular ones by a simplified ventricular geometry. The satisfactory agreement between the theoretical and measured extracellular potential distribution shows that the complex changes which occur throughout the ST-T wave are predicted well on the basis of changes in the intracellular potential distributions. In contrast to the well known lability of the T wave from beat, the results show that for any single beat the events of repolarization proceed in an entirely repeatable and deterministic fashion. The results present a way to directly represent cardiac extracellular events during the ST-T wave, a method analogous to the use of isochromes during QRS, and they imply that in the future it will by possible to achieve a more precise quantitative understanding of the events of the ST-T wave than thus far has been possible for QRS.


A comparison of aortic baroreceptor discharge in normotensive and spontaneously hypertensive rats. Electrophysiological characteristics of individual aortic baroreceptors from normotensive rats (NTR) and spontaneously hypertensive rats (SHR) were compared. Impulse activity of afferent fibers was examined following the application of pressure steps to an in vitro aortic arch-aortic nerve preparation. Thirty-one fibers including seven unmyelinated fibers were studied completely over the range of 0-260 mm Hg, using steps 1-30 seconds in duration. The pressure threshold for peak transient discharge of baroreceptors of SHR's was 88.4 +/- 10.1 (mean +/- SE) mm Hg, whereas for baroreceptors of NTR's it was 77.5 +/- 9.3 mm Hg. The pressure threshold for steady state discharge was 137.3 +/- 5.2 mm Hg for SHR baroreceptors and 103.5 +/- 7.1 for NTR baroreceptors. These values compare favorably to measurements in vivo in the rat. The relationship between peak transient impulse frequency and pressure was linear, whereas the relationship between steady state impulse frequency and pressure was hyperbolic. The curvature of the steady state frequency-pressure curves was significantly reduced for baroreceptors of SHR's. The steady state pressure-volume curves of the aortas of SHR's and NTR's were similar, so that a reduction in distensibility could not account for the larger, significant differences in threshhold and sensitivity. Therefore resetting cannot be attributed simply to reduced vascular compliance.


Inhibition of adrenergic neurotransmission in canine vascular smooth muscle by histamine: mediation by H2-receptors. Histamine depressed the contractions of dog saphenous vein strips caused by stimulation of their sympathetic nerves. This was due to a decrease in the release of norepinephrine which appears to be mediated by histamine H2-receptors. The evidence for this is as follows: (1) Contractions of the strips caused by activating the nerve endings electrically or by depolarization with potassium ions were depressed by histamine, whereas contractions caused by tyramine and norepinephrine were either unchanged or augmented. (2) Strips were incubated with norepinephrine[7-3H] and mounted for superfusion and isometric tension recording. The perfusate was collected for estimation of total radioactivity and for column chromatographic separation of norpinephrine and its metabolites. Histamine (0.9 muM) depressed the release of norepinphrine[7-3H] during contractions caused by electric stimulation, whereas the release of radioactive compounds caused by tyramine was unaffected. (3) The depression by histamine of the contractions and the efflux of radioactive compounds caused by electric stimulation were inhibited by an H2-receptor antagonist (metiamide), but were unaffected by an H1-receptor antagonist (pyrilamine). (4) Contractions caused by electric stimulation were inhibited by an H2-receptor agonist (4-methylhistamine) and augmented by an H1-receptor agonist (2-methylhistamine). These findings suggest the possibility that histamine, which is abundant in sympathetic nerves, might have a regulatory role in the release of the neurotransmitter.


Intact vesicles of canine cardiac sarcolemma: evidence from vectorial properties of Na+, K+-ATPase. Most biological membranes are functionally asymmetric. To study biochemical control of cardiac transsarcolemmalion fluxes, it would be of obvious advantage to use isolated vesicles of sarcolemma which retains the low passive permeability characteristics of intact sarcolemma because in such vesicles the membrane should exhibit its normal asymmetric character with respect to enzymic activities. The purpose of this investigation was to attempt identify such vesicles in a cardiac microsomal (membrane vesicular) preparation. We studied activation by Na+ and K+ of Na+, K+-ATPase and its associated K+-phosphatase activities, using as substrates ATP or p-nitrophenylphosphate (pNPP) in the presence of Mg2+. Optimal concentrations of K+ alone (10 mM) stimulated p-nitrophenylphosphatase (pNPPase) activity 1.8-fold, and over 80% of the increase could be inhibited by ouabain. Optimal Na+ plus K+ concentrations (100 mM and 10 mM, respectively) stimulated the rate of ATP hydrolysis 2-fold, but only 11 +/- 1.1% of the increased activity was ouabain-sensitive. Optimal pretreatment with sodium dodecyl sulfate (SDS) (0.3 mg/ml) rendered both activities completely sensitive to inhibition by ouabain and reduced the basal Mg2+-ATPase activity by 70-90%. The K+-stimulated pNPPase activity doubled after preincubation in SDS, but the ATPase activity stimulated by Na+ plus K+ fell by 50% under these conditions. A similar pattern of apparent activation was produced by preincubation with deoxycholate (DOC), except that basal Mg2+-dependent activities were resistant to destruction by this detergent. The incremental responses to activation by ions and substrates, and inhibition by oubain, are consistent with the hypothesis that permeability-intact vesicles of sarcolemma are present in the isolated preparation, and that detergent activation renders the vesicles highly permeable to the ions, substrates, and ouabain.


Experimental myocardial ischemia: dynamic alterations in ventricular contractility and relaxation with dissociation of speed and force in the isovolumic dog heart. Although the time course of changes in myocardial function during ischemia has been demonstrated for the papillary muscle, this time course in the intact heart is less well understood. Accordingly, in 24 isolated, isovolumic, perfused dog hearts, coronary perfusion pressure (PP) was lowered to various fixed levels. Left ventricular developed pressure (LVP) rapidly fell and reached 63 +/- 3% of control at 1 minute of ischemia 50 +/- 5% at 6 minutes; this was due primarily to an abbreviation of time to peak tension (TPP). dP/dt was 70 +/- 3% of control at 1 minute and 56 +/- 5% at 6 minutes. The rate of relaxation as reflected by negative dP/dt declined as well to 49 +/- 4% of control at 1 minute of ischemia and to 41 +/- 4% control at 6 minutes. These changes were directly correlated with the decrease in PP. When PP was restored to normal, an overshoot of LVP and dP/dt was noted, peaking at 1 minute, returning to control by 5 minutes, and then gradually declining to 90 +/- 2% of control following 25 minutes of recovery. Depression of the rate of relaxation was reduced, but persisted throughout recovery. Diminution of force development early in ishcemia is due primarily to decreased duration of contraction accompanied by a decrease in relaxation rate. Later, the rate of force development also falls, but some preservation of force development may result from the return toward normal of the duration of contraction.


Relationships between myocardial perfusion, myocardial necrosis, and technetium--99m pyrophosphate uptake in dogs subjected to sudden coronary occlusion. The quantitative relationship between abnormalities seen on technetium-99m pyrophosphate (99mTc-PYP) infarct scintigrams and the size of the myocardial infarction is unclear. We evaluated two possible determinants of 99mTc-PYP accumulation: myocardial perfusion measured with 7-10 mu microspheres and the extent of necrosis determined histologically. Hemodynamics and myocardial perfusion to small segments of the left ventricle were measured prior to, 5-10 min, and 44-48 hours following sudden occlusion of the left anterior descending coronary artery in ten awake dogs. 99mTc-PYP was injected i.v. following the third injection of microspheres and the animals were killed 2 hours later. The important findings were as follows: 1) there is a close relationship between the extent of myocardial necrosis observed and the perfusion of segments 5-10 min following coronary occlusion; and 2) that segmental myocardial perfusion is an important determinant of 99mTc-PYP accumulation by myocardial segments which contain areas of necrosis. Although the present data preclude statistical analysis of the relationship between the level of necrosis in a segment and the accumulation of 99mTc-PYP by that segment, the two do not appear to be related, a finding which would discourage use of intensity of 99mTc-PYP images for infarct size. The distribution of an abnormality on the scintigram may provide an estimate of infarct size. However, the geometry of the infarct and the resolving power of the scanning equipment will significantly limit this in many clinical situations.


