Is the cyclic AMP in psoriatic epidermis low?

A deficiency of epidermal cyclic adenosine 3', 5'-monophosphate (cyclic AMP) has been implicated as the cause of the incomplete differentiation, increased mitosis and increased glycogen concentration which characterize the psoriatic lesion. This communication contains our data on the epidermal concentration of cyclic AMP in the lesional and 'normal' (non-involved) skin of patients with psoriasis. In these studies we have introduced the following improvement in methodology: (1) Local ischaemia in the biopsy specimens was prevented by not giving any injections prior to removal of the tissues and by freezing the tissues prior to removal. (2) The epidermis was microdissected from the dermis prior to cyclic AMP assay as as to give as pure an epidermal specimen as possible. Epidermal cyclic AMP was found to be 20-25% higher in the skin lesion than in the uninvolved skin when compared on a dry weight or protein basis but essentially when compared on the basis of DNA content. Our conclusion is that the cyclic AMP level in the psoriatic lesion is not lower than that in the 'normal' skin and thus these characteristics in psoriatic lesions cannot be related simply to a cyclic AMP deficiency.


A method of liberating living cells from the dermal infiltrate.

A new technique is described whereby viable infiltrating cells can be freed from skin biopsy specimens. The specimens are incubated with collagenase and then mechanically disaggregated. The liberated cells are still suitable for immunological and morphological study. Using this method, the nature of the dermal infiltrate in patients with skin reticuloses was compared with that in lichen planus. A predominance of T cells was found in mycosis fungoides, the Sezary syndrome, and lichen planus, and of B cells in non-Hodgkin lymphomas.


Further studies of scratching during sleep.

Fifteen patients with a variety of itching skin diseases (atopic eczema, dermatitis herpetiformis, lichen planus, urticaria and psoriasis) have been studied in the sleep laboratory. Recordings were made of all-night electroencephalogram, electro-oculogram, submental electromyogram, and muscle potentials from both forearms. Bouts of scratching during orthodox (NREM) sleep occurred more frequently in stages 1 and 2 than in stages 3 and 4. The frequency in paradoxial (REM) sleep was close to that in stage 2 sleep. This pattern was similar for all the diseases studied and seems to be related to the physiology of the sleep stages rather than to the skin diseases themselves. The mean duration of the bouts of scratching was not related to the sleep stage in which they started.


Unusual cutaneous associations of a malignant carcinoid tumour of the bronchus--erythema annulare centrifugum and white banding of the toe nails.

The case history of a patient who presented with erythema annulare centrifugum and white banding of the toe nails is recorded. On investigation for an underlying malignancy the patient was found to have an opacity in the lower lobe of the right lung. Subsequently lobectomy was performed and histology of the operative specimen revealed a malignant carcinoid tumour of the bronchus.


Absorption and biotransformation of topically applied 8-(methylthio) cyclic AMP. Studies in dogs and humans.

8-(Methylthiocyclic AMP-35S was applied topically to the intact or abraded skin of dogs at a dose of about 2-5 mg/kg. In this species, an average of 7% of the dose was absorbed through the intact and 62% through abraded skin. Half-lives for radioactivity in plasma averaged 3-6 h. After the topical application of the drug to the intact skin of dogs, the average concentration in plasma of glucose, but not of insulin, was increased by as much as 21%; after application to abraded skin, glucose and insulin were increased, on average, by as much as 84 and 221%, respectively. 8-(methylthio)adenosine and two unidentified compounds were present as metabolites in the urine of dogs; some unchanged 8-(methylthio)cyclic AMP-35S was also excreted. 8-(methylthio)cyclic AMP-35S was also applied topically (25 mg; 0-32 mg/kg) to the intact and stripped skin of normal human subjects under conditions similar to those used for dogs. Three subjects with intact skin did not absorb any of the drug, whereas three subjects with stripped skin absorbed 0-5, 8-3, and 23-3% of the dose. The half-life of radioactivity in the plasma of the subject with the greatest absorption was 0-5 h during the first 2 h, and 11 h for the next 10 h. During the first 2 h, this same subject excreted unchanged drug and 8-(methylthio)adenosine in his urine. No changes in glucose or insulin concentrations in plasma were observed in any of the subjects nor was there any apparent irritation of the skin.


Plane xanthomata associated with multiple myeloma and hyperlipoproteinaemia.

A case of plane xanthomatosis associated with multiple myeloma and hyperlipoproteinaemia in a 62-year-old woman is reported. The patient had IgG type lambda paraproteinaemia and type II A hyperlipoproteinaemia. The IgG paraprotein showed binding activity against high-density lipoprotein. It could not be demonstrated that these paraprotein-lipoprotein complexes were responsible for the hyperlipidaemia. Microchemical analysis of pathological skin demonstrated a high content of triglycerides, similar to that in eruptive xanthomata. No correlation was found between the lipid composition of the xanthomatous skin and the serum lipid composition. The results obtained with an in vitro culture method showed that the paraprotein present in the patient's serum was synthesized in the bone marrow but not in the normal or pathological skin.


Granulocyte function in chronic granulocytic leukaemia. I. Bactericidal and metabolic capabilities during phagocytosis in isolated granulocytes.

The ingestion, bactericidal activity and metabolism of isolated mature neutrophil leucocytes during phagocytosis was studied in 17 patients with chronic granulocytic leukaemia (CGL) with the simultaneous use of normal controls. Seven patients had received no treatment and the others had been treated previously with Busulphan. The phagocytic indices for killed yeast cells did not differ from those of the controls. A diminished bactericidal activity against E. coli was found in nine CGL cases. The bactericidal capacity closely correlated with the degree of leucocytosis since patients with a WBC count of 90 000/mul or higher with one exception showed decreased bactericidal activities while patients with WBC counts below 90 000/mul with two exceptions showed normal bactericidal activities. The [I-14C]-glucose oxidation during phagocytosis was increased in four patients and decreased in three patients. Some correlation was found between abnormally high or low [I-14C]glucose oxidation and diminished bactericidal activity. The intracellular iodination reaction during phagocytosis was decreased in 10 cases while the extracellular iodination was increased in six cases and decreased in one case. The data for granulocyte iodination did not correlate with WBC count, bactericidal capacity or [I-14C]glucose oxidation. The time course for the bactericidal activity and granulocyte iodination seemed to deviate from the controls indicating a slow initial ingestion and/or degranulation phase. The CGL granulocyte content of myeloperoxidase was normal or increased, the lysozyme content was decreased in half of the cases while the amount of antibacterial cationic proteins was increased, normal or low. The present findings indicate a variety of abnormalities in the mature CGL granulocyte, which are not closely interrelated.


Partial myeloperoxidase deficiency in a case of preleukaemia. I. Studies of fine structure and peroxidase synthesis of promyelocytes.

The ultrastructural localization of peroxidase activity has been studied in the circulating neutrophils and in a neutrophilic series of bone marrow cells from a patient with preleukaemia. Light microscopic examination showed 36% of the polymorphonuclear leucocytes to be totally devoid of myeloperoxidase, while 50% were normally stained and 14% were slightly positive for this enzyme. Electron microscopic studies revealed considerable heterogeneity in the promyelocyte population, since the number of peroxidase-deficient azurophil granules was seen to vary from 0 to 100% in these cells. Thus, several types of promyelocytes were identified. One cell type, which resembled that seen in normal subjects, contained myeloperoxidase within its azurophil granules and also within the cisternae of the rough endoplasmic reticulum and Golgi complex. A second type of promyelocyte, which was at an early stage of development, lacked myeloperoxidase in its secretory apparatus. These cells contained two species of azurophil granules, the first of which was devoid of peroxidase activity whereas the other reacted positively. These observations suggest that the premature arrest of myeloperoxidase synthesis in the promyelocytes from a preleukaemic patient may give rise to several populations of circulating neutrophils which can exhibit varying contents of myeloperoxidase.


Partial myeloperoxidase deficiency in a case of preleukaemia. II. Defects of degranulation and abnormal bactericidal activity of blood neutrophils.

A patient with a refractory anaemia preceding acute myeloblastic leukaemia had an increased susceptibility to infection due to Staphylococcus aureus. 36% of neutrophils lacked myeloperoxidase (MPO) activity and, in vitro, these polymorphonuclear neutrophils (PMN) had a defect of bactericidal activity against Staphylococcus aureus. Cytochemical studies of phagocytosis with the electron miscroscope have shown that the degranulation of primary granules (MPO+ or MPO-) was normal after phagocytosis of Escherichia coli which were normally lysed. A defective destruction of Staphylococcus aureus and Candida albicans was observed in some PMN with or without MPO activity, suggesting that MPO deficiency itself was not the only cause of this defect. In PMN which appeared normal, most MPO(+) granules were unable to fuse with the phagocytic vacuole containing intact germs even after 90 min of contact. There was, therefore, in addition to a partial MPO deficiency, a defect in cellular degranulation. This defect, the mechanism of which is unknown, may be in part responsible for the defective bacterial degradation.


Sodium periodate modification of factor VIII procoagulant activity.

Chemical treatment of human factor VIII by sodium periodate resulted in increased levels of procoagulant activity when measured by the two-stage assay. Increased levels of factor-VIII activity were observed following periodate treatment of factor-VII concentrates and normal, haemophilic and von Willebrand's disease plasmas. However, sodium periodate treatment of haemophilic plasmas which were completely deficient in factor-VIII activity did not cause any reduction in the clotting times of two-stage assays. No increase in procoagulant activity following oxidation of factor VIII could be detected by the one-stage assay method. Gel filtration of factor-VIII concentrates before periodate treatment showed that the protein exhibiting factor-VIII activity had an apparent molecular weight in excess of 1 000 000, whilst after periodate treatment the apparent molecular weight was reduced to 690 000.


Primary carcinoma of stomach with uterine metastasis.

Curettings from a 42-year-old woman who suffered from heartburn, weight loss and irregular vaginal bleeding showed apparently metastatic mucoid adenocarcinoma in the endocervix. The primary site of tumour was not evident. At laparotomy a gastric adenocarcinoma was found and total hysterectomy with bilateral salpingo-oophorectomy and partial gastrectomy were performed. Histological examination showed a diffuse mucoid adenocarcinoma of stomach with metastases to lymph nodes, uterine body, cervix and one ovary. The literature about metastasis of extragenital cancer to the uterus is reviewed and possible mechanisms are discussed.


