Comparison of the substrate specificity of adenosine 3':5'-monophosphate- and guanosine 3':5'-monophosphate-dependent protein kinases. Kinetic studies using synthetic peptides corresponding to phosphorylation sites in histone H2B. The substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinases have been compared by kinetic analysis using synthetic peptides as substrates. Both enzymes catalyzed the transfer of phosphate from ATP to calf thymus histone H2B, as well as to two synthetic peptides, Arg-Lys-Arg-Ser32-Arg-Lys-Glu and Arg-Lys-Glu-Ser36-Tyr-Ser-Val, corresponding to the amino acid sequences around serine 32 and serine 36 in histone H2B. Serine 38 in the latter peptide was not phosphorylated by either enzyme. Cyclic GMP-dependent kinase  and cyclic AMP-dependent kinase catalyzed the incorporation of 1.1 and 2.0 mol of phosphate/mol of histone H2B, respectively. The phosphorylation of histone H2B, respectively. The phosphorylation of histone H2B by cyclic GMP-dependent kinase showed two distinct optima as the magnesium concentration was increased. However, the phosphorylation of either synthetic peptide by this enzyme was depressed at high magnesium concentrations. As the pH of reaction mixtures was elevated from pH 6 to pH 9, the rate of phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase continually increased. Acetylation of the NH2 terminus of the peptide did not qualitatively affect this pH profile, but did increase the Vmax value of the enzyme 3-fold. The apparent Km and Vmax values for the phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase were 21 microM and 4.4 mumol/min/mg, respectively. The synthetic peptide Arg-Lys-Glu-Ser36-Tyr-Ser-Val was a relatively poor substrate for cyclic GMP-dependent kinase, exhibiting a Km value of 732 microM, although the Vmax was 12 micromol/min/mg. With histone H2B as substrate for the cyclic GMP-dependent kinase, two different Km values were apparent. The Km values for cyclic AMP-dependent kinase for either synthetic peptide were approximately 100 microM, but the Vmax for Arg-Lys-Arg-Ser32-Arg-Lys-Glu was 1.1 mumol/min/mg, while the Vmax for Arg-Lys-Glu-Ser36-Tyr-Ser-Val was 16.5 mumol/min/mg. These data suggest that although the two cyclic nucleotide-dependent protein kinases have similar substrate specificities, the determinants dictated by the primary sequence around the two phosphorylation sites in histone H2B are different for the two enzymes.


Partial characterization of the acidic and basic polypeptides of glycinin. The subunits of the 11 S storage protein from soybean cultivar CX635-1-1-1 were purified and characterized. Six polypeptides with acidic isoelectric points and four with basic isoelectric points were isolated from the purified storage protein. The acidic polypeptides had phenylalanine, leucine, isoleucine, and arginine at the NH2 termini, while the basic polypeptides all had glycine at the NH2 termini. Amino acid analysis indicated that certain acidic and basic polypeptides contained 3 to 6 times more methionine than the other polypeptides. Since the low nutritional quality of legume storage proteins is due to a deficiency in methionine, this observation will have significance in efforts to improve soybean quality. The purified polypeptides were further characterized by NH2-terminal sequence analysis. Considerable homology was found between the members of individual families of acidic and basic polypeptides, indicating that the members of each family arose from a common ancestral gene. This study showed that the glycinin polypeptide composition is more complex than previous reports indicated, and for the first time characterized the various polypeptides of the 11 S storage protein by structural analysis.


Natural abundance carbon-13 nuclear magnetic resonance study of anthopleurin-A, a cardiac stimulant from the sea anemone Anthopleura xanthogrammica. Natural-abundance 13C NMR spectra (at 15.04 MHz) of the polypeptide cardiac stimulant Anthopleurin-A are presented. The spectra contain many resolved one- and two-carbon resonances from carbonyl and aromatic carbons and a few resolved resonances from aliphatic carbons. Most of these have been assigned to individual carbons in the protein. The effect of pH on the 13C spectrum has been investigated. In conjunction with the resonance assignments, this yields estimates for the pK alpha values of the COOH-terminal and NH2-terminal residues, the side chain carboxylate of 1 of the 2 aspartic acid residues, and the imidazolium groups of the 2 histidine residues. The effects of the lanthanides La3+ and Gd3+ on the spectrum have also been studied. The results suggest that there are at least two binding sites, and further studies will be required to characterize these before they can be utilized as an aid in structural mapping. Finally, the results are discussed in relation to a postulated model for the mode of action of Anthopleurin-A.