Evaluation of a serum protein in pregnant rats. The increase of a serum protein in the circulation of rats during pregnancy has been described. The protein is different from the human pregnancy zone protein previously described. This new serum protein factor is present in normal rat serum and accounts for approximately 1% of the total serum protein. The protein factor disappears from the serum of animals bearing Morris hepatomas 7777 and 7800. The amount of factor diminishes as the tumors increase in size. The levels of this protein in the serum increased several fold during pregnancy with highest levels found shortly before parturition. Levels remained elevated for at least 12 days post partum. No corresponding increase in the levels of this serum factor in the fetal circulation was observed. Increased levels in the maternal circulation were due to an increased synthesis of this serum protein during pregnancy. A corresponding band was observed on polyacrylamide gels for normal human female and pregnant human sera or plasma. However, there was no increase during pregnancy as judged by polyacrylamide gel electrophoresis. Quantitative determination of the protein in human sera was not possible since the band corresponding to the serum protein in rats did not cross react with a rabbit anti-rat serum factor.


A comparison of methods for the immunoassay of serum apolipoprotein B in man. Three assays for serum apolipoprotein B, radioimmunoassay, automated immunoprecipitation and rocket immunoelectrophoresis were compared. The antiserum used was raised against lipoprotein of density 1.040-1.053 g/ml (lipoprotein B). Each of the methods had a high degree of specificity when tested against potential interfering substances. The lowest levels of apolipoprotein B could be measured with radioimmunoassay but this sensitivity entailed the use of high dilutions of serum and resulted in a lower precision. Concentration response curves of lipoprotein B solution and serum were parallel for radioimmunoassay and automated immunoprecipitation but were not for rocket immunoelectrophoresis. Serum apolipoprotein B could be assayed by immunoelectrophoresis, however, if serum calibrated against the protein concentration of lipoprotein B solution by either of the other two methods was used as a secondary standard. Such a secondary serum standard also proved advantageous for all the methods because of the relative stability of the apolipoprotein B content of serum as compared to aqueous solutions of lipoprotein B. The mean apolipoprotein B concentration in 29 normolipaemic subjects aged 20-30 years was 0.84 +/- 0.12 g/1 (mean +/- S.D.) by radioimmunoassay, 0.85 +/- 0.11 g/1 by automated immunoprecipitation and 0.88 +/- 0.11 g/1 by rocket immunoelectrophoresis. The correlation between apolipoprotein B levels measured by the three methods was good. The ratio of serum cholesterol to serum apolipoprotein B was unaffected by individual differences in serum cholesterol or triglyceride levels. There was no significant difference between fasting and post-prandial serum apolipoprotein B concentrations. Radioimmunoassay is particularly suited to the measurement of low levels of apolipoprotein B, automated immunoprecipitation for large numbers of samples and rocket immunoelectrophoresis, when care is taken in the interpretation of results, for small numbers of samples.


Zero electrophoretic mobility of very low density lipoproteins in type III hyperlipidemic patients during treatment. In some cases of type III hyperlipoproteinemia reduction of lipid levels by diet and/or drugs results in a complete loss of mobility of the VLDL fraction on paper electrophoresis.


The separation of plasma lipoproteins using gel electrofocusing and polyacrylamide gradient gel electrophoresis. Polyacrylamide gel electrofocusing and gradient electrophoresis have been used to separate the lipoproteins in whole plasma and in fractions prepared by sequential flotation in the ultracentrifuge and by precipitation with dextran sulphate and manganous chloride. After the two-dimensional separation, high density lipoproteins appear as a zone showing noticeable heterogeneity with respect to both isoelectric point and molecular weight. Low density lipoproteins are resolved as a compact spot while very low density lipoproteins are visible as a long horizontal streak across the top of the electrophoresis gel. The implications of the technique for the analysis of lipoprotein patterns in pathological plasmas are discussed.


Urate production in heterozygotes for glucose-6-phosphatase deficiency. Urate production and excretion were studied in heterozygous parents of a child with glucose-6-phosphatase deficiency. Both parents demonstrated glucose-6-phosphatase concentrations in platelets intermediate between those in the homozygote and the normal. The miscible urate pool and turnover rate, the rate of incorporation of [14C]glycine into urate, the renal clearance of urate and the percentage excretion of labelled urate by the renal route were within the normal range in both heterozygotes, as were the serum cholesterol and triglyceride concentrations. Thus, a partial deficiency of glucose-6-phosphatase was not associated with the abnormalities of urate or lipoprotein metabolism which are features of homozygous glucose-6-phosphatase deficiency. Erythrocyte phosphoribosyl-pyrophosphate concentration, an increased concentration of which has been postulated as the mechanism responsible for the increased de novo purine biosynthesis in glucose-6-phosphatase deficiency, was found to be within the normal range in erythrocytes from both a homozygote and a heterozygote for this condition.


Lipoprotein fractionation by a precipitation method. A simple quantitative procedure. A method is described for quantitation of the three major classes of serum lipoproteins. After precipitation of very low density lipoprotein (VLDL) using sodium dodecyl sulphate, the cholesterol and triglyceride content of this lipoprotein class is directly measured. In a second aliquot serum high density lipoprotein (HDL) lipids are measured after precipitation of VLDL and low density lipoprotein (LDL). LDL cholesterol and triglyceride contents are calculated by difference. The procedure requires 2 ml serum, and sensitivity is adequate to permit lipoprotein analyses on umbilical cord serum. Close agreement is observed between this precipitation method and preparative ultracentrifugation.


Nyctohemeral variation and suppressibility of plasma ACTH in various stages of Cushing's disease. In order to define nyctohemeral plasma ACTH secretory patterns, frequency plasma ACTH samples were obtained in seven patients with untreated Cushing's disease (i.e. pituitary-dependent Cushing's syndrome), five Cushing's patients treated by bilateral adrenalectomy, four of whom had Nelson's syndrome, and one patient with 21-hydroxylase deficiency (congenital adrenal hyperplasia). A nyctohemeral rhythm of plasma ACTH concentration was apparent in the one patient with the adrenogenital syndrome but not in those with Nelson's syndrome or Cushing's disease. The effect of graded doses of dexamethasone, 2,8 or 32 mg per 24 h period, on plasma ACTH concentrations was studied in patients with untreated or treated Cushing's disease or Nelson's syndrome. In all of these hypercorticotrophic states, the mean plasma ACTH concentraton was not significantly affected by the smallest dose of dexamethasone, was partially suppressed by the intermediate dose, and further suppressed by the largest dose. In contrst, the patient with congenital adrenal hyperplasia and elevated plasma ACTH concentrations showed complete suppression of plasma ACTH levels following the smallest dose of dexamethasone. These findings indicate that there is resistance to ACTH suppression by dexamethasone in all stages of Cushing's disease and suggest that negative feedback of glucocorticoids may be involved in the pathogenesis of this disease.


Low-density lipoproteins in patients homozygous for familial hyperbetalipoproteinaemia. 1. The low-density lipoproteins (LDL; density 1-007-1-063 g/ml) from two patients homozygous for familial hyperbetalipoproteinaemia have been submitted to chemical and physicochemical analysis. 2. The presence of an anomalous lipoprotein with a low proportion of triglyceride and a raised proportion of cholesterol has been confirmed. 3. In one patient, this lipoprotein accounted for about 85% of the LDL, but in the second, the amount varied from about 85% to a point at which it could not be detected among the coexisting normal lipoproteins. 4. The protein moiety of this anomalous LDL has effectively the same amino acid composition as that derived from the LDL of healthy subjects. 5. The proportions of carbohydrate, phospholipid and fatty acids could not be reliably distinguished from those of normal LDL. 6. The molecular weight and diffusion constant of the abnormal lipoprotein, even in the purest preparation, were close to the values determined for normal LDL of similar flotation rate.