Mechanism and specificity of rhodopsin phosphorylation.

Partial separation of protein kinase activity from rhodopsin in isolated bovine retinal photoreceptor outer segments was accomplished by mild ultrasonic treatment followed by ultracentrifugation. Residual kinase activity in the rhodopsin-rich sediment was destroyed by chemical denaturation which did not affect the spectral properties of the rhodopsin. The retinal outer segment kinase was found to be specific for rhodopsin, since in these preparations it alone of several bovine protein kinases was capable of phosphorylating rhodopsin in the light. The phosphorylation reaction apparently requires a specific conformation of the rhodopsin molecule since it is abolished by heat denaturation of rhodopsin, and it is greatly reduced or abolished by treatment of the visual pigment protein with potassium alum after the rhodopsin has been "bleached" by light. When kinase and rhodopsin or opsin fractions were prepared from dark-adapted and bleached outer segments and the resultant fractions were mixed in various combinations of bleached and unbleached preparations, the observed pattern of light-activated phosphorylation was consistent only with the interpretation that a conformational change in the rhodopsin molecule in the light exposes a site on the visual pigment protein to the kinase and ATP. These results rule out the possibility of a direct or indirect (rhodopsin-mediated) light activation of the kinase. Finally, phosphorylation of retinal outer segment protein in monochromatic lights of various wavelengths followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that both rhodopsin and the higher molecular weight visual pigment protein reported by several laboratories have the same action spectrum for phosphorylation. This result is consistent with the suggestion that the higher molecular weight species is a rhodopsin dimer.


The subunit structure of the cytochrome c oxidase complex.

The subunit structure of the cytochrome c oxidase complex has been obtained for three preparations each isolated by a different detergent procedure. Six polypeptides were present in all samples with the following molecular weights: subunits I, 36000; II, 22500, III, 17100; IV, 12500; V, 9700; and VI, 5300. These subunits have been purified by gel filtration in sodium dodecyl sulfate or in 6 M guanidine hydrochloride and their amino acid compositions have been determined. Subunit I is hydrophobic in character with a polarity of 35.7%. Subunits II through VI are more hydrophilic with polarities of 45.5, 48.6, 47.8, 49.7, and 53.7%, respectively.


Requirement for protein synthesis in the regulation of protein breakdown in cultured hepatoma cells.

The modes of action of insulin and of inhibitors of protein synthesis on the degradation of labeled cellular proteins have been studied in cultured hepatoma (HTC) cells. Protein breakdown is accelerated upon the deprivation of serum (normally present in the culture medium), and this enhancement is inhibited by either insulin or cycloheximide. An exception is a limited class of rapidly turning over cellular proteins, the degradation of which is not influenced by insulin or cycloheximide. Alternative hypotheses to explain the relationship of protein synthesis to the regulation of protein breakdown, viz., control by  the levels of precursors of protein synthesis, regulation by the state of the ribosome cycle, or requirement for a product of protein synthesis, have been examined. Protein breakdown was not influenced by amino acid deprivation, and measurements of valyl-tRNA levels in HTC cells subjected to various experimental conditions showed no correlation between the levels of charged tRNAVal and the rates of protein degradation. Three different inhibitors of protein synthesis (puromycin, pactamycin, and cycloheximide) suppressed enhanced protein breakdown in a similar fashion. A direct relationship was found between the respective potencies of these drugs to inhibit protein synthesis and to block enhanced protein breakdown. When cycloheximide and insulin were added following a prior incubation of HTC cells in a serum-free medium, protein breakdown was maximally suppressed within 15-30 min. Actinomycin D inhibited protein breakdown only after a time lag of about 90 min. It is suggested that the regulation of protein breakdown in hepatoma cells requires the continuous formation of a product of protein synthesis, in a manner analogous to the mode of the control of this process in bacteria.


Kinetics of hydrogen-deuterium exchange in adenosine 5'-monophosphate, adenosine 3':5'-monophosphate, and poly(riboadenylic acid) determined by laser-Raman spectroscopy.

Pseudo-first-order rate constants governing the deuterium exchange of 8-CH groups in adenosine 5'-monophosphate, adenosine 3':5'-monophosphate, and poly(riboadenylic acid) (poly(rA)) were determined as a function of temperature in the range 20-90 degrees C by means of laser-Raman spectroscopy. For 5'-rAMP, the logarithm of the rate constant exhibits a strictly linear dependence on reciprocal temperature, i.e., kpsi = Ae-Ea/RT, with A = 2.3 X 10(14) hr-1 and Ea = 24.2 +/- 0.6 kcal/mol. For cAMP, above 50 degrees C, kpsi is nearly identical in magnitude and temperature dependence to that of 5'-rAMP. However, below 50 degrees C, isotope exchange in cAMP is much more rapid than in 5'-rAMP, characterized by a lower activation energy (17.7 kcal/mol) and frequency factor (9.6 X 10(9) hr-1). Exchange in poly(rA) is considerably slower than in 5'-rAMP at all temperatures, but like cAMP the in k vs. 1/T plot may be divided into high temperature and low temperature domains, each characterized by different Arrhenius parameters. Above 60 degrees C, poly(rA) gives Ea = 22.0 kcal/mol and A = 3.2 X 10(12) hr-1, while below 60 degrees C, Ea = 27.7 kcal/mol and A = 1.8 X 10(16) hr-1. Thus, increasing the temperature above 60 degrees C does not diminish the retardation of exchange in poly(rA) vis a vis 5'-rAMP. These results indicate that the distribution of electrons in the adenine ring of cAMP is altered by lowering the temperature below 50 degrees C, although no similar perturbation occurs for 5'-rAMP. Retardation of exchange in poly(rA) is most probably due to base stacking at lower temperatures and to steric hindrance from the ribopolymer backbone at higher temperatures. We also report the spectral effects of deuterium exchange on the vibrational Raman frequencies of 5'-rAMP, cAMP, and poly(rA) and suggest a number of new assignments for the 5' and cyclic ribosyl phosphate groups.


Activation of liver alcohol dehydrogenases by imidoesters generated in solution.

Various omega-halogenated carboxy acids and amides were evaluated as potential active-site-directed reagents for alcohol dehydrogenase. 2-Bromoacetamide and bromoacetic and 3-bromopropionic acids inactivated the enzyme; AMP, NAD+, and NADH markedly decreased the rate of inactivation. Some omega-halogenated carboxyamides, X(CH2)nCONH2, increased the activity of the enzyme with the rate and extent of activation depending on the number of methylene units (n) in the order 3 greater than 4 greater than 2 and on X in the order Br greater than Cl. 4-Chlorobutyramide (0.1 M) activated the horse liver enzyme 20-fold in 24 hr at pH 8.0 and 25 degrees. The activation was not prevented by AMP or 2,2-bipyridine, but was by NADH. The kinetic constants and turnover numbers for human and horse liver alcohol dehydrogenases treated with chlorobutyramide were increased markedly compared to those for native enzymes. Alcohol dehydrogenase treated with chlorobutyramide was not further activated by methyl picolinimidate, an imidoester which activates native enzyme by modifying amino groups in the active sites. Chlorobutyramide does not appear to react directly with the enzyme but cyclizes in the reaction medium to form an intermediate imidoester, 2-iminotetrahydrofuran, which reacts with most of the amino groups of the enzyme.


Activation of acetyl- and butyrylcholinesterase by enzymatic removal of sialic acid from intact neuroblastoma and astroblastoma cells in culture.

Removal of sialic acid from intact mammalian nervous system cells in tissue culture is accompanied by an immediate increase in cellular cholinesterase activity. Treatment of hamster astroblast cells (clonal line NN) and mouse neuroblastoma cells (clonal lines S21, N18, and N115) for brief periods with a low level of Clostridium perfringens sialidase, 5 X 10(-3) units/ml, removed 1-15 mug of sialic acid per mg of cell protein and brought about a large increase in v0 and Vmax of cellular acetylcholinesterase (EC 3.1.1.7). Butyrylcholinesterase (EC 3.1.1.8) activities also increased upon careful enzymatic removal of cellular sialic acid, and cells with characteristically low butyrylcholinesterase activity, e.g., adrenergic clonal line N115 neuroblasts displayed relatively high activity after treatment with sialidase. These findings open the possibility that adaptive regulation of cholinesterases in mammalian cells may be mediated rapidly through changes in their sialic acid content.


The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: chemiluminescence and peroxidation.

Reaction of bovine erythrocyte superoxide dismutase with H2O2 was accompanied by a luminescence whose intensity was a function of the concentration of H2O2 and whose duration was coincident with the inactivation of the enzyme by this reagent. Oxygen, which protected against inactivation, also diminished the luminescence. Several other compounds which prevented the inactivation by H2O2 also modified the luminescence. Thus urate, formate, and triethylamine inhibited luminescence whereas imidazole and xanthine augmented it. These seemingly contrary effects can be explained by assuming that the compounds which protected the enzyme were peroxidized in competition with the sensitive group on the enzyme. The luminescence arises because that group on the enzyme was oxidized to a product in an electronically excited state, which could return to the ground state by emitting light. Imidazole and xanthine gave electronically excited products whose quantum efficiency was greater than that of the group on the enzyme, whereas urate, formate, and triethylamine gave products with much lower luminescent efficiencies. This superoxide dismutase could catalyze the peroxidation of a wide range of compounds, including ferrocytochrome c, luminol, diphenylisobenzofuran, dianisidine, and linoleic acid. In control experiments, boiled enzyme was inactive. This peroxidative activity can lead to unexpected effects when superoxide dismutase is added to H2O2-producing systems, as a probe for the involvement of O2-. Several examples from the literature are cited to illustrate the misinterpretations which this previously unrecognized peroxidative activity can generate.


Inactivation of rabbit, pig, and carp adenylate kinases by N6-o- and p-fluorobenzoyladenosine 5'-triphosphates.