A resonance Raman and electronic absorption probe of membrane energization. Quinaldine red in cells of Streptococcus faecalis. Resonance Raman and electronic absorption spectra were used to show that the state of an amphiphilic cation, relative to dilute aqueous solution, changes when it is accumulated by cells of Streptococcus faecalis when they are energized. The general characteristics of the cation employed, quinaldine red, closely paralleled those of other amphiphilic cations which have been used to measure membrane potential. A major aspect of the change is that in sodium-loaded cells, essentially all of the quinaldine red accumulated as the result of energization forms a strong bond with an anionic group. This binding is similar to that which occurs for the basal level of quinaldine red taken up in nonenergized cells. Ionic binding was detected using resonance Raman spectroscopy through shifts associated with a N+ parallel C--C parallel C stretching vibration to lower frequency on uptake. Another aspect of the change in state is that the cell-localized probe cation can aggregate while ionically bonded in a card pack fashion, the transition dipoles being parallel. A combination of resonance Raman and electronic absorption spectroscopy was used to characterize this aggregation. The aggregates were estimated to contain at least five quinaldine red cations at or near van der Waals contact, and the presence of other molecules, such as phospholipids, could not be excluded. Aggregation effects are complex depending on the ratio of cells to probe cation, and on energization. The site of binding is suggested to be the lipid bilayer region of the plasma membrane on the basis of experiments with liposomes and other model systems. In addition, some quinaldine red may be present in the cytoplasm in an aggregated, ionically bound form. The change in state on uptake following energization seems to be associated with a membrane potential, similar spectral and uptake effects being produced by an artificially generated membrane potential in cells and liposomes. The results show that membrane potential cannot be computed in a simple manner from the distribution of quinaldine red between cells and medium, assuming that the thermodynamic activity coefficient of cell-localized material is identical with that in dilute aqueous solution. However, uptake as well as subsequent ionic binding of quinaldine red seems to be related to potential in an as yet undefined manner.


Novel mannitol-containing oligosaccharides obtained by mild alkaline borohydride treatment of a chondroitin sulfate proteoglycan from brain. Mannitol-containing oligosaccharides have been isolated from a rat brain proteoglycan after mild alkaline borohydride treatment under conditions which prevent "peeling." Their structural properties were studied by gas-liquid chromatography-mass spectrometry of disaccharides as their trimethylsilylated and permethylated derivatives, methylation, analysis, specific degradations, and CrO3 oxidation. The following components were identified: Gal(beta 1 leads to 4) [Fuc(alpha 1 leads to 3)]GlcNAc(beta 1 leads to 3)Manol,GlcNAc(beta 1 leads to 3)Manol, and Manol. Evidence was also obtained for the occurrence of a sialylated oligosaccharide and another (possibly sulfated) acidic oligosaccharide, both having the sequence GlcNAc(beta 1 leads to 3)Manol at their proximal ends. These mannitol-containing oligosaccharides constitute a novel group of alkali-labile oligosaccharides in mammalian glycoconjugates. The origin of the oligosaccharides and the possible occurrence of a carbohydrate-peptide linkage involving mannose are discussed.


Energy cost of walking with lesions of the foot. The oxygen consumption during walking was measured in patients affected by chronic lesions of the foot. Only subjects with rigidity of the talocalcaneal joint showed increased oxygen consumption, which reached 5 to 20 per cent above normal. They also showed an increased step frequency, probably as a compensatory mechanism to reduce the mechanical work performed during each step.