Intravenous fat-tolerance test in ischaemic heart disease and peripheral vascular disease. 1. The intravenous fat-tolerance test and serum lipid and lipoprotein measurements were carried out in ninety-three normal subjects, fifty-one patients with ischaemic heart disease and thirty patients with peripheral vascular disease. 2. The fractional turnover rate of exogenous triglyceride was significantly slower in patients with ischaemic heart disease and in patients with peripheral vascular disease than in normal men. The rate was also slower in normal men than normal women. 3. Serum triglyceride and cholesterol concentrations were higher in both vascular disease groups than in control subjects. 4. The proportion of both groups of patients who had a subnormal fractional turnover rate of exogenous triglyceride was 35%, and 32% of patients had hypertriglyceridaemia in the fasting state; 27% of patients were hypercholesterolaemic. 5. Although the intravenous fat-tolerance test did not provide significantly better discrimination between cardiovascular patients and control subjects than did measurement of serum triglyceride, the results suggest that hypertriglyceridaemia in such patients may be separable into a group in which impaired triglyceride clearance may be partly responsible, and a group in which overproduction of serum triglyceride may be the major mechanism of the hyperlipidaemia.


Calcium-activated ATPase activity in a plasma membrane-rich preparation of rat pancreas. 1. Calcium-stimulated ATPase activity was studied in a plasma membrane rich fraction of rat pancreas homogenate. 2. The enzyme is stimulated to the same maximum rate of ATP hydrolysis by either calcium or magnesium, but the apparent requirement for calcium is five-fold lower than for magnesium. 3. Maximum hydrolytic activity of the enzyme is not increased when additional magnesium is added to the optimal amount of calcium. 4. The enzyme does not require Na+ or K+ for its activation by Ca2+ and is not inhibited by ouabain or 2,4-dinitrophenol. 5. Pancreozymin, in concentrations which evoke secretion of zymogen protein, inhibits the calcium-stimulated ATPase. 6. Carbachol and dibutyryl cyclic AMP, in concentrations which increase the release of digestive proteins, do not alter the activity of the calcium-stimulated enzyme. 7. It is suggested that the plasma membrane calcium-activated enzyme, is not involved in the active calcium extrusion, previously reported to occur with use of the various pancreatic secretagogues tested.


Effects of some catecholamines on noradrenergic transmission in the rabbit ear artery. 1. The effects of noradrenaline, adrenaline, isoprenaline, dopamine and the N-methyl and alpha-methyl homologoues of dopamine were studied on tritium release and on vasoconstrictor responses to sympathetic nerve stimulation in isolated arteries of the rabbit ear after labelling noradrenergic transmitter stores with 3H-noradrenaline. These observations were made in the presence of cocaine (100 muM). 2. Noradrenaline (0-5 and 5 muM) inhibited transmitter release and abolished vasoconstrictor responses. A concentration of 0-05 muM had no effect on transmitter release, although the vasoconstrictor response was reduced and there was sometimes a vasodilator response. 3. Adrenaline in concentrations of 0-05, 0-5 and 5 muM decreased transmitter release and vasoconstrictor responses; sometimes there was a vasodilator response. 4. Isoprenaline in concentrations of 0-05, 0-5 and 5 muM did not affect transmitter release. Vasoconstrictor responses were either unaffected or enhanced by all concentrations of isoprenaline when infusions had been in progress for 15 min; but immediately after beginning the infusion of 5 muM isoprenaline, the response was reduced. 5. Dopamine (0-05, 0-5 and 5 muM) produced a concentration-dependent decrease in transmitter release, but vasoconstrictor responses were not reduced with infusions of 0-05 or 0-5 muM; during infusions of 5 muM, stimulation produced a vasodilator response.


Intracranial pressure and rapid eye movement sleep in hydrocephalus. Intracranial pressure (ICP) was monitored for 24 h in 30 hydrocephalic patients (21 infants, 9 children) representing borderline cases. The need for surgery was uncertain because their hydrocephalus seemed to be more or less arrested. In 13 cases an electroencephalogram, an electro-oculogram, an electromyogram and an actogram were simultaneously recorded. During sleep related to a period of rapid eye movement (REM) fairly regular steep-rising waves of raised ICP recurred every 50-75 min, decreasing slowly to previous levels in 25-40 min. No concomitant clinical symptoms were exhibited. The cerebral pulse wave amplitude increase during REM sleep, but might vary during one period of REM sleep. The relationship between these raised ICP waves and an increase in cerebral blood flow occurring during REM sleep is studied.


[Computer-supported after-care system for patients with tumors using a cancer-register]. A computerized follow-up system for patients with malignant tumors is described. This system is in use in the "Chirurgische Klinik der Universitat Munster" and in the "Fachklinik Haus Hornheide". As a special service to these clinics a central cancer registry furnishes periodically lists of patients. These lists are used as surveys of treated cancer patients. It is possible to contact those patients by letter, who do not present themselves for clinical examination. Goals of the system are the documentation of the course of disease, the analysis of survival and the follow-up of the patients. Thus, the clinics manage a comprehensive cancer care program with a minimum lost of contact.


[Angiodysplasia and the skeletal system]. 1. Congenital angiodysplasias of the extremities are often associated with skeletal disorders, i.e., overgrowth (local giantism, hyperplasia) or reduced growth (hypoplasia). From a clinical point of view three types of combined lesions may be differentiated: (a) a secondary local giantism induced by hemodynamically active congenital a.v. fistulas, usually of intra- and extraosseous location (Weber-type); - (b) local giantism in combination with venous and/or lymphatic angiodysplasias; in this group skeletal overgrowth seems not to be caused by the vascular anomalies but represents more probably a coordinated "inborn error" of tissue composition and distribution (Klippel-Trenaunay-type); - (c) hemangiomatosis usually of venous (cavernous) type, affecting soft tissues as well as bones in association with skeletal hypoplasia. There is a retardation of bone growth by substitution and destruction of the epiphysial cartilages by the intraosseous hemangioma (Servelle-Martorelltype). In most cases (a-c) the use of special angiological investigations allows a clear diagnosis particularly concerning the pathogenesis of skeletal disorders. 2. In looking for an unequivocal terminology the clinical classification of these anomalies should be based on the leading clinical symptome, i.e., the giantism or the angiodysplasia. In this respect the vascular findings take on a predominant position. Paying regard to historical aspects the classification may be completed by the additional term "Klippel-Trenaunay" type, "Weber" type, "Servelle-Martorell" type. 3. An exact angiologic diagnosis gives further informations about prognosis and choice of treatment (group I: Early operative elimination or reduction of the a-v shuntvolume; group II and III: "wait and see!"; usually conservative treatment with compression bandages).


Colorectal adenocarcinoma in patients less than 40 years old. Carcinoma of the colorectum knows no age barrier. To date, more than 1,400 cases have been reported to occur in persons less than 40 years old. Our experience with 70 patients treated over a ten-year interval is reviewed. Signs and symptoms in the young are not distinctive. Diagnosis depends on a high index of suspicion and appropriate investigative procedures. Survival times are shorter in the patients who have mucinous and anaplastic tumors, and their incidences seem to be increased in this age segment. Overall survival does not significantly differ from our general experience (41 per cent, 5-year survival). Early diagnosis and prompt institution of aggressive surgical treatment can be expected to produce survival equivalent to that in patients of other ages.