N6-O- and p-fluorobenzoyladenosine 5'-triphosphates (IIIc and IIc, respectively) have been synthesized as potential adenosine 5'-triphosphate (ATP) site-directed reagents for enzymes. IIc and IIIc were substrates of yeast hexokinase; neither they nor the corresponding ADP derivatives inactivated yeast hexokinase or rabbit pyruvate kinase. IIc rapidly inactivated rabbit and carp muscle adenylate kinases; the effect is probably ATP site directed because N6-benzoyl-ATP did not inactivate and was a substrate (Vmax = 28 and 10%, respectively, that of ATP), and because of ATP retarded the inactivation. The inactivations followed pseudo-firsr-order kinetics; in the presence of 2.64 mM ATP at 0 degrees the half-life of the rabbit kinase was 210 min with 50 muM IIc and the half-life of the carp kinase was 130 min with 100 muM IIc. Adenylate kinase of pig muscle was inactivated by IIc in a manner similar to the rabbit and carp enzymes except that the rate of inactivation exhibited an inflexion. IIIc inactivated rabbit, pig, and carp adenylate kinases by pseudo-first-order kinetics; the rate constants for inactivation at 0 degrees were 9.1 X 10(-3), 1.3 X 10(-3), and 1.9 X 10(-3) min-1 and the apparent dissociation constants (K) of the IIIc-enzyme complexes were 710, 970, and 720 muM, respectively. From the substrate properties of IIIc alone and in admixture with ATP its dissociation constants (Ki) from the ATP sites of the enzymes were found to be 500, 700, and 845 muM, respectively. The similarity between the K and Ki values, together with marked retardation of the inactivations by ATP, indicates that IIIc is an ATP-site-directed reagent for the three adenylate kinases.


Uridine diphosphate glucose synthase from calf liver: determinants of enzyme activity in vitro.

The reaction catalyzed by calf liver uridine diphosphate glucose synthase (pyrophosphorylase) (EC 2.7.7.9; UTP + glucose 1-phosphate = UDP-glucose + PPi) is an example of an enzymic reaction in which a nucleoside triphosphate other than ATP is the immediate source of metabolic energy. Kinetic properties of the enzyme, acting in the direction of UCP-glucose formation were investigated in vitro. The reaction was inhibited by UDP-glucose (0.072), Pi (11), UDP (1.6), UDP-xylose (0.87), UDP-glucuronate (1.3), and UDP-galacturonate (0.95). The numbers in parentheses indicate the concentration (mM) required for half-maximal inhibition under the conditions used. Other compounds tested, including ATP, ADP, and AMP, had no effect. Over a range of concentrations of UTP (0.04-0.8 MM) and UDP-glucose (0.05-0.03 mM), the reaction rate was more dependent on the concentration ratio [UDP-glucose]/[UTP] than on the absolute concentration of either compound. Comparison of the kinetic properties in vitro with estimates of metabolite levels in vivo suggests that (1) the enzyme operates in a range far from its maximal rate, and (2) the concentrations of glucose 1-phosphate and Pi and the ratio [UDP-glucose]/[UTP] may be the most important determinants of UDP-glucose synthase activity.


Rotational correlation times and partition coefficients of a spin label solute in lecithin vesicles.

Di-tert-butylnitroxide dissolved in an aqueous suspension of egg yolk lecithin vesicles is distributed between the two phases. Partition coefficients of the nitroxide between the lipid and the water, calculated from the nitroxide electron paramagnetic resonance (EPR) spectra, decrease with decreasing temperature until approximately the freezing point of the solvent. Below this temperature the nitroxide is detected only in the lecithin. The rotational correlation times of the spin label present in the lecithin were calculated for the temperature range from +45 to -60 degrees C. At low temperatures, the EPR spectra are characteristic of a superposition of two spectra resulting from the nitroxide dissolved in the lipid in two environments with different rotational correlation times.


Quantitative aspects of hormone-receptor interactions of high affinity. Effect of receptor concentration and measurement of dissociation constants of labeled and unlabeled hormones.

It is demonstrated that because of limitations in the magnitude of the specific activity of radiolabeled hormone derivatives, direct binding studies of hormone-receptor interactions of high affinity (10(-9) -10(-11) M, depending on whether 3H- or 123I-labeled hormones are used) will be subject to artifactual distortions due to the need to utilize high concentrations of the receptor. If the concentration of the receptor is not ten times lower than the true affinity constant, the apparent dissociation constant obtained from direct concentration binding curves will vary as a linear function of the receptor concentration. In addition, at high receptor concentrations saturability becomes difficult to demonstrate experimentally and the binding data yield apparently non-hyperbolic, sigmoidal curves which can be mistakenly interpreted to depict cooperative interactions. Similar artifacts related to receptor concentration are predicted for measurements of the hormone concentration dependence of biological proce-ses (e.g. activation of adenylate cyclase, transport processes, etc.). Methods for detecting these effects, and correctly measuring affinities for labeled and unlabeled hormones under these conditions, are described. The implications for measuring the binding properties of hormone-receptor interactions are discussed, especially in reference to studies of the comparative analysis of receptor function in altered metabolic states and to studies relating the biological and binding properties of hormones.


Redox state of respiratory chain enzymes and potassium transport in silkworm mid-gut.

The midgut of Hyalophora cecropia actively transports potassium from hemolymph to lumen and the energy for this process appears to be intimately linked to oxidative metabolism. In the present investigation, we monitored concurrently the rate of active transport and the redox levels of the components of the respiratory chain in the intact tissue under a variety of experimental conditions. Approximately equal concentrations of cytochromes a3, a, c and b-557 were found. Other investigators (Pappenheimer, Jr, A.M. and Williams, C.M. (1954) J. Biol. Chem. 209, 915, Shappirio, D.G. and Williams, C.M. (1957) Proc. R. Soc. Lond. Ser. B 147, 233 and Chance, B. and Pappenheimer, Jr, A.M. (1957) J. Biol, Chem, 209, 931) have indentified cytochrome b-557 with b5 and found that it exists primarily in an extramitochondrial location. Steady-state experiments demonstrated that all these cytochromes were approximately 50% reduced while active transport proceeded at a high rate in regular cecropia Ringer containing 32 mM KCl. When the potassium concentration was reduced, the active transport decreased and all the cytochromes became more oxidized. Addition of 1 mM cyanide inhibited active transport by 90% and caused a 100% reduction of all cytochromes. Redox state and short circuit current (Isc) kinetics measured as the tissue was made anoxic showed that all the respiratory enzymes, except cytochrome b-557, became fully reduced at a faster rate than the rate of inhibition of the Isc. The rate of cytochrome b-557 reduction followed kinetically the Isc. These observations are interpreted in a scheme where cytochrome b-557 (possibly b5) branches off cytochrome c from the conventional resporatory chain, utilizing cytochrome a3 as the terminal oxidase for both branches. Cytochrome b-557 may be involved in providing a direct link between oxidative metabolism and active transport in the midgut of the silkworm.


Symmetry, orientation and rotational mobility in the a3 heme of cytochrome c oxidase in the inner membrane of mitochondria.

The photoinduced linear dichroism of absorption changes resulting from photolysis of the complex between heme a3 of the cytochrome oxidase and CO is studied. The experiments started from isotropic solutions or suspensions of the enzyme both in its isolated form and in mitochondria. The anisotropy responsible for the linear dichroism was induced by excitation with a flash of linearly polarized light. The dichroic ratios observed with various systems; polymerized enzyme in solution, enzyme in mitochondria and in submitochondrial particles (at 20 degrees C as well as at liquid N2-temperature) all approached a value of 4/3 which characterizes a chromophore which is circularly degenerate. Therefrom we conclude that the interaction of heme a3 with its microenvironment within the protein does not break its four-fold symmetry. The experiments with mitochondria and submitochondrial particles suspended in aqueous buffer revealed similarly high dichoric ratios without any dichroic relaxation other than a rather slow one which could be attributed to the rotation of the whole organelle in the suspending medium. Therefrom we conclude that the cytochrome oxidase either is totally immobilized in the membrane, or that it carries out only limited rotational diffusion around a single axis coinciding with the symmetry axis of heme a3. In the light of independent evidence for a transmembrane arrangement of the oxidase and for the general fluidity of the inner mitochondrial membrane we consider anisotropic mobility of the cytochrome oxidase around an axis normal to the plane of the membrane as the most likely interpretation. Then our experimental results imply that the plane of heme a3 is coplanar to the membrane.


ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver.

1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enyzme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-Pi exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin. 4. The decrease in ATP synthesis and ATP-Pi exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase. 5. The following electron transport activities in the megmitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc1-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.


On the nature of the iron sulfur cluster in a deuterated algal ferredoxin.

A protonated and a completely deuterated two-iron algal ferredoxin from Synechococcus lividus have been studied by optical, electron paramagnetic resonance, electron-nuclear double resonance, proton magnetic resonance and Mossbauer spectroscopies; temperature dependent magnetic susceptibility measurements are reported as well. These studies have confirmed the electron localized model of the active center in the two-iron ferredoxins, as previously deduced from studies of spinach ferredoxin, have yielded much more precise spectroscopic parameters for this center, and have thus greatly increased the confidence in this model.


Respiratory control in isolated perfused rat heart. Role of the equilibrium relations between the mitochondrial electron carriers and the adenylate system.

The effects of KCl-induced cardiac arrest on the redox state of the fluorescent flavoproteins and nicotinamide nucleotides and on that of cytochromes c and a were studied by surface fluorometric and reflectance spectrophotometric methods. These changes were compared with measurements of the concentrations of the adenylate system, creatine phosphate, some intermediates of the tricarboxylic acid cycle and reactants of the glutamate dehydrogenase system. KCl-induced cardiac arrest caused reduction of the fluorescent flavoproteins and nicotinamide nucleotides, oxidation of cytochromes c and a, inhibition of oxygen consumption and an increase in the ATP/(ADP X Pi) ratio. The increase in the latter was due mainly to a decrease in the concentration of Pi and an equivalent increase in creatine phosphate. The cytochromes c and  a were maintained at equal redox potential and changed in parallel. When the redox state of the mitochondrial NAD couple was calculated from the glutamate dehydrogenase equilibrium, the free energy change (deltaG) corresponding to the potential difference between the NAD couple and cytochrome c was 115.8 kj/mol in the beating heart and 122.2 kj/mol in the arrested heart. The deltaG values of ATP hydrolysis calculated from the concentrations of ATP, Pi and ADP, corrected for bound ADP, were 111.1 kj/2 mol and 115.4 kj/2 mol in the beating and arrested heart respectively. The accumulation of citrate and the direction of the redox changes in the respiratory carriers indicate that the tricarboxylic acid cycle flux is controlled by the respiratory chain. The data also show a near equilibrium between the electron carriers and the adenylate system and suggest that the equilibrium hypothesis of mitochondrial respiratory control is applicable to intact myocardial tissue.