Effects of low electrolyte media on salt loss and hemolysis of mammalian red blood cells. Cation loss and hemolysis of various mammalian red cells suspended in isotonic non-electrolyte media were investigated. Sucrose buffered with 10 mM Tris-Hepes, pH 7.4 was used as the non-permeable non-electrolyte. Mammals from which the red cells were derived include the human, guinea pig, rat, rabbit, newborn calf, newborn piglet and pig, all of which contain K as the predominant cation species (HK type) and the dog, cat, sheep and cow, all of which possess Na as the predominant cation species (LK type). Of HK cells, a rapid efflux of K takes place from humans, rats and guinea pigs. Of LK type cells, the dog and cat exhibit an augmented membrane permeability to Na. The governing factors which influence cation permeability are the change in pH, temperature, and ionic strength. In response to increase in pH, the red cells of humans, dogs and cats become more permeable to cations, whereas the red cells of rat and rabbit are unaffected. In response to increase in temperature, HK type cells exhibit augmented K efflux, while the Na loss from the dog and cat cells manifest a well-defined maximum at near 37 degrees C. In all cases, a small substitution of sucrose by an equal number of osmoles of salts results in a dramatic decrease in cation loss. By contrast, the red cells of the rabbit, newborn calf, adult cow, newborn piglet, adult pig and sheep display no discernible increase in ion-permeability under the conditions alluded to above. In some species including the newborn calf, dog, and cat, an extensive hemolysis occurs usually within an hour in isotonic buffered sucrose solution. The osmolarity of sucrose solution affects these cells differently in that as the osmolarity increases from 200--500 mM, hemolytic rates of the calf and dog reach a saturation near 300 mM sucrose, whereas the hemolytic rate of the cat decreases progressively. Common features pertaining to this hemolysis are (1) the intracellular alkalinization process; and (2) the diminution of the cell volume which take place prior to and onset of hemolysis. SITS, a potent anion transport inhibitor, completely protects the cells from hemolysis by inhibiting chloride flux and the concomitant rise in intracellular pH.


[Malignant tumour of the testis abdominal cryptorchism revealed by torsion of the spermatic cord and diagnosed by arteriography (author's transl)]. The authors present a case of torsion of an intra-abdominal choriocarcinoma of the testis, in which the pre-operative diagnosis was made by aortic arteriography. The association of the two remains rare, as a review of literature proved. The orchidopexy if it does not tend to prevent the possibility of malignant change gives an easier follow-up and an earlier diagnosis.


Radioimmunoassay of a basic somatomedin: comparison of various assay techniques and somatomedin levels in various sera. Purification of a basic somatomedin (SM), with similarity to SM-C and insulin-like growth factor, from human plasma Cohn fraction IV-1 enabled development of a RIA based on this SM.SM antiserum was produced by immunizing rabbits with partially purified SM. This antiserum (final dilution, 1:50,000) specifically bound approximately 40% of added [125I]-SM in this RIA. The RIA sensitivity was 2 x 10(-4) U immunoreactive SM (IRSM). Highly purified SM-C, insulin-like growth factor 1, and our SM revealed parallel and approximately equipotent dose-response curves in this RIA; rat SM and multiplication stimulation activity revealed less cross-reactivity. IRSM was detected in sera of all species tested except fish. Acidification of sera, without subsequent chromatography, before assay permitted measurement of total IRSM with either an equilibrium or nonequilibrium RIA technique. Acidification of serum appears to increase SM-binding capacity while decreasing binding affinity of the 20,000--50,000 mol wt proteins in serum. The mean (+/- SEM) IRSM concentrations in sera from normals and patients with acromegaly, hypopituitarism, GH deficiency before/after treatment, and Laron dwarfism were 1.45 +/- 0.17, 5.49 +/- 0.48, 0.19 +/- 0.07, 0.10 +/- 0.02/0.64 +/- 0.45, and 0.25 +/- 0.11 U/ml, respectively, compared to a pooled normal human serum reference standard which was designated to contain 1 IRSM U/ml. Measurements of total IRSM (bound and free) in serum may not accurately reflect SM bioactivity and will require interpretative caution.


Enzyme-linked immunosorbent assay for detection of Streptococcus pneumoniae antigen. An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Streptococcus pneumoniae polysaccharide antigen in cerebrospinal fluid and serum. Sensitivity and specificity were determined for purified antigen preparations. Specificity was also evaluated in the rabbit meningitis model, and the sensitivity was compared to counterimmunoelectrophoresis, using the infected rabbits' cerebrospinal fluid and serum. The ELISA was a specific technique for detecting S. pneumoniae antigen. ELISA was 25 times more sensitive than counterimmunoelectrophoresis for purified antigen and resulted in an increased positivity of the cerebrospinal fluid and serum from infected rabbits. ELISA should prove very useful in the diagnosis of pneumococcal infections.