[Treatment of essential hypertension with propranolol (author's transl)]. Effect of long-term administration of propranolol was studied in 90 unselected patients with essential hypertension. At a dose of 180 and 360 mg daily it dropped the blood pressure to at least 155/95 mm Hg in 31 patients (34.4%). Those who responded to propranolol had lower systolic and diastolic blood pre-treatment pressures than those who failed, were younger by an average of eight years, and in no case had decreased plasma-renin activity and demonstrated greater plasma-renin activity after furosemide. After a titration phase with increasing doses of propranolol those who responded were treated in a crossed double-blind test with propranolol or placebo. There was a significant difference in systolic blood pressure (standing and lying) and diastolic pressure (standing) between the two. There was also a highly significant difference in heart rate. During chronic beta blockade the basal and stimulated plasma-renin activity fell. But percentage stimulation of renin secretion was fully maintained. However, aldosterone response was decreased. Serum potassium rose slightly but significantly, cAMP  24-hour urine excretion fell significantly. Electrolyte excretion did not differ between the two treatment periods.


Induction of lactogenic receptors. I. In the liver of hypophysectomized female rats. To identify the hormones which affect lactogenic receptors in the liver of chronically hypophysectomized female rats, hormones were injected s.c. for 7 days. Specific binding (%, SB) of labelled ovine prolactin (PRL) in liver membrane preparations (1000,000 X g pellet) of controls was 1%. Estradiol (E2), cortisone (Con), ACTH or bovine growth hormone (bGH) treatment did not induce hepatic binding sites for PRL. Human GH and a single dose of 2mg PRL (but not lower doses) increased SB of PRL. Treatment with oPRL plus ACTH was less effective than hGH plus ACTH (13 vs 28%); combinations of oPRL plus Con as well as administration of oPRL plus ACTH to hypophysectomized and adrenalectomized female rats did not induce SB for PRL. Therapy with oPRL plus hGH (26%) was more potent than oPRL plus bGH (2%). These studies suggest that PRL, GH, and ACTH induce and in concert with sex steroids, modulate the lactogenic receptors in the female rat liver. The effect of ACTH is not due to increased adrenal corticoid secretion.


Induction of lactogenic receptors. II. Studies on the liver of hypophysectomized male rats and on rats bearing a growth hormone secreting tumor. The role of growth hormone (GH), as well as prolactin and ACTH, in the induction of the PRL receptor was investigated both in hypophysectomized male rat livers and in the livers of male rats bearing a GH secreting tumor. After 7 days of s.c. injections, specific binding (% SB) of PRL in controls and rats treated with oPRL, hGH, ACTH, hCG, estradiol (E2), or testosterone (T) was approximately 1%. Treatment with oPRL plus ACTH increased SB to 4%; adding E2 to this combination produced a further increase to 8%, whereas the addition of T decreased hepatic binding to 1%. Combination of hGH with ACTH was most effective, giving a SB of 33%, which is similar to that observed in the liver of rats bearing a GH secreting tumor (36%). These studies suggest that GH acts synergistically with PRL and/or ACTH to increase lactogenic binding sites in the male rat liver and that sex steroids have a modulating effect on this action.


Nuclear poly(A) polymerase from rat liver and a hepatoma. Comparison of properties, molecular weights and amino acid compositions. Poly(A) polymerase was extracted from isolated nuclei of rat liver and a rapidly growing solid tumor (Morris hepatoma 3924A). The enzyme from each tissue was purified by successive chromatography on DEAE-Sephadex, phosphoecllulose, hydroxyapatite and QAE-Sephadex. Purified enzyme from both liver and tumor was essentially homogeneous as judged by polyacrylamide gel electrophoresis. Under nondenaturing conditions, enzyme activity corresponded to visible protein and, upon denaturation, a single polypeptide was detected. The enzymes had absolute requirements for Mn2+ as the divalent ion, ATP as the substrate and an oligonucleotide or polynucleotide as the primer. Both enzymes were inhibited by sodium pyrophosphate, N-ethylmaleimide, Rose Bengal, cordycepin 5'-triphosphate and several rifamycin derivatives. The reactions were unaffected by potassium phosphate, alpha-amanitin and pancreatic ribonuclease. However, the liver and hepatoma enzymes differed from each other with respect to apparent Km, primer saturation levels and sensitivity to pH changes. The most striking differences between the enzymes were in their calculated molecular weights (liver, 48000; hepatoma, 60000) and amino acid compositions. Finally, the level of the hepatoma enzyme relative to that of the liver enzyme was at least 1.5-fold higher when expressed per mg DNA.


The inhibition of isocitrate oxidation by palmitoyl-l-carnitine and palmitoyl-C0 A in rat liver mitochondria. Palmitoyl-L carnitine decreases the oxidation of isocitrate in rat liver mitochondria in state 3 by 25-30%. Palmitoyl-L-carnitine acts as an additional substrate raising the rate of oxidative phosphorylation, NAD reduction and ATP/ADP ratio in mitochondria. Palmitoyl-CoA added to mitochondria oxidizing isocitrate in state 3 causes a strong inhibition of isocitrate oxidation and of oxidative phosphorylation and a considerable elevation of intramitochondrial NADH/NAD and ATP/ADP ratios. The effect of palmitoyl-CoA is dependent on its concentration and is competitive with ADP. Carnitine restores only oxidative phosphorylation, but the oxidation of isocitrate remains inhibited. Evidence is presented that the transport of isocitrate is not affected by palmitoyl-CoA is due to the inhibition of adenine nucleotide translocation. The kinetic studies of NAD-dependent isocitrate dehydrogenase in the soluble fraction of sonicated mitochondria revealed that the enzyme is very sensitive towards the inhibition by NADH and only very slightly affected by ATP (Ki for NADH and ATP are 0.017 and 3.6 mM respectively). On the basis of the kinetic data the relative contribution of NADH and ATP in the inhibition of isocitrate oxidation by fatty acids was calculated. It is concluded that the inhibition of isocitrate oxidation caused by palmitoyl-L-carnitine and palmitoyl-CoA is primarily due to the increased reduction of NAD, whereas the increase of ATP/ADP ratio is much less important.


Coenzyme properties of NAD+ bound to different matrices through the amino group in the 6-position. A method for the synthesis of N6-(2-aminoethyl)-NAD+ is given. The binding of this NAD+ derivative to different soluble and insoluble supports and the direct coupling of NAD+ to epoxyactivated Sepharose are described. Proofs are given that NAD+ is bound through the amino group in 6- position and the NAD+ derivative through the aliphatic amino group of the side chain. Non-enzymic reduction of the bound coenzyme to an almost quantitative extent is possible in all cases, but the enzymic reduction is largely influenced by the support. While N6-(2-aminoethyl)-NAD+ coupled to soluble dextran is nearly completely reducible by different dehydrogenases with a velocity of about 40% of that for free NAD+, the coenzyme bound to different insoluble matrices is very slowly reduced. Only 5% of the coenzyme derivative bound to BrCN-activated Sepharose are reducible, but 40% when it is bound through a spacer. From capacity determinations evidence is given that, even in this coenzyme gel, only those coenzyme molecules are useful in affinity chromatography which are on the surface of the gel grains; it is supposed that this may be due to the slow diffusion of an enzyme into the inner parts of an affinity gel.


Evidence for a function of core protein in complex III from beef-heart mitochondria. Puried complex III ) ubiquinol-cytochrome c reductase) from beef heart mitochondria was alkylated with iodol [1-14C]acetamide. After 6-8 h of incubation with iodo[1-14C]acetamide, duroquinol and ubiquinol-2-cytochrome c reductase activites were inhibited approximately 50%. During this time 4.5 +/- 1.6 nmol of iodo[1-14C]acetamide reacted per mg of complex III protein. Experiments carried out over 24 h indicated that enzyme activity could be inhibited to 70% and the alkylation of complex III was proportional to inhibition. The rates of cytochrome b and c1 reduction by duroquinol are also decreased upon treatment of complex III with iodoacetamide. Separation of the peptides of complex III by electrophoresis in sodium dodecylsulfate shows that all of the radioactivity is located in a single peptide of 50 000 molecular weight, which has been identified as one of the two core proteins. The possible functions of core protein are discussed.