Water proton relaxation as a monitor of membrane-bound manganese in spinach chloroplasts.

First measurements of proton relaxation on chloroplast membranes are presented here. Experiments show that the water proton spin-lattice relaxation rate in chloroplast thylakoid membrane suspensions can be used to monitor membrane-bound manganese. The relaxation effect is reduced to 0.4 of its original value upon manganese extraction by washing with either alkaline Tris buffer or NH2OH/EDTA solution. Large increases in the proton relaxation rate are measured in the presence of reductants such as tetraphenylboron and NH2OH; oxidants such as potassium ferricyanide or 2,6-dichlorophenolindophenol lead to an decrease in this rate. These results suggest that manganese exists as a mixture of oxidation states in dark-adapted chloroplasts.


Phosphatidylglycerol in lung surfactant. II. Subcellular distribution and mechanism of biosynthesis in vitro.

Lamellar inclusion bodies, apparent precursors for alveolar surfactant lining, have remarkably similar phospholipid composition to surfactant from alveolar lavage, but distinctly different from other fractions studied: mitochondria, microsomal fraction containing endoplasmic reticulum membranes, plasma membranes and nuclei. Surfactant contained (as % of total phospholipid phosphate): 75.5-77.0% lecithin, 11.0-11.2% phosphatidylglycerol, 4.2-4.6% phosphatidylethanolamine, 3.0-3.2% phosphatidylinositol, 1.5-1.7% bis-(monoacylglycerol) phosphate, 1.2-1.9% phosphatidylserine, and 0.7-1.5% sphingomyelin. Fatty acids of phosphatidylglycerol from lamellar bodies were similar to those from microsomes but different from those in mitochondria. Lung homogenate in continuous sucrose density gradient displayed two major activity peaks of phosphatidylglycerol synthesis: the heavier from mitochondria; the lighter from endoplasmic reticulum. Studies on mechanism of phosphatidylglycerol synthesis in vitro revealed (in these two fractions) CDP-diglyceride and sn-glycerol phosphate precursors to phosphatidylglycerol phosphate, that hydrolysed to phosphatidylglycerol. In microsomes disaturated CDP-diglycerides were 1.6-1.9 times more active substrates than in mitochondria, whereas CDP-diglycerides from egg lecithin were almost equally active. In contrast to lung mitochondria no cardiolipin synthesis was detected in microsomes. The highest specific activities for phosphatidate cytidyltransferase, CDP-diglyceride-inositol phosphatidyltransferase, choline phosphotransferase, and phosphatidylethanolamine methyltransferase were all found in microsomes. The present in vitro studies and additional evidence (M. Hallman and L. Gluck, (1975) Fed. Proc. 34, 274) support the hypothesis that de novo synthesis of surfactant lecithin phosphatidylinositol and phosphatidylglycerol takes place in the endoplasmic reticulum of alveolar cells.


Differential effects of denaturing agents on acetylcholinesterase: insensitivity of the reaction of methanesulfonyl fluoride compared to diisopropylphosphorofluoridate and p-nitrophenyl acetate.

The effects of various agents that could be expected to perturb enzyme structure in a non-specific and reversible manner (alcohols, dimethylsulfoxide, dimethylformamide, dinitrobenzene, urea and guanidine - HCl) have been determined on reaction of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) with a substrate, p-nitrophenyl acetate, and two irreversible inhibitors, diisopropylphosphorofluoridate and methanesulfonyl fluoride. In all three of these reactions an acyl group (acetyl, phosphoryl or sulfonyl respectively) bonds covalently with the active center of the enzyme. As expected, the reactions of p-nitrophenyl acetate and diisopropylphosphorofluoridate were severely retarded by most of these agents. By contrast, reaction of methanesulfonyl fluoride was usually depressed to a far smaller degree, and in two cases was faster. These findings are of interest in connection with: (1) differing requirements for the integrity of tha active center in catalysis with various substrate analogs, and (2) the mechanism by which cationic substrate analogs accelerate reaction of the enzyme with methanesulfonyl fluoride.


Cyclic guanosine 3'-5'-monphosphate. High levels in the male accessory gland of Acheta domesticus and related crickets.

Guanosine 3',5'-monophosphate (cyclic GMP) was found in the accessory gland of reproductively mature male house crickets (Acheta domesticus (L.)) up to the exceptionally high level of 500 pmol/mg protein (10(-4) mol/kg wet weight). The identity of cricket cyclic GMP was confirmed by enzymatic and spectral analysis. A survey of 10 closely related species of Orthoptera indicated that high levels of cyclic GMP in the accessory gland occur in the subfamily Gryllinae, to which A. domesticus belongs. In these crickets, cyclic GMP in the accessory gland increases together with protein content during two weeks after the final molt. Levels are not augmented by dissection, and are independent of the presence of sperm in the seminal vesicles and of the production of spermatophores by the gland. The function of cyclic GMP in the accessory gland is not yet understood.


Ascorbate increases the synthesis of procollagen hydroxyproline by cultured fibroblasts from chick embryo tendons without activation of prolyl hydroxyla.

An improved procedure was developed to extract prolyl hydroxylase from tendon cells of chick embryos with detergent, and improved assays were developed for both the activity of the enzyme and the amount of enzyme protein. Freshly isolated tendon cells were found to contain approx. 100 mug of enzyme protein per 10(8) cells and 40-50% of the enzyme protein was active. When the cells were cultured, they were found to contain the same amount of enzyme protein but only 15-20% of the enzyme protein was active. Gel filtration of cell extracts indicated that the active form of prolyl hydroxylase in freshly isolated tendon cells and incultured tendon cells had the same apparent size and the same activity per mug of immunoreactive protein as enzyme which was shown to be a tetramer. The inactive form was found to have about the same apparent size as subunits of the enzyme. When freshly isolated cells were incubated for 2 h in the presence of 40 mug per ml of ascorbate, there was a slight increase in the rate of hydroxyproline synthesis. In cultured cells, ascorbate at a concentration of 40 mug per ml caused a 2-fold increase in the rate of hydroxyproline synthesis within 30 min. However, ascorbate did not icrease the activity of prolyl hydroxylase in extracts from either cell system. Therefore it appears that the influence of ascorbate on synthesis of procollagen hydroxyproline by the cells studied here must be ascribed to a cofactor effect on the hydroxylation reaction similar to that observed with purified enzyme, and it does not involve "activation" of inactive enzyme protein to active enzyme as has been observed in cultures of L-929 and 3T6 mouse fibroblasts.


Dependence of gonadotropin-induced steroidogenesis upon RNA and protein synthesis in the interstitial cells of the rat testis.

The effects of inhibitors of RNA and protein synthesis upon gonadotropic stimulation of testosterone and cyclic AMP production by the  Leydig cellwere investigated in vitro with enzyme-dispersed interstitial cells of the rat testis. The testosterone response to human chorionic gonadotropin was abolished by cycloheximide and puromycin, and was markedly reduced by actinomycin D and cordycepin. During 3-h time studies, cycloheximide caused complete inhibition of subsequent steroid production when added at times up to 90 min after the commencement of incubation with human chorionic gonadotropin. Actinomycin D did not completely abolish the testosterone response when added at zero time, and became progressively less effective when added at later times during the incubation period. The stimulation of steroidogenesis by dibutyryl cyclic AMP was also completely abolished by cycloheximide and puromycin, and was significantly reduced by acinomycin D and cordycepin. By contrast with the marked inhibition of steroid production by cycloheximide and actinomycin D, the formation of cyclic AMP during human chorionic gonadotropin stimulation was relatively unaffected by either inhibitor. These results indicate that the stimulation of testosterone production by the Leydig cell in response to gonadotropins and dibutyryl cyclic AMP is dependent upon the synthesis of new RNA and protein moleucles. Thus, effects of gonadotropin and cyclic AMP upon both transcriptional and translational processes appear to be essential intermediate steps in the activation of testicular steroidogenesis. The rapid and complete abolition of subsequent steroid synthesis following addition of cycloheximide or puromycin to human chorionic gonadotropin-stimulated interstitial cells suggests that a relatively labile protein is formed during gonadotropin action.


Amylase secretion by rabbit parotid gland. Role of cyclic AMP and cyclic GMP.

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10(-4) M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by alpha-adrenergic blockade with phenoxybenzamine. Epinephrine (4 - 10(-5) M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the beta-blocking agent, propranolol. Pure alpha-adrenergic stimulation with methoxamine (4 - 10(-4) M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 - 10(-6) M, isoproterenol (a beta-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 - 10(-5) M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cyclic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 - 10(-6) M). These data strongly suggest that cholinergic muscarinic agonists and alpha-adrenergic agonists stimulate amylase output in rabit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by alpha-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this tissue to the effects of cholinergic stimuli.


Collagenolytic activity of carrageenin granuloma in rats.

Collagenolytic activity at various phases of the development of carrageenin granuloma was investigated by measuring the amount of dialysable hydroxyproline formed during incubation in vitro of minced granulation tissue. Collagenolytic activity reached a maximum at day 8 after carrageenin injection and then decreased gradually, while collagen synthetic activity was rapidly decreased from day 4 to day 11. The significance of dialysable hydroxyproline in native collagen breakdown of carrageenin granuloma is discussed.


Effect of adenine nucleotides and pyrophosphate on the exchange of transferrin-bound carbonate.

1. ATP, ADP and pyrophosphate accelerate the exchange of carbonate of the transferrin-iron-carbonate ternary complex, while AMP, cyclic AMP and phosphate have no effect. 2. ATP promotes carbonate exchange without removing iron from transferrin, whereas pyrophosphate effectiely attacks both the anion and iron components of the ternary complex. 3. Transferrin readily takes over iron from its ATP or pyrophosphate complex. 4. Neither ATP nor pyrophosphate can substitute for carbonate of the ternary complex. These results fit in well with the concept that ATP may play a direct role in the iron uptake by reticulocytes.


Effect of hydrocortisone and glucagon on glycogen metabolism in the fetal rat liver.

1. Hydrocortisone increases in vivo incorporation of [14C] glucose into fetal liver glycogen in the last days of gestation, whereas in glucagon-treated fetuses, a slight decrease in the incorporation rate was found. 2. Hydrocortisone increases total synthetase activity as that of synthetase a but was without effect on fetal liver glycogen phosphorylase. 3. Glucagon causes a slight increase in phosphorylase a activity on days 19-21, and was without effect on the activities of synthetase a and total synthetase. 4. Dibutyryl cyclic AMP had no effect on the key enzymes of glycogen metabolism 1 h after injection in utero, whereas after 6 h an increase in phosphorylase a activity was found without any change in synthetase a activity.


A comparison of the structure of hamster pancreatic insulin and insulin extracted from a transplantable hamster islet-cell carcinoma.

Insulin has been isolated from pancreases of the Syrian hamster and from a transplantable islet-cell tumor of the hamster. Acid/ethanol extraction, ether precipitation, ion exchange and gel filtration chromatography gave preparations of suitable purity for structural studies. Using trypsin cleavage, automatic Edman degradation and manual Edman degradation, a complete sequence of the pancreatic insulin B chain was determined. By automatic Edman degradation, the amino-terminal 10 residues of the pancreatic A chain were assigned and the sequence of carboxy-terminal eleven residues could be deduced by homology to other mammalian and avian insulins. The sequence assigned to hamster insulin A chain is identical to that of the rat, mouse and spiny mouse. The sequence of hamster insulin B chain is identical to rabbit and spiny mouse B chain. In terms of protein evolution, hamster insulin thus appears to occupy an intermediate position between rabbit and rat insulins. Amino acid composition, tryptic peptide composition and partial sequence analysis of the hamster tumor insulin showed no differences from hamster pancreatic insulin.


Proton magnetic resonance studies of peroxidases from turnip and horseradish.

Proton NMR spectra at 270 MHz have been measured for horseradish peroxidase and turnip peroxidase isoenzymes (P1, P2, P3 and P7) in both their high spin ferric native states and as the low spin ferric cyanide complexes. Resonances of amino acids near the heme have been identified and used to investigate variations in the structure of the heme crevice amongst the enzymes. Ligand proton resonances have been resolved in spectra of the cyanide complexes of the peroxidases and these provide information on the heme electronic structure. The electronic structure of the heme and the tertiary structure of the heme crevice are essentially the same in the acidic turnip isoenzymes, P1, P2 and, to a lesser extent, P3 but differ in the basic turnip enzyme, P7. The heme electronic structure and nature of the iron ligands in peroxidases are discussed. Further evidence is presented for histidine as the proximal ligand. A heme-linked ionizable group with a pK of 6.5 has been detected by NMR in the cyanide complex of horseradish peroxidase.


Size and charge heterogeneity of rat tissue ferritins.

Ferritins purified from horse spleen and from rat liver, kidney, heart and hepatoma were analyzed by quantitative polyacrylamide gel electrophoresis. From the migration characteristics of these ferritins at several gel concentrations, Ferguson plots were constructed and the molecular sizes and charges (apparent valences) together with their statistical variability were obtained by applying Rodbard computer programs to the data. Finally, ellipses were drawn describing the 95% confidence limits of these data for size and charge and were used to identify those ferritins that differed in size and/or charge. By these criteria, many of the tissue ferritins were differentiated from one another in terms of their molecular size and/or charge. Among the various tissue ferritin monomers, the molecular sizes were essentially similar (420 000-490 000) except for the two heart ferritins which were larger (530 000 and 626 000, respectively). However, the estimated charges on rat liver, kidney and hepatoma monomers (30-38 net protons per molecule) differed from that of spleen monomer (51 net protons per molecule) while the larger rat heart ferritin also had a greater charge (83 net protons) than the smaller (40 net protons). Apoferritins prepared  chemically by removal of iron from the holoferritins had migration properties indistinguishable from the parent holoferritins. The migration properties of minor (dimeric) ferritin bands on the gels were compared with those of the monomer bands. The molecular sizes of the minor bands were larger than those of the major bands, and were not inconsistent with a doubling in size. However, charge differences varied, being either similar for major and minor forms (spleen ferritin), approximately twice for the minor form (rat hepatoma ferritin) or five times greater for the minor form (rat liver ferritin). These differences in behavior were confirmed by using minimally sieving gels, on which the major bands of horse spleen ferritin failed to separate whereas those of rat liver ferritin were readily separable. It is concluded that dimers of ferritins from different tissues may associate in different ways.


Studies on epsilon-prototoxin of Clostridium perfringens type D. Physicochemical and chemical properties of epsilon-prototoxin.

epsilon-Prototoxin of clostridium perfringens type D consists of one polypeptide chain of 311 amino acids with the following composition: Asp52 Thr31 Ser25 Glu28 Pro12 Gly17 Ala14 Val28 Met5 Ile15 Leu18 Tyr17 Phe8 Lys31 His2 Arg5 Tyr2. It has no free cysteine but contains one blocked cysteine. The N-terminal as well as the C-terminal residue is lysine. The ultracentrifuge pattern gave one single boundary having S020,w = 2.15 S and Do20,w = 5.56-10(-7) cm2/s. Calculation of the molecular weight from D020,w and S020,w gave a value of 34 250. The molecular weight determined from sedimentation equilibrium using ultraviolet optics gave a value of 33 000 +/- 1000. On the other hand molecular weights calculated from a calibrated Sephadex G-100 column in borate buffer was 25 000 and that in sodium phosphate, ionic strength 0.2, was 27 500. This discrepancy between values obtained in the ultracentrifuge and by gel filtration is attributed to adsorption of epsilon-prototoxin by Sephadex.


Effect of cholesterol content on some physical and functional properties of mitochondria isolated from adult rat liver, fetal liver, cholesterol-enriched liver and hepatomas AH-130, 3924A and 5123.

The cholesterol to phospholipid ratio in mitochondria from hepatomas AH-130, 3924A and 5123 is higher than in the particles isolated from adult or fetal rat livers. Nearly all the cholesterol of hepatoma mitochondria is located in membranes. As in liver mitochondria, in the particles isolated from hepatoma AH-130 there is more cholesterol in the outer than in the inner membrane. In mitochondria from cholesterol-enriched liver and hepatomas, there occurs a decrease in extent of hypoosmotic and phosphate-induced swelling and a decrease of conformational changes linked to energy states. The phenomenon is more marked in particles which exhibit higher cholesterol to phospholipid ratios. A statistically significant negative correlation exists between the cholesterol to phospholipid ratio and extent of volume or conformational changes. No significant modifications of these parameters were found in fetal liver mitochondria. Cholesterol content does not influence K+ uptake by cholesterol-enriched or hepatoma mitochondria. Nor does cholesterol content affect the respiratory increment related to this uptake. As a consequence of K+ uptake, total mitochondrial water exchangeable with tritiated water rises 20% while sucrose-impermeable water rises 42-48% in both adult rat liver and hepatoma AH-130 mitochondria. Absorbance changes linked to ion uptake do not correspond merely to variations in mitochondrial water content. Water content is apparently not influenced by the cholesterol to phospholipid ratio. However, the ratio is significantly correlated to both extent and initial rate of absorbance decrease of mitochondrial suspensions during K+ uptake. The higher the ratio, the lower the extent and initial rate of absorbance decrease.


Subfractionation of rat liver plasma membrane. Uneven distribution of plasma membrane-bound enzymes on the liver cell surface.

Plasma membranes were isolated from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5'-nucleotidase and (Na+ + K+)-ATPase were used. The yield of plasma membrane was 0.6-0.9 mg protein per g wet weight of liver. The recovery of 5'-nucleotidase and (Na+ +K+)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the activity of glucose-6-phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5'-nucleotidase, alkaline phosphatase, (Na+ +K+)-ATPase and Mg2+-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na2+ +K+)-ATPase and Mg2+-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphate was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.


Comparison of the secretory processes in the parotid and sublingual glands of the mouse. 1. Regulation of the secretory processes.

1. Secretion from the mucous sublingual gland of the mouse has been investigated and compared with the serous parotid gland. The influence of acetylcholine, noradrenalin and adrenalin on the secretion of glycoproteins (e.g. mucins) and proteins (e.g. amylase) from these glands in vitro, and the involvement of cyclic AMP and Ca2+ has been studied. 2. Secretion from the parotid gland could be stimulated by both acetylcholine and the catecholamines. It appears that cyclic AMP plays an important role in the adrenergic secretory process, but not in the cholinergic-induced secretion. In the latter case, exogenous Ca2+ strongly increased the secretion. 3. Mucin secretion from the sublingual gland could be affected by acetylcholine in the presence of exogenous Ca2+. Noradrenalin and adrenalin induced only a slow mucin secretion and, for this secretory process, exogenous Ca2+ is also required. Though cyclic AMP is present in the sublingual gland, no influence on its level could be detected in this gland after stimulation of the adrenergic beta-receptor, whereas, in contrast to the parotid gland, dibutyryl cyclic AMP induced only a slow secretion. Because it was observed that the sublingual gland of the mouse is not innervated sympathetically, it seems reasonable to suppose that the catecholamines stimulate the mucin secretion from this gland via hormonal receptors and not via the adrenergic beta-receptor. 4. The protein secretion from the sublingual gland could be stimulated by both acetylcholine and the catecholamines. An involvement of cyclic AMP in this process was not observed. Addition of exogenous Ca2+ is less important, as was found for the mucin secretion. So it has been concluded that protein and mucin secretion from the sublingual gland are regulated via different pathways.


Distribution of ferritin-conjugated lectins on sialidase-treated membranes of human erythrocytes.

The labeling of sialidase-treated, human erythrocyte membranes with ferritin-conjugates of four plant lectins, concanavalin A, Ricinus communis hemagglutinin, Bauhinia purpurea hemagglutinin and Arachis hypogoea hemagglutinin, is reported. Among these ferritin-conjugated lectins, ferritin-conjugated concanavalin A and ferritin-conjugated R. communis hemagglutinin were found in clusters on the sialidase-treated membranes, whereas ferritin-conjugated B. purpurea hemagglutinin and ferritin-conjugated A. hypogoea hemagglutinin were found in a random distribution on the membranes. Furthermore, when the membranes were labeled with a mixutre of concanavalin A and ferritin-conjugated B. purpurea hemagglutinin, ferritin particles were found in clusters, indicating that the membrane receptors for B. purpurea hemagglutinin were forced to more together with those for concanavalin A. A method for the quantitative estimation of the clustering of ferritin particles on the membranes was also devised and applied to the labeling of sialidase-treated, human erythrocyte membranes with the above four ferritin-conjugated lectins.