Primary and tertiary structure of the principal human adenylate kinase. 1. Human adenylate kinase (isoenzyme AK-1-1) from skeletal muscle is a single polypeptide chain of 194 amino-acid residues with an acetylmethionine at the N-terminus and a lysine at the C-terminus. 2. The primary structure of the enzyme was determined: Ac-Met-Glu-Glu-Lys-Leu-Lys-Lys-Thr-Lys-Ile-Ile-Phe-Val-Val-Gly-Gly-Pro-Gly-Ser-Gly-Lys-Gly-Thr-Gln-Cys-Glu-Lys-Ile-Val-Gln-Lys-Tyr-Gly-Tyr-Thr-His-Leu-Ser-Thr-Gly-Asp-Leu-Leu-Arg-Ser-Glu-Val-Ser-Ser-Gly-Ser-Ala-Arg-Gly-Lys-Lys-Leu-Ser-Glu-Ile-Met-Glu-Lys-Gly-Gln-Leu-Val-Pro-Leu-Glu-Thr-Val-Leu-Asp-Met-Leu-Arg-Asp-Ala-Met-Val-Ala-Lys-Val-Asn-Thr-Ser-Lys-Gly-Phe-Leu-Ile-Asp-Gly-Tyr-Pro-Arg-Glu-Val-Gln-Gln-Gly-Glu-Glu-Phe-Glu-Arg-Arg-Ile-Gly-Gln-Pro-Thr-Leu-Leu-Leu-Tyr-Val-Asp-Ala-Gly-Pro-Glu-Thr-Met-Thr-Arg-Arg-Leu-Leu-Lys-Arg-Gly-Glu-Thr-Ser-Gly-Arg-Val-Asp-Asn-Glu-Glu-Thr-Ile-Lys-Lys-Arg-Leu-Glu-Thr-Tyr-Tyr-Lys-Ala-Thr-Glu-Pro-Val-Ile-Ala-Phe-Tyr-Glu-Lys-Arg-Gly-Ile-Val-Arg-Lys-Val-Asn-Ala-Glu-Gly-Ser-Val-Asp-Glu-Val-Phe-Ser-Gln-Val-Cys-Thr-His-Leu-Asp-Ala-Leu-Lys. 3. When the primary structure of the human enzyme was fitted to the electron density map of porcine adenylate kinase, all nine amino acids which are different in the homologous enzymes from pig and man were located on the surface of the molecule. 4. Precession photographs of crystalline human and of crystalline porcine adenylate kinase corroborated the result that the polypeptide chains of the two enzymes are folded in a closely related manner. 5. The structure of human adenylate kinase incorporates the so-called nucleotide-binding domain which is present in a wide variety of proteins in nature. Some implications of this phenomenom for the molecular biology and the molecular pharmacology of man are discussed.


The phosphorylation of rabbit skeletal muscle glycogen synthase by glycogen synthase kinase-2 and adenosine-3':5'-monophosphate-dependent protein kinase. Purified glycogen synthase is contaminated with traces of two protein kinases that can phosphorylate the enzyme. One is protein kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) and the second is an activity termed glycogen synthase kinase-2 [Nimmo, H.G. and Cohen P, (1974)]. Glycogen synthase kinase-2 has been found to be localized relatively specifically in the protein-glycogen complex. It has been purified 4000-fold by two procedures, both of which involve disruption of the complex, followed by the DEAE-cellulose and phosphocellulose chromatographies. However the salt concentration at which glycogen synthase kinase-2 is eluted from DEAE-cellulose depends on the method that is used to disrupt the complex. The results indicate that glycogen synthase kinase-2 is firmly attached to a protein component of the complex. The isolation procedures separate glycogen synthase kinase-2 from phosphorylase kinase, cyclic AMP-dependent protein kinase and other glycogen-metabolising enzymes. Glycogen synthase kinase-2 is the major phosvitin kinase in skeletal muscle, although  glycogen synthase is a six to eight-fold better substrate than phosvitin under the standard assay conditions. Phosphorylase kinase and phosphorylase b are not substrates for glycogen synthase kinase 2. Following incubation with cyclic-AMP-dependent protein kinase, cyclic AMP and Mg-ATP, the phosphorylation of glycogen synthase reaches a plateau at 1.0 molecules of phosphate incorporated per subunit and the activity ratio measured in the absence and presence of glucose 6-phosphate falls from 0.8 to a plateau of 0.18. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthase b1, is the 0.6 mM. Following incubation with glycogen synthase kinase-2 and Mg-ATP, the phosphorylation reaches a plateau of 0.92 molecules of phosphate incorporated per subunit and the activity ratio decreases to a plateau of 0.08. The Ka for glucose 6-phosphate of this phosphorylated species, termed glycogen synthetase b2, is 4 mM. In the presence of both cyclic-AMP-dependent protein kinase and glycogen synthase kinase-2, the phosphorylation of glycogen synthase reaches a plateau when 1.95 molecules of phoshophate have been incorporated per subunit. The activity ratio is 0.01 and the Ka for glucose 6-phosphate is 10 mM. The results indicate that glycogen synthase can be regulated by two distinct phosphorylation-dephosphorylation cycles. The implication of these findings for the regulation of glycogen synthase in vivo are discussed.


Separation of two phosphorylase kinase phosphatases from rabbit skeletal muscle. Cyclic-AMP-dependent protein kinase catalyses the activation of phosphorylase kinase and the phosphorylation of two serine residues on the alpha subunit and beta subunit of phosphorylase kinase [Cohen, P., Watson, D.C. and Dixon, G.H. (1975)]. The dephosphorylation of phosphorylase kinase has been shown to be catalysed by two distinct enzymes, termed alpha-phosphorylase kinase phosphatase and beta-phosphorylase kinase phosphatase. These two enzymes show essentially absolute specificity towards the alpha and beta subunits respectively. The two phosphatases copurified through ethanol fractionation, DEAE-cellulose chromatography and ammonium sulphate precipitation, but were separated from each other by a gel filtration on Sephadex G-200. alpha-Phosphorylase kinase phosphatase was purified 500-fold from the ethanol precipitation step, and beta-phosphorylase kinase phosphatase 320-fold. The molecular weights estimated by gel filtration were 170--180 000 for alpha-phosphorylase kinase phosphatase and 75--80 000 for beta-phosphorylase kinase phosphatase. Since the activity of phosphorylase kinase correlates with the state of phosphorylation of the beta subunit (Cohen, P. (1974)), beta-phosphorylase kinase phosphatase is the enzyme which reverses the activation of phosphorylase kinase. alpha-Phosphorylase kinase phosphatase is an enzyme activity that has not been recognised previously. Since the role of the alpha-subunit phosphorylation is to stimulate the rate of dephosphorylation of the beta subunit (Cohen, P. (1974)), alpha-phosphorylase kinase phosphatase can be regarded as the enzyme which inhibits the reversal of the activation of phosphorylase kinase. The implications of these findings for the hormonal control of phosphorylase kinase activity by multisite phosphorylation are discussed.


Pathophysiology and therapy of hypercalciuria in patients who form recurrent stones. There are two alternative mechanisms that might be responsible for idiopathic hypercalciuria in recurrent stone formers: increased intestinal absorption of calcium with parathyroid suppression and overflow hypercalciuria (primary intestinal hyperabsorption) or renal calcium leak with compensatory hyperparathyroidism and intestinal hyperabsorption (primary renal-tubular hypercalciuria). In this study, urinary excretion of cAMP, the intracellular effector substance synthetised under parathyroid hormone stimulation, was found to be in the normal range. This finding would argue against intestinal hyperabsorption of calcium as the primary cause of hypercalciuria.


Piribedil in Parkinson's syndrome: a clinical study. The effect of a new dopamine receptor stimulating agent, piribedil (ET 495), was studied in 10 patients with Parkinson's syndrome, who had had no or a poor response to previous L-DOPA treatment, or had displayed marked side effects during L-DOPA administration. Piribedil produced significant improvement of the functions of activity of daily living (ADL), and appeared to have a preferential effect on parkinsonian tremor. However, treatment was difficult to control primarily because of severe psychiatric side effects.