Biochemical characteristics of the outer membranes of plant mitochondria.

Like the outer membranes of liver mitochondria, those of plant mitochondria are impermeable to cytochrome c when intact and can be ruptured by osmotic shock. Isolated plant outer mitochondrial membranes are also similar to the corresponding liver membranes in terms of phospholipid and sterol content. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis experiments indicate that a single class of proteins (apparent molecular weight 30 000) comprises the bulk of the plant outer membrane protein. There are also considerable amounts of polysaccharide associated with these membranes, which may contribute to their osmotic stability.


Effect of colchicine on the osmotic water flow across the toad urinary bladder.

Osmotic water movement across the toad urinary bladder in response to both vasopressin and cyclic AMP was inhibited by 10(-5) to 10(-4) M colchicine on the serosal but not on the mucosal side. This inhibitory effect was found to be time- and dose-dependent. Colchicine alone did not change basal osmotic flow and a baseline of the short-circuit current (Isc) and also did not affect a vasopressin-induced rise of the Isc. The inhibitory effect was not prevented by the addition of pyruvate. The osmotic water movement produced by 360 mM Urea (mucosal), 360 mM mannitol (serosal) or 2 mug/ml amphotericin B (mucosal), was not affected by 10(-4) M colchicine. These results  suggest that colchicine inhibits some biological process subsequent to the formation of cyclic AMP except a directional cytoplasmic streaming process where microtubules may be involved.


Choleragen stimulates steroidogenesis and adenylate cyclase in cells lacking functional hormone receptors.

Choleragen stimulates steroid secretion and adenylate cyclase in three cell lines, adrenal tumor line (Y-1), a corticotropin-resistant mutant derived from Y-1 called OS-3, and a receptor-deficient Leydig tumor line (I-10). Sensitivity for half-maximal stimulation varies from 3 to 36 pM choleragen, the I-10 line being the most sensitive. Latency before the onset of steroidogenesis is longer in OS-3 and I-10 cells than in the Y-1 line. In both OS-3 and I-10 cells choleragen stimulates adenylate cyclase whether ITP or 5'-guanylylimidodiphosphate is the regulatory cofactor used. In addition to the responses of the receptor-deficient lines, choleragen does not, during its latency, block the response to corticotropin in Y-1 cells; corticotropin does not block binding of 125I-labeled choleragen to Y-1 cells; gangliosides do not interfere with the corticotropin-induced stimulation of Y-1 cells. We conclude that the corticotropin and choleragen receptors are different.


Endotoxic lipopolysaccharides stimulate steroidogenesis and adenylate cyclase in adrenal tumor cells.

Lipopolysaccharides (endotoxins) from Escherichia coli, Serratia marcesens and Salmonella typhosa stimulated steroid production in Y-1 adrenal tumor cells in culture with a latent period of 3-4 h. Lipid A, derived from Escherichia coli lipopolysaccharide, also stimulated steroidogenesis. Lipopolysaccharides and lipid A also stimulate adenylate cyclase activity and cause rounding of the cells. In contrast, lipopolysaccharides do not stimulate steroidogenesis in receptor-deficient adrenal tumor cells (OS-3) or Leydig tumor cells (I-10). This tends to rule out contamination by enterotoxin to which these lines respond. Although both hormone and lipopolysaccharide responses are lost in these lines, there was no interaction between these sites as judged by the failure of lipopolysaccharides to block, during their latency, the response to corticotropin in Y-1 cells. The possibility that the lipopolysaccharide effect is one on membrane conformation is discussed.


Changes in lipid metabolism and cell morphology following attack by phospholipase C (Clostridium perfringens) on red cells or lymphocytes.

When intact human erythrocytes were treated with phospholipase C (Clostridium perfringens), up to 30% of the membrane phospholipids were broken down without significant cell lysis. Only phosphatidylcholine and sphingomyelin were attacked. Ceramide (derived from sphingomyelin) accumulated, but 1,2-diacylglycerol (derived from phosphatidylcholine) was largely converted into phosphatidate. Up to 12% of the cell phospholipid could be converted into phosphatidate in this way. Pig erythrocytes and lymphocytes showed a similar but smaller synthesis of phosphatidate after phospholipase C attack. Phospholipase C also caused a marked morphological change in erythrocytes, giving rise to spherical cells containing internal membrane vesicles. This change appeared to be due to ceramide and de and diacylglycerol accumulation rather than to increased phosphatidate content of the cells.


A comparison of DNA from free living and endosymbiotic Rhizobium leguminosarum (strain PRE).

1. Bacteroids of Rhizobium leguminosarum (strain PRE) purified from root nodules of Pisum sativum (var. 'Rondo') by the standard procedure of differential centrifugation contained considerable contamination of mitochondrial material. This could be removed by incubation of the bacteroid preparation with 1 M KCl/1% deoxycholate. 2. The DNA content of bacteroid cells of R. leguminosarum was found to have increased about three fold in comparison with the DNA content of free living R. leguminosarum bacteria. 3. No significant difference in DNA composition of free living R. leguminosarum bacteria and bacteroids could be detected by CsCl equilibrium centrifugation, RNA - DNA hybridization and DNA - DNA reassociation studies.


[Photoinduced reduction of ferriperoxidase. I. Reaction with reduced nicotinamide-adenine dinucleotide].

Photoinduced reduction of peroxidase with reduced NAD is revealed.; its kinetics is studied. The kinetic scheme of this process is suggested and calculated. It is shown that metastable excited state appears after NAD-N irradiation. This state participated in the reaction with ferriperosidase. It is also shown that the NAD radical which appears in the course of this reaction reduces the peroxidase; the rate constant of this process is (5 +/- 0,5) -10(4) M-1 sec-1.


[Kinetics of dark reduction of P700 centers in photosynthesis].

Relationship between the rate of dark fall of ESR I signal in the chloroplasts and light intensity 710 nm was studied. It is shown that the kinetics of the signal drop at sufficiently high levels of intensity is a biphase one. The fast phase (2 sec) disappears in the presence of an inhibitor of noncyclic electron transfer. It is concluded that the kinetics of electron transport is not described within the concept of the linear chain of electron carriers and a suggestion is put forward concerning a possible mechanism of this process which depends on the participation of plastoquinone in electron transport.


Partial characterization of the 1-anilino-8-naphthalene sulfonate-adrenochrome semicarbazide interaction site in erythrocyte ghost membrane fragments.

The effect of adrenochrome semicarbazide on the conformation of erythrocyte ghost membranes has been studied by ANS fluorescence, lipid and sulfhydryl spin labels and circular dichroism. No large conformational alterations in the membrane were detected by these techniques. Noncompetitive quenching of ANS fluorescence by ADCS suggests ADCS to interact with the membrane at sites close to the ANS binding domain.


[Propagation of spikes in statistical neuron ensembles. I. Concept of phase transitions].

A system of two coupled integro-differential equations for the propagation of sipkes is presented. A qualitative consideration of the system shows possibility of concentrational phase transition in the neuron ensemble from the state of spontaneous firing to the strong periodic oscilltation activity. Naer the point of the phase transition the neuron ensemble becomes labile, which maintains appropriate conditons for the existence of mosaic structures in the neuron network.


[Basis for the use of Pierre's method for the determination of ACTH in human blood plasma].

Adrenal glands of rats were immersed in Ringer-Lock solution (used as control), other rat adrenal glands were immersed in human blood plasma. The amount of corticosterone in the extracts received was estimated after two hours of incubation. The activity of adrenocorticotropic function of the pituitary gland of the patients examined was judged by the difference in the corticosterone indices in these extracts. ACTH (1.5 and 10 mkg) was added to the plasma to determine the sensitivity of this technique. Regular and significant increase in corticosterone was revealed. Modified Pierre's technique can be applied for ACTH determination in man.


[Interrelationship of the mediator receptors in the terminals of the afferent nerves of the cornea of the eye].

In local application of substances with a mediator action (adrenaline, acetylcholine, histamine) and blockers of specific receptors (dihydroergotoxin, inderal, atropine and of subcutaneous dimedrol) together with dicaine it was possible to reveal a distinct presence in the cornea of the eye of the mediator receptors capable of influencing one another. Pharmacological analysis permitted a supposition to be made that there were two receptor systems in the sensory nerve endings of the cornea; these systems were in reciprocal inhibitory relations; due to this their regular influence on the processes of excitation and inhibition is effected in the sensory nerve ending.


[Some indices of the activity of the brain in "rapid" sleep preceeded or not preceeded by delta-sleep].

In order to study the functional interaction between the delta sleep and the REM sleep some psychophysiological features of REM sleep were examined in REM-onset (without any preceding delta sleep--"early REM period") and in the REM period (REMP) terminating the normal sleep cycle (with the preceding delta sleep) of 92 daytime sleep attacks in 10 narcoleptic patients. Under these conditions the significant differences exist in the characteristics of the dream reports and in subjective estimations of sleep quality and duration. Sleep was evaluated as "superficial" and underestimations of sleep duration took place after an early REMP. Correct estimations of sleep duration and evaluations of sleep as "deep" dominated after REMP enging sleep cycles. The results obtained indicate the functional interaction between the delta sleep and REM sleep existing in the sleep cycle and largely determining the psychic content of the brain activity in the REM sleep.


Determination of isozymes of phosphoglycerate-mutase (E.C.2.7.5.3) and enolase (E.C.4.2.1.11) in human erythrocytes.

525 human hemolysates were tested for the isozymic patterns of phosphoglycerate mutase and enolase. Genetic models for interpreting the pherograms are suggested.


Dorsal thumb flap for release of adduction contracture of the first web space.

A local flap, utilizing the skin over the dorsum of the thumb, for release of a first web space contracture has been described. Its construction allows for full integumentary release, adequate visualization of underlying contracted tissues, and a closure that provides full thickness sensory skin coverage over the entire web space and key pinch contact area.


[Cytological aspects of various particular varieties of breast epitheliomas].

Needle aspiration for cytological investigation has been used at the Foundation Curie since 1954. Until 1973, no less than 3,176 mammary tumours have been studied by this method and comparison made with the histological findings. In the whole group some relatively rare carcinomas have been encountered -- mucoid, medullary, adenoid cystic, squamous and apocrine. Each category is described from the cytologic viewpoint and illustrated with an appropriate cyto/histologic example to show what a high degree of precision may be attained by diagnostic cytology in mammary cancer.


[Immunovirological factors in epitheliomas of the rhinopharynx].

The epidemiology of nasopharyngeal carcinoma (NPC) suggests the intervention of genetic factors. These factors may be represented by a specific HL-A pattern (leucocyte antigens) as found in Chinese NPC patients (deficit of antigens corresponding to the second sublocus and discovery of a new antigen, named SIN-2). The association between NPC and a herpesvirus (the EBV) is based on elevated antibodies against a variety of EBV coded antigens, on cell mediated immune reactions, and on the regular presence of viral fingerprints in the epithelial tumour cells (viral DNA and nuclear antigen -- EBNA). The serological profile should help to establish the diagnosis of NPC in certain cases. The association between genetic factors, a herpesvirus and a human tumour represents a unique model in human cancer research.


Effects of "natural oestrogen" replacement therapy on menopausal symptoms and blood clotting.

In a double-blind study on the value of equine ("natural") oestrogens 30 patients presenting with menopausal symptoms in a group practice were monitored for possible adverse effects on blood clotting, weight, and blood pressure. The women were randomly allocated to two groups and given either three months' hormone treatment followed by three months' placebo or vice versa. An appreciable amelioration of all symptoms on placebo made it difficult to asses the genuine value of oestrogen treatment during the period of study. Both groups made a dramatic clinical improvement during the first three months. Nevertheless, the symptoms of the 15 women who received oestrogen first returned after the cross-over to placebo without any suggestion of a placebo response. In contrast, the other group who took placebo first did not deteriorate after changing to oestrogen. The menopausal index and the karyopyknotic index were not reliable guides to the need for oestrogen treatment. Hot flushes, however, were proportionately reduced on oestrogen and they seemed to be more readily eliminated in individual cases by oestrogen. The results of blood clotting studies indicated that natural oestrogen administration raised the levels of the extrinsic clotting factors VII and X and accelerated the prothrombin time. The findings were similar to those observed after three months synthetic oestrogen administration with oral contraception. Long-term studies and epidemiological surveys of the clinical incidence of thrombotic and other sequelae are needed before large-scale oestrogen replacement treatment can be recommended.


Viruses and the aetiology of diabetes: a study in identical twins.

Sera were collected from 49 pairs of identical twins, 27 of whom were discordant (only one twin affected) and 22 concordant (both diabetic) for insulin-dependent diabetes. All were tested for antibodies to mumps, cytomegalovirus, rubella, Coxsackie virus types B1-5, and Mycoplasma pneumoniae. The diabetic co-twins had no more antibodies to any of the viruses than the non-diabetic co-twins of the discordant pairs. Antibodies to Coxsackie B2, rubella virus, and M pneumoniae were found more often in the discordant than in the concordant twins. In 30 of the 71 diabetic twins symptoms began when they were aged 4-6 years or 10-15 years. More concordant than discordant twins were diagnosed during the months January to March. Hence there was no direct evidence of a virus aetiology of juvenile onset diabetes in these twins, and the difference in antibody titres between the concordant and discordant twins was in keeping with a genetic difference between them. The age and time of onset suggested that environmental factors may be important in causing diabetes in the twins.


Histological evidence of carcinoma in a hepatic tumour associated with oral contraceptives.

A primary hepatic tumour occurred in a 21-year-old woman who had been taking oral contraceptives for two years; she was treated by partial hepatectomy. Part of the neoplasm showed features suggestive of focal nodular hyperplasia, while the remainder had the histological characteristics of a well-differentiated hepatocellular carcinoma. This is the first report of malignant transformation of a tumour in a patient taking oral contraceptives.


Regulation of 3-hydroxy-3-methylglutaryl coenzyme-A reductase activity in type II hyperlipoproteinaemia.

The regulation of 3-hydroxy-3-methylglutaryl coenzyme-A reductase activity in leucocytes was studied in a family with familial type II hyperlipoproteinaemia (WHO classification). The propositus was heterozygous and 16 other members of the family were studied. Leucocytes were isolated by a relatively quick and simple procedure and used for enzyme estimations. The results suggest that measurement of the regulation of this enzyme in leucocytes may be helpful in the diagnosis and management of the condition.


Investigations on in vitro survival and virulence of T. pallidum under aerobiosis.

Motility of pathogenic T. pallidum was maintained in aerobic in vitro cultures for several weeks using a special medium. The latter consisted of McCoy's 5a medium supplemented with glutathione, sodium pyruvate, HEPES buffer, gentamycin (garamycin), and fetal calf serum. The virulence of the organisms was lost in 5 to 6 days. No multiplication of the organisms was observed. Four antibiotics (viomycin, kanamycin, gentamycin (garamycin), and neomycin) were tested for their bactericidal action and possible toxicity to T. pallidum. Gentamycin proved to be superior to the other three antibiotics in being non-toxic to the treponemes and showing a possible stimulatory effect on their motility and longevity. Cultivation of T. pallidum in cultured cells in the presence of the enzymes, superoxide dismutase and catalase, in a special medium showed possibilities for future experimentation under monitored, reduced oxygen pressure with a continuous system to dismutate superoxide radicals.


Incidence of Herpesvirus hominis antibodies among blood donor populations.

The microneutralization test was used to determine the occurrence of antibodies to Herpesvirus hominis Type 1 and Type 2 in sera from patients attending the Special Clinic, Bristol Royal Infirmary, with proven herpes genitalis, and in sera taken from blood donors in Bath, Dursley, and Bristol, as well as from donors in three different prison populations. The findings in patients with herpes genitalis indicate that the test accurately reflects the antibody response expected in relation to the type of herpes virus isolated from the lesions. The incidence of Type 2 antibodies among the blood donors ranged from 5 per cent. for donors from the Bath area up to 60 per cent. among donors from Dartmoor prison. The findings suggested that Type 2 herpes infection could spread among longterm prison populations, and it is postulated that this may be due to both homosexual contact, and also by non-sexual contact, either directly or via fomites.


An androgen receptor in rat brain and pituitary.

Dihydrotestosterone (DHT) binding was measured in cytosols from brain regions and pituitary of adult female rats and, with the addition of ventral prostate, in adult male rats. Two types of binding were distinguished: one, saturable at concentration of DHT greater than or equal to 5 X 10(-9) M and an unsaturable component. In intact males saturable (limited capacity) binding was detected only in ventral prostate cytosol; 3 days after orchidectomy the saturable binding sites increase 3-fold in prostate and in pituitary, hypothalamus, amygdala and cortex to detectable levels in approximately the same abundance as in females. There were significant differences in the affinities of the limited capacity binding reactions in cytosols of different tissues though all were in the order of magnitude, 10(-9) M DHT. The affinity in pituitary cytosol was lower than in brain regions with the single exception of female amygdala in which the affinity was significantly lower than in cytosol of the same region from 3-day castrate males. The specificity of the limited capacity binding was investigated by competition between [3h]DHT and unlabelled steroids; the most effective competitors were potent androgen agonists and antagonists.


Direction-selective units in the rabbit's nucleus of the optic tract.

A class of direction-selective (DS) units, histologically localized within the nucleus of the optic tract (NOT) was isolated in the rabbit's pretectum. These units typically had a maintained discharge of 25-50 action potentials/sec and large receptive fields (up to 40 X 150 degrees) in the visual streak area of the contralateral eye. They were excited by a visual pattern moving in one direction and inhibited by motion in the opposite direction. The reactions were sustained. Excitatory and inhibitory acceptance angles were each 180 degrees. Most units were excited by anterior motion of the stimulus and reacted to a wide range of velocities (0.01-20 degrees/sec). Random checkerboard patterns (elements 0.8 degrees), grids of black and white stripes (1, 2 and 4 degrees wide) and single black and white edges were all effective, with a decreasing response magnitude in roughly this order. A stimulus area of 2 X 2 degrees was already effective; response increased with area and was maximal for 15 X 15 degrees and larger stimuli. Latency for visual stimulation was 60 +/- 10 (S.D.) msec, for electrical stimulation of the chiasm 2.2 +/- 0.3 (S.D.) msec. Synaptic latency and presynaptic conduction velocity were estimated at 0.7 msec and 13m/sec, respectively. A strong convergence of retinal DS fibers upon NOT units is postulated. Since most properties of NOT units are compatible with those of optokinetic nystagmus, and electrical stimulation of the NOT elicits vigorous nystagmus, these data suggest that these cells are the essential afferent link in the optokinetic reflex arc.


[Evolution of visual evoked responses during various states of vigilance in Papio papio (author's transl)].

Averaged evoked responses (AER) to light flashes were recorded in baboons (Papio papio) during wakefulness, slow-wave sleep and rapid eye movement (REM) periods, at the visual cortex, retrocalcarine sulcus, optic tract (OT), lateral geniculate (LG) and pulvinar. Waking AERs were composed: in the OT, of a negative, low amplitude wave at 13.3 msec (I), a high amplitude wave at 34.8 msec (II), a negative wave at 72 msec (III) and a late component at 151 msec; in the LG, a small positive wave (II) with a peak latency of 40 msec, a high amplitude negative wave (III) with a latency of 70 msec and a late component; in the pulvinar of two low amplitude short latency waves (I and II), respectively negative and positive at 25 msec and 40 msec, then a high amplitude negative wave (III) at 75 msec and a late component; in the retrocalcarine sulcus 3 positive waves (I, II and III) were recorded at 25, 45 and 100 msec and a late component; in the visual cortex, 3 low amplitude negative waves (II, III and IV at 40, 50 and 54 msec, then a positive wave at 80 msec and some late components. In slow-wave sleep, AERs did not change in the OT, but in the LG and pulvinar, they showed an increase in the amplitude of wave II from stage 1 to stage 3. At the cortical level, early waves (II for the retrocalcarine sulcus, II and IV for the visual cortex) presented a marked increase in amplitude during stages 2 and 3, but only a slight increase for stage 1. Peak latency increase of each wave in cortical and subcortical AERs was seen during slow-wave sleep. REM AERs resembled, in amplitude and peak latency, those recorded in the LG and pulvinar during wakefulness; in the visual cortex and retrocalcarine sulcus, they were similar to those obtained during wakefulness and stage 1. In conclusion, a different evoked response was found between visual cortex and deep structures (except for the OT): firstly, during slow-wave sleep (the AERs showed a difference for stage 1 between the visual cortex or the retrocalcarine sulcus and the LG or the pulvinar), secondly, in REM (on the cortex, REM AERs looked like wakefulness and stage 1 responses); on the contrary, in the LG and pulvinar, REM AERs were similar only to those recorded during waking. Finally, it can be said that for Papio papio the differentiation and structural responses between the various stages of sleep (particularly light sleep and REM) were greater in the cortex than in the thalamic structures.