Studies on the maturation of thymus stem cells. The effects of catecholamines, histamine and peptide hormones on the expression of T cell alloantigens. Studies have been performed in order to investigate the effects of catecholamines. histamine, peptide hormones and various other agents known to increase cellular adenosine 3':5'-cyclic monophosphate (cAMP) levels on the expression of Thy-1 and TL antigens on 14-day fetal thymic stem cells. It has been shown that the proportions of Thy-1 and TL positive cells, as detected by dye exclusion cytotoxicity tests, can be significantly increased by some of these agents and that these effects may be inhibited by beta adrenergic and H2 antagonists. The value of these tests as a means for studying normal thymic stem cell maturation and for investigating T cell deficiency is discussed.


Alterations in the efficacy of naloxone induced by stress, cyclic adenosine monophosphate, and morphine tolerance. Pretreatment of mice with a single injection of morphine or by chronic implantation of morphine pellets increased the ability of naloxone to antagonize the analgetic effects of morphine. However, this increased effectiveness of naloxone was also produced by pretreatment with diethylether, ACTH, corticosterone or dexamethasone. Thus, the increased potency of naloxone observed after pretreatment with narcotics may be due, at least in part, to those pretreatments on the pituitary--adrenal axis. In addition, in animals made highly tolerant and dependent by cAMP administration during morphine pellet implantation, the narcotic antagonist potency of naloxone was similar to that of untreated animals.


A comparison of the effects of angiotensin II and heptapeptide on smooth muscle (vascular and uterine). On the rabbit isolated aorta, dose-dependent contractions to both angiotensin II and heptapeptide ([des-Asp1]-angiotensin II) were obtained. The curves were parallel, and reached the same maximum level. On the rat isolated uterus, angiotensin II and the heptapeptide displayed non-parallel dose-response curves. Results obtained with angiotensin-analog antagonists and cross-tachyphylaxis experiments suggest that the heptapeptide and angiotensin II act, preferentially, on different populations of receptors in the uterus. The difference in action of indomethacin on recovery from tachyphylaxis to angiotensin II and heptapeptide on rat isolated aorta suggests that the mechanism of induction of tachyphylaxis by these two peptides may differ. SQ 20881, the angiotensin converting enzyme inhibitor, totally inhibited uterine responses to both decapeptide and nonapeptide, while slightly potentiating those to angiotensin II and heptapaptide. Indomethacin had no significant effect on uterine responses to either angiotensin II or the heptapeptide.


Interaction of clonidine with pre- and post-synaptic adrenergic receptors of rat brain: effects on cyclic AMP-generating systems. The locus and mechanism of interaction of clonidine with catecholamine-elicited accumulations of cyclic AMP has been investigated in brain slices from control and 6-hydroxydopamine-treated rats of the F-344 and Sprague-Daeley strains. The inhibitory effects of clonidine on the norepinephrine-stimulated accumulation of cyclic AMP and the potentiative effects of clonidine on the isoproterenol-stimulated accumulation of cyclic AMP are present to the same extent in cerebral cortical slices from control and 6-hydroxydopamine-treated Sprague-Dawley rats. Clonidine, at concentrations between 0.0001 and 100 muM has no intrinsic stimulatory activity on cortical cyclic AMP-generating systems from either control or 6-hydroxydopamine-treated rats. In F-344 rats phenoxybenzamine (100 muM) elicits a significant accumulation of cyclic AMP in cerebral cortical slices which can be abolished by pretreatment of rats with 6-hydroxydopamine, or by incubation of tissue slices with either clonidine or sotalol. The hyperresponsiveness to catecholamines usually observed following central administration of 6-hydroxydopamine failed to develop in the F-344 rat. Concentrations of phenoxybenzamine which have no significant stimulatory effects on cyclic AMP accumulation are capable of abolishing the potentiative effects of clonidine on the isoproterenol-stimulated formation of cyclic AMP. The beta-antagonist, sotalol, is a less effective antagonist of isoproterenol-stimulated accumulation of cyclic AMP in the presence of clonidine. Clonidine has no significant effect on the accumulation of cyclic AMP elicited by submaximal concentrations of isoproterenol in cerebellar slices. In toto, the data are consistent with the hypothesis that both the inhibitory effects on norepinephrine-stimulated accumulation of cyclic AMP and the stimulatory effects of clonidine on isoproterenol-elicited accumulation of cyclic AMP are exerted at postsynaptic alpha-adrenoceptors. No evidence was found for a presynaptic generation of cyclic AMP, although clonidine does reverse the stimulatory effects of phenoxybenzamine on cyclic AMP accumulation, presumably by interaction with a presynaptic site controlling norepinephrine release.


Effect of cirrhosis on pulmonary fibrogenesis caused by silica dust in rats. The cirrhosis in male albino rats was produced after repeated subcutaneous injections of carbon tetrachloride, in dosage schedule as described. A fully developed cirrhosis was observed after 150 days. The silicosis in lungs was produced after single intratracheal injection of silica dust of particle size less than 5 microns and studied up to 180 days. The effect of damaged liver on the development of silicosis in the lungs was studied. It was found that pulmonary fibrosis caused by silica dust appeared to be a tissue reaction confined to the lung tissue alone. The dysfunction of liver did not exert any influence on fibrogenesis in the lungs.


Effect of quartz dust on the lungs of mice. Intratracheal inoculations of quartz dust (250 mg/kg body weight) of a particle size less than 5 mum were given in mice and pulmonary tissue reactions studied over a period of 210 days. Acute inflammatory reaction in the lung parenchyma was observed at early periods and later the aggregates of dust laden macrophages encountered around bronchi and blood vessels. Towards the termination of the experiment at 210 days the fibrotic reaction comprised chiefly of thick compactly arranged reticulin fibers with few collagen fibers which remained restricted to the peribronchial and perivascular areas. There was no silicotic nodule formation in the parenchyma of the lung. The atypical pulmonary tissue response to quartz dust in mice has been attributed to different tissue reactivity.


[Effect of Solanum melongena on experimental atheromatosis. VI. Enzyme histochemical, physiopathological and chemical studies on cholesterol-induced atheromatosis in rabbits. Conclusions (author's transl)]. The mode of action of Solanum Melongena (Sol. Mel.) in its inhibitory effect on the development of atheromatous plaques in cholesterol-induced atheromatosis in rabbits is still unexplained. In this paper the author reports his enzyme histochemical, physiopathological and chemical observations drawing conclusions for future experiments. Animals and experimental conditions were the same as described in the recent studies (see literature no. 2, 15, 16, 17, 18). 1. Enzyme histochemistry: The aortas were removed immediately after death, placed in Holt's solution, freed from the advential fatty tissue, cut into 5-6 mm high cylinders and either processed instantly or preserved in the same fluid at +2 degrees C. From the individual portions cryostat sections (10 mum thick) were cut after the method of Pearse. They were mounted to cover glasses (22 X22) and placed in substrate-filled specially produced cuvettes. The capacity of such a cuvette was 10 ml with room for 4 cover glasses each 4 mm apart. For protection against vaporization small caps of plastic foil were used. The method given by GLOCKNER and SCHMIDT (7) who recently reported their studies in the rat aorta, yielded satisfactory results in small arteries. In thereabbit aorta, however, the results varied markedly, which may be due to greater thickness of the vascular wall. Consequently, despite of all its advantages, this technique could not be employed in this study. The following enzymes were demonstrated: hydrolases: phosphomonoesterase I--3.1.3.1. with naphthol AS-sulphate after BURSTONE in the modification of LOJDA phosphomonoesterase II--3.1.3.2. after BURSTONE as modified by LOJDA with naphthol AS-phosphate and hexazonium pararosanilin leucine aminopeptidase--3.4.1.1. technique according to NACHLAS et al. with leucine beta-naphthylamide lipase--3.1.1.3. after GOMORI (Tween 80) adenosine-5-phosphatase (5'nucleotidase)--3.1.3.5. after GOMORI succinic dehydrogenase--1.3.99.1.--method according to NACHLAS et al. NAD (NADH and NADP (NADPH) diaphorase--1.4.1.2.--1.4.1.4., technique of SCARPELLI, HESS and PEARSE as modified by LOJDA (see article) Substratefree or heatinactivated controls were prepared for each histochemical demonstration. Several additional series with phenazine methosulphate (PMS) as an intermediate electron acceptor or such without PMS as well as preparations with KCN or without KCN were examined for the demonstration of anaerobic dehydrogenases. However, to avoid the nothing dehydrogenase effect only PMS-processed specimens were evaluated. For histological information several additional sections of the tissue samples were stained with haematoxylin-eosin or with elastica-van Gieson and Weigert's iron haematoxylin in combination. The enzyme histochemical preparations were embedded in Kaiser's glycerine gelatin, the histological sections were embedded in Caedox. The test animals were grouped like in the recent experiments (see literature no. 2, 15, 16, 17, 18). 2...


Comparative karyometric studies on small pseudolobuli and hepatomas in thioacetamide induced liver cirrhosis. In the tumorous phase of TAA-induced cirrhosis of the liver comparative studies on the mitotic index, on the frequency of binucleated liver cells and on nuclear volume showed approximately similar values in the small pseudolobuli and in several hepatomas. Obviously "minimum deviation hepatomas" had developed. Decrease of ploidy characteristic for hepatomas was observed. In thioacetamide hepatomas the phenomenon of quadrupling of the cell nuclei due to dysfunctional nuclear edema occurred whereas the normal nuclei of liver cells can only double in volume in all phases of thioacetamide-intoxication. Particular karyometric aspects of the nuclear-nucleolar metabolism of hepatoma cells following thioacetamide intoxication are discussed.


Interaction of Lens culinaris lectin, concanavalin A, Ricinus communis agglutinin and wheat germ agglutinin with the cell surface of normal and transformed rat liver cells. The observation of BOREK et al. (1973) on nonagglutinability of transformed rat liver cells by Lens culinaris lectin and our ultrastructural findings of a greater mobility of the Lens culinaris lectin receptors on transformed rat liver cells as compared to normal rat liver cells (ROTH 1975) initiated the present agglutination experiments on liver cells with lectins. For agglutination assay the microhemadsorption technique after FURMANSKI et al. (1973) was used with exception of several tests on EDTA-detached cells. The transformed rat liver cells exhibited, in contrast to the findings of BOREK et al. (1973), a positive microhemadsorption with Lens culinaris lectin as well as with Concanavalin A, Ricinus communis lectin and wheat germ agglutinin whereas the normal rat liver cells became positive only after a brief trypsin treatment. The significance of the difference in agglutinability of rat liver cells with Lens culinaris lectin and the other lectins used is discussed with regard to the cell-cell interaction mediated by lectins.


Structural patterns and histological behaviour of experimental sarcomas. I. General considerations, histology and histochemistry. Methylcholanthrene-induced rat sarcomas were used as a model for the examination of morphological and cytological differentiations in tumours of the connective tissue. Particular attention was paid to the question how far these sarcomas are comparable to human connective tissue tumours. The histological examination yields a broad spectrum of mesenchymal differentiations ranging from undifferentiated anaplastic sarcomas over less differentiated fibrosarcomas and malignant  fibrous histocytomas to well-differentiated fibrosarcomas, myosarcomas and haemangiopericytomas. It is worth mentioning that different histological structures can be encountered with one and the same tumour.


Oxygenation of a dipyrromethene model for bilirubin: formation of a singlet oxygen-like product. An oxodipyrromethene model compound for bilirubin is found to undergo oxidation to a blue tetrapyrrole and a water-propentdyopent on a silica gel thin layer chromatography plate. The reaction involves ground state oxygen and requires silica gel, although the propentdyopent is an expected product from reaction with singlet oxygen.


Some properties of external NADH oxidation by human placental mitochondria. Isolated human term placenta mitochondria catalyse oxidation of external NADH in the presence of cytochrome c. This reaction is insensitive to the respiratory chain inhibitors such as rotenone and antimycin A, and is not coupled to phosphorylation. Comparison of the effect of Mg++ ion on NADH plus cytochrome c oxidation by human term placental, human skeletal muscle and rat skeletal mitochondria showed that Mg++ ion exerts an inhibitory effect in the case of human mitochondria and a stimulatory effect in the case of rat skeletal muscle mitochondria.


Metabolic requirements for entry of DNA in Branhamella catarrhalis. Although requirements for transformation in Branhamella catarrhalis are quite complex, DNA synthesis does not appear to be one of these needs, as indicated by the inability of nalidixic acid to interefere with transformation. Exogenous sources of energy, such as cAMP and cGMP also failed to enhance frequency, suggesting cells may actively engage in energy production to achieve uptake of DNA, or lack a transport mechanism for these compounds.


3-Hydroxy-3-methylglutaric acid and triton-induced hyperlipidemia in rats. 3-Hydroxy-3-methylglutaric acid (HMG) significantly decreased cholesterol, triglyceride and phospholipid levels in whole serum, serum beta-lipoproteins and liver of Triton-induced hyperlipidemic rats. Therapeutically 50 mg HMG/kg is equivalent to 200 mg nicotinic acid/kg in lowering all these lipid parameters. HMG may exert its hypolipidemic effect through inhibition of lipoprotein synthesis.


Correlation between leuteinizing hormone responses to luteinizing hormone-releasing hormone (LH-RH) and to D-leu-6-LH-RH-ethylamide in patients with hypothalamo-pituitary disease. Seventeen patients (eleven females and six males) with organic hypothalamo-pituitary disease were subjected to a test consisting of a rapid intravenous injection of 50 mug of luteinizing hormone-releasing hormone (LH-RH) at 8:00 A.M., with blood sampling before and 30 and 60 minutes afterward. Two to four weeks later, a rapid intravenous injection of 50 mug of D-Leu-6-des-Gly-10-LH-RH-ethylamide (D-Leu-6-LH-RH-EA) was given under similar conditions, with blood sampling before and 30, 60, and 90 minutes afterward. Serum LH levels were determined by radioimmunoassay. D-Leu-6-LH-RH-EA caused a greater and more sustained rise in serum LH levels than did an equal dose of LH-RH. However, functional classifications of patients were similar with either preparation. This finding suggests that acute administration of D-Leu-6-LH-RH-EA does not cause a higher number of relevant responses as compared with LH-RH, but only a greater stimulation of LH releases in responsive patients.