An ultrastructural study of the articular cartilage in calcium pyrophosphate dihydrate (CPPD) crystal deposition disease (chondrocalcinosis articularis).

An ultrastructural study of articular cartilage from five patients with calcium pyrophosphate dihydrate (CPPD) crystal deposition disease was performed. The CPPD crystals, identified by micro X-ray diffraction, were usually found in clusters, located in intercellular areas of the intermediate cartilage layer. The matrix surrounding the clusters either showed a normal morphology or a homogeneous appearance, within which faint cross striations but no distinctly fibrillar outlines could be identified. Another change in the matrix, characterized by an increased electron density and longitudinally fragmented collagen fibres, was also seen. This latter change was generally seen without concomitant CPPD crystal deposition. No specific relationship between the crystals and the collagen fibres or the granular background material of the matrix was encountered.


Comparative enzymatic properties of avian liver and skeletal muscle D-fructose-1,6-bisphosphate 1-phosphohydrolase.

The enzymatic properties of purified preparations of chicken liver and chicken skeletal muscle fructose bisphosphatases (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) were compared. Both enzymes have an absolute requirement for Mg2+ or Mn2+. The apparent Km for MgCl2 at pH 7.5 was 0.5 mM for the muscle enzyme and 5 mM for the liver enzyme. Fructose bisphosphate inhibited both enzymes. At pH 7.5, the inhibitor constants (Ki) were 0.18 and 1.3 mM for muscle and liver fructose bisphosphatases, respectively. The muscle enzyme was considerably more sensitive to AMP inhibition than the liver enzyme. At pH 7.5 and in the presence of 1 mM MgCl2, 50% inhibition of muscle and liver fructose bisphosphatases occurred at AMP concentrations of 7 X 10(-9) and 1 X 10(-6) M, respectively. EDTA activated both enzymes. The degree of activation was time and concentration dependent. The degree of EDTA activation of both enzymes decreased with increasing MgCl2 concentration. Ca2+ was a potent inhibitor of both liver (Ki, 1 X 10(-4) M) and muscle (Ki, 1 X 10(-5) M) fructose bisphosphatase. This inhibition was reversed by the presence of EDTA. Ca2+ appears to be a competitive inhibitor with regard to Mg2+. There is, however, a positive homeotropic interaction among Mg2+ sites of both enzymes in the presence of Ca2+.


Polypeptide hormone receptors: characteristics and applications.

Major developments in the area of polypeptide hormone receptors have been reviewed. Receptors are high affinity, high specificity binding sites which appear to be located largely, if not entirely, on the plasma membrane of cells. Receptors are proteins intimately associated with and influenced by lipids. Receptor sites and degrading sites appear to be readily distinguishable entities. The binding of hormone to receptor is distinct and has been dissociated from subsequent steps leading to hormonal response. There is no direct relationship between receptor occupancy and the magnitude of target response to hormone. So called 'spare' receptors can be viewed thermodynamically as enhancing target tissue sensitivity to hormone. The binding of hormone to receptor appears to be a point at which regulation of tissue sensitivity can be influenced either through altering the affinity for hormone or the number of receptors. One factor apparently involved in the regulation of receptor levels is the hormone itself. Receptors have been used to develop assay procedures which have significantly complemented the bioassay and radioimmunoassay. Finally, the measurement of receptor levels in disease has provided new insights into pathophysiology.


Effect of temperature on the activity and synthesis of glucose-catabolizing enzymes in Pseudomonas fluorescens.

The activity of the enzymes of the oxidative non-phosphorylated pathway, glucose and gluconate dehydrogenases, were not significantly affected by changes in the assay temperature. Both enzymes demonstrated only a threefold difference in activity when compared at assay temperatures of 30 degrees C and 5 degrees C. In contrast, the enzymes involved in the direct phosphorylation and catabolism of glucose or its oxidation products, gluconate and 2-ketogluconate, exhibited a more pronounced response to decreasing assay temperatures. At least one enzyme in each pathway, involved in the direct phosphorylation and catabolism of glucose or 2-ketogluconate (2KG), demonstrated an eightfold decrease in activity with a decrease in assay temperature from 30 degrees C to 5 degrees C. A similar decrease in assay temperature resulted in a fivefold decrease in activity of the enzymes involved in the direct phosphorylation and catabolism of gluconate. The observed differential effect of temperature on the activity of the enzymes of glucose catabolism and on the accumulation of direct oxidation products during growth with glucose in P. fluorescens E-20 is discussed. Growth with glucose at 5 or 20 degrees C resulted in high induced levels of all glucose-catabolizing enzymes examined when compared with the levels of these same enzymes in pyruvate-grown cells. However, only low levels of glucose dehydrogenase were detected during growth at 30 degrees C with glucose, gluconate, or 2-KG. Similarly, only low levels of gluconate dehydrogenase were detected during growth with glucose at 30 degrees C, although a weak induction was observed during growth with gluconate or 2-KG at 30 degrees C. The levels of 2-KG kinase plus KPG reductase during growth at 30 degrees C were undetectable with glucose, weakly induced with gluconate, and fully induced with 2-KG. High induced levels of glucose dehydrogenase, gluconate dehydrogenase, and 2-KG kinase plus KPG reductase were present during growth at 20 degrees C with glucose or 2-KG. The low levels of glucose and gluconate dehydrogenases present at a growth temperature of 30 degrees C was not due to heat lability of the enzymes at this temperature. The low amounts of these two enzymes during growth with glucose at 30 degrees C probably prevented sufficient inducer(s) formation from glucose to allow induction of enzymes of 2-KG catabolism. The results demonstrated that temperature may regulate the pathways of glucose dissimilation by regulating, either directly or indirectly, the activity and synthesis of the enzymes involved in these pathways.


Immunochemical studies of the surface and cytoplasmic membranes of Entamoeba invadens (Rodhain 1934).

The antigenic properties of cell fractions prepared from axenically grown Entamoeba invadens were investigated using various immunoelectrophoretic methods. All membrane fractions displayed varying degrees of antigenicity, the most predominant being a cytoplasmic light-vesicle fraction. The surface membrane showed the least diversified range of antigenic components and was also the least immunogenic fraction as judged by the reactivity of the antisera produced. Using tandem-crossed immunoelectrophoresis the antigenic profiles of the membrane fractions were compared. No evidence was obtained for the presence of either lipid or carbohydrate in the antigenic moieties of any of the membrane fractions. Using a series of sequential solvent extractions it was concluded that both divalent metallochelate linkages and interiorly located hydrophobic associations were principally involved in binding the major antigenic components within the light-vesicle membrane. An enzymic function was assigned to certain of the membrane antigens, the immunoprecipitates obtained showing both acid phosphohydrolase and non-specific esterase acitivity toward a variety of substrates.


Pathways of carbohydrate metabolism in mycobacterium tuberculosis H37Rv1.

Radiorespirometric studies using glucose labelled at 1, 2, 3-4, and 6 positions and enzymatic studies were conducted to determine the primary pathways of glucose dissimilation in Mycobacterium tuberculosis H37Rv. The pattern of 14CO2 recovery was C3-4 greater than C1 greater than C6 = C2. The Embden-Meyerhof pathway was found to be the predominant pathway for glucose oxidation, operative to the extent of 94%. The pentose phosphate pathway accounted for the remaining 6%. Maximum incorporation of 14C into cellular components was from C2 and C6 labelled glucose.


A comparative study on the heat stability of triosephosphate isomerase in psychrophilic, psychrotrophic, and mesophilic clostridia.

The temperature stability of triosephosphate isomerase (EC 5.3.1.1) in the cell-free extracts of psychrophilic, psychrotrophic, and mesophilic Clostridium species has been investigated. Even though this enzyme was found to be heat labile in the psychrophilic isolates, no detectable loss in activity was evident when cell-free extracts were heated for 1/2 h at the maximum temperature of growth for the organisms. Two organisms, each with a maximum growth temperature of 23 degrees C, showed different heat stability of triosephosphate isomerase. However, a trend is evident that as the maximum growth temperature increases, the thermostability also increases. It is suggested that the heat liability of this enzyme is not a controlling factor in psychrophilism, but rather an adaptation to the cold habitat of these organisms.


Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. II. Intracellular hyphae and vesicles.

Intracellular hyphae and vesicles in mycorrhizal roots of yellow poplar were examined by electron microscopy. An investing layer of host wall material and cytoplasm enclosed the endophyte within the cells. Young developing hyphae contained abundant cytoplasm and few vacuoles. As hyphae matured, they became highly vacuolated and accumulated carbohydrate (glycogen) and lipid reserves. Mature vesicles were engorged with lipid droplets, possessed a trilaminate wall and were also enclosed by host wall material and cytoplasm. Compared with uninfected cells, infected cortical cells showed an increase in cytoplasmic volume, enlarged nuclei, and a reduction of starch reserves. Host nuclei were always proximal to the hyphae during hyphal development and deterioration. While other cytoplasmic components of infected and uninfected cells were comparable large electron-dense bodies occurred in vacuoles of most cells containing hyphae. Deterioration of intracellular hyphae occurred throughout the samples examined. Septa separated functional and degenerating portions of the hyphae. Hyphal deterioration involved degeneration and ultimate disappearance of fungal cytoplasm as well as collapse of hyphal walls. Based on these observations, the authors hypothesize that deterioration of the endophyte may release significant quantities of mineral nutrients, via hyphal contents, which are absorbed by the host.