[Interaction of norethindrone on estrogen and progesterone receptors in the rabbit uterine cytosol (author's transl)]. Norethindrone (ENT), which is a representative in estrane series of progestogen, is not only strongly progestational but also estrogenic and in some cases, antiestrogenic. To understand progestational effect and antiestrogenic effect, the interactions of ENT on estrogen and progestogen receptors were studied in the uterine cytosol of white female rabbit. The 274,200 X G supernatant of uterine homogenate was used as cytosol. 3H-Estradiol, 3H-Progesterone, 3H-ENT or cold ENT were incubated with uterine cytosol at 4 degrees C for 2 hours. Results are as follows: 1. Sucrose gradient centrifugation [5 approximately 20% linear and 40,000 rpm (159,200 X G) for 16 hours at 4 degrees C]: ENT was bound to extrogen 8S receptor in immature rabbit uterus (Fig. 2 & 3), and to progestogen 8S receptor in estrogen primed rabbit uterus (Fig. 5). 2. Kinetic study, determined by dextran coated charcoal (0.001% dextran and 0.1% charcoal): (1) In the uterine cytosol of immature rabbit, 3H-estradiol-receptor binding was observed with Kd divide by 3.6 X 10-9 M and it was revealed that ENT was a competitive inhibitor to this binding with Ki divide by 2.6 X 10-6 M, as in Fig. 6. (2) 8S component, obtained by centrifugation of uterine cytosol (Fig. 1) in estrogen primed rabbit, binds 3H-progesterone with Kd divide by 8.1 X 10-10 M and Bm (maximal binding sites) divide by 5.0 X 10-8 M/mg of protein, and ENT was a competitive inhibitor in this binding with Ki divide by 2.3 X 10-9 M (FIG. 7 & 8). 3H-ENT-8S binding was demonstrated with Kd divide by 1.1 X 10-9 M and Bm divide by 8.7 X 10-8 M/mg of cytosol protein (Fig. 8). These results indicate: (a) ENT is bound to both estrogen and progestogen receptors in 8S macromolecules of uterine cytosol, (b) competitive inhibition of ENT to these bindings indicated that ENT is bound to these receptors at the steroid binding sites where estradiol and progesterone bind to, (c) ENT has much more affinity to progestogen receptor (Ki divide by 2.3 X 10-9 M) than to estrogen receptor (Ki divide 2.6 X 10-6 M), (d) while ENT is bound to progestogen and estrogen receptors at the same time, Bm of ENT (8.7 X 10-8 M/mg of cytosol protein) is more than Bm of progesterone (5.0 X 10-9 M/mg of cytosol protein), and Kd of ENT (1.1 X 10-9 M) was less than Ki of ENT (2.3 X 10-9 M) in the binding to progesterone-receptor. Biologically, while ENT is bound to progestogen -receptor with high affinity and to estrogen receptor with low affinity, ENT is actually progestational in low dose and antiestrogenic in high dose but the anti-estrogenicity seems to be incomplete in vivo as ENT may be metabolized to a potent estrogenic compound, ethinyl estradiol


[Studies on progesterone receptor in rabbit uterine cytosol -- nuclear translocation and chromatin binding-- (author's transl)]. Estrogen priming increases uterine 8S macromolecule which binds progesterone specifically. Progesterone-8S complex in the cytoplasm enters into nucleus and is bound to chromatin finally. In this paper, the mode of nuclear translocation of steroid in exchange assay of receptor introduced by Anderson et al., and the mode of binding to chromatin were studied on the progesterone-receptor complex in the uterus of estrogen primed female rabbit. 1. After intravenous administration of 200 mug progesterone into the estrogen primed immature rabbit, uterine nuclei were prepared by the method in Table 1. These nuclei were incubated with 3H-progesterone and cold steroids at 4 degrees C for 30 minutes, and then washed with buffer A. The radioactivity of the nuclei was counted. This experiment was performed at 4 degrees C because progesterone receptor and chromatin were observed to be degraded at 37 degrees C for 20 minutes. The effect of cold steroids in vitro on the incorporation of 3H-progesterone into the uterine nuclei of rabbit pretreated with progesterone was found to be similar to their effect on progesterone-receptor binding in cytosol or chromatin (Fig. 1). 2. The effect of cold steroids on 3H-progesterone-receptor-chromatin triplex (Table 2 and Fig. 2) was examined. Once 3H-progesterone-receptor-chromatin triplex was formed, it was difficult to exchange 3H-progesterone to other steroids at 4 degrees C. These results (1 & 2) indicate that progesterone-receptor complex enters into nucleus and is bound to chromatin. Exchange of steroid may occur in the nuclear progesterone-receptor complex, which is free from the binding with chromatin. And thus exchange assay cannot represent quantitative data on receptor content. 3. 3H-progesterone-8S or 5S complexes were obtained by 5 approximately 20% sucrose linear gradient centrifugation (Fig. 3). The same molar concentration of these complexes from estrogen primed or castrated rabbit uterus were incubated with primed uterine chromatin for 30 minutes. Then the chromatin was washed with buffer A and the radioactivity was counted. It was shown in Fig. 4 that 3H-progesterone-8S complex was bound to chromatin much more tightly than 3H-progesterone 5-S complex in preparations obtained from both castrated or primed uterine cytosols. All these results indicate that 8S may be the biologically active form of the receptor. 4.3H-progesterone uptake into uterine nuclei was observed in very limited amount following the injection into uterine artery. The radioactivity in nuclei decreased easily by washing with buffer A as in Fig. 5. The small amount of residual radioactivity after washing, that is, very limited number of binding sites with high affinity is considered to be indicative of biologically active binding.


Studies on glucagon secretion using isolated islets of Langerhans of the rat. Glucagon secretion and its control have been studied in perifused isolated islets of Langerhans of the rat. It was shown that a low concentration of glucose per se does not cause increased glucagon secretion, but that at low glucose concentrations the amino acid arginine stimulates a biphasic secretory response. Such amino acid stimulated glucagon secretion can be suppressed by increasing the glucose content of the perifused media from 1.67 to 5.5 or 16.7 mM; insulin secretion is also then increased. Since high concentrations of added porcine insulin (10 mU/ml) did not affect amino acid stimulated glucagon secretion at low glucose concentration, it was concluded that high concentrations of glucose and not insulin secreted in response to that glucose are probably responsible for suppression of glucagon secretion. At low concentrations of glucose, epinephrine (2.5 X 10(-7) M) also stimulated glucagon secretion. It is concluded that isolated rat islets of Langerhans can be used for the study of glucagon secretion in vitro, and that substances appearing in the blood in vivo at low glucose concentrations are probably responsible for increased glucagon secretion under conditions associated with hypoglycemia.


Plasma immunoreactive glucagon fractions in four cases of glucagonoma: increased "large glucagon-immunoreactivity". Immunoreactive glucagon (IRG) fractions from plasma of 8 normal subjects and 4 patients with glucagon secreting tumors were studied by gel filtration techniques on Bio Gel P--30 and Sephadex G--50 columns. The pancreatic glucagon specific anti serum (30K) of Unger was utilized to measure IRG. Columns were calibrated with labelled albumin, proinsulin, insulin and glucagon. Four peaks were defined in normal and tumor bearing patients: peak I (greater than 20 000 mol. wt.), peak II (primarily 9000 mol. wt.), peak III pancreatic glucagon (3500 mol. wt.) and peak IV small gucagon (less than 3500 mol. wt.). Glucagonoma patients differed from our normal and reported normal subjects in that peak II contained most of the circulating IRG. The percent of IRG associated with peak II was 9.5--31.5% in normals and 39.1--61.2% in glucagonomas. Glucagon-like biological activity in an isolated hepatocyte system was demonstrated for all peaks. However, relative to immunoreactivity, peak II showed reduced activity (25--33%). Immunoassay of dilutions of all peaks revealed the probability of immuno determinants identical with procine pancreatic glucagon. The presence of heterogenous IRG peaks with biological glucagon-like activity suggest that the larger molecules may be prohormones. Further, it is possible that specific elevation of peak II may be a diagnostic feature of glucagonomas.


Catecholamine receptor sensitivity and the regulation of lipolysis in adult diabetes. The sensitivity of alpha- and beta-adrenergic receptors, and the antilipolytic actions of prostaglandin E1 or insulin on adipose tissue of obese diabetic and non-diabetic subjects have been studied. Accumulation of cyclic AMP in adipose tissue and release of glycerol in response to several catecholamines (adrenaline, noradrenaline and isoprenaline) in the presence or absence of an alpha-adrenergic blocker (phentolamine) have been used to assess catecholamine receptor sensitivity. No differences in beta-receptor activity were observed between diabetics and non-diabetics, either on glycerol release or accumulation of cyclic AMP; alpha-receptor activity was also similar, except for significantly less accumulation of cyclic AMP in diabetic tissue incubated with noradrenaline and phentolamine (p less than 0.01). The antilipolytic action of prostaglandin E1 (at concentrations of 30 fM to 30 pM) on lipolysis (stimulated submaximally with isoprenaline, 10(-7) M) was similar in diabetic and control groups. The antilipolytic action of insulin (from 10(-10) to 10(-6) M) on lipolysis was also similar between the groups. It is concluded that neither disorders of the catecholamine receptor nor of the antiliolytic actions of prostaglandin E1 or insulin are responsible for the abnormalities of fatty acid metabolism in adult diabetes.