Graded imagination and relaxation as components of experimental desensitization: a psychophysiological evaluation. Studied the effects of aversive imagery gradation and muscular relaxation as procedural variables within desensitization by using (pre-vs. posttreatment) peripherally cued electrodermal responsivity as the dependent-variable measure of fear. There was no evidence that experimental desensitization or any of its procedural components yielded attenuated electrodermal responsivity to the peripheral fear cue. Because this result derives from a relatively well-done study, it poses a challenge to widespread notions about the vehicle of therapeutic effects from desensitization.


The desensitization derby: in vivo down the backstretch, imaginal at the wire? Recontacted 17 Ss who had undergone successful imaginal or in vivo desensitization for snake phobia and had been evaluated pre- and posttreatment for a 9-month follow-up. Main findings were that: (a) overall treatment gains were maintained through the follow-up period; (b) differential gains in favor of the in vivo technique on one behavioral and two self-report snake-specific criteria were dissipated; and (c) imaginal Ss registered a reduction in self-rated fear during an approach task from posttesting to follow-up. Results are discussed in relation to the notion of cognitive rehearsal as a means of self-reinforcing treatment gains.


The abrupt discontinuation of antihypertensive treatment. Although deleterious events following abrupt withdrawal of antihypertensive treatment are relatively uncommon, considerable attention has recently been focused on this problem. A withdrawal syndrome may occur after termination of almost all types of antihypertensive drugs, but most experience has been with the centrally acting agents and with beta-adrenoreceptor blockers. Abrupt discontinuation of high doses of centrally acting drugs such as alpha-methyldopa, clonidine, and guanabenz can produce a syndrome of sympathetic overactivity that includes agitation, headache, sweating, and nausea and less commonly can provoke rapid upswings in blood pressure. If beta blockers are suddenly stopped, a similar pattern can occur that may be related to excessive activity of thyroid hormones as well as sympathetic factors. Additionally, patients with ischemic heart disease may be susceptible to an acute exacerbation of their cardiac disease when beta-blocker treatment is stopped. It seems likely that discontinuation events can be particularly severe when combinations of different types of antihypertensive medications are sud-disease when betablocker treatment is denly stopped. This problem can be dealt with by educating patients to avoid sudden drug cessation and when elective discontinuation is planned, by gradual dose reduction.


Nonspecific suppressor cells in patients with chronic graft-vs-host disease after marrow grafting. Forty-four human long-term survivors after marrow transplantation for aplastic anemia or hematologic malignancy were studied for the presence of circulating nonspecific suppressor cells. Twenty-two of the patients were healthy and 22 had mild to moderately severe chronic graft-vs-host disease (GVHD). Patient mononuclear cells (of donor origin) were tested for their ability to suppress the responses of lymphocytes obtained from the respective marrow donors to alloantigens in mixed leukocyte culture (MLC) and/or to concanavalin A (Con A). Tests were carried out between 199 and 2393 (median 376) days after transplantation. Cells from only 1 of 22 patients without chronic GVHD showed suppression of donor cell blastogeneis responses. In contrast, cells from 11 of 22 patients with chronic GVHD showed more than 30% suppression of donor cell responses in MLC and/or to Con A. The finding of suppressor cells was not related to the time of testing after grafting nor to immmunosuppressive therapy. Nonspecific suppressor activity was abrogated by irradiation with 1600 rads in vitro in five of six cases tested. Nonspecific suppressor cells may be one explanation for the severe combined immunodeficiency and the recurrent infectious complications characteristic of patients with chronic GVHD.


[The influence of present therapeutic methods and especially of beta-blockers on fetal and maternal prognosis in hypertensive syndromes in pregnancy. 43 case records (author's transl)]. The authors studied 43 cases of arterial hypertension in pregnancy in an attempt to determine the efficiency and safety of different anti-hypertensive drugs. The patients were divided into two major groups: arterial hypertension which revealed itself during pregnancy (true toxaemias of pregnancy and relapsing toxaemias), and arterial hypertensions which were added on to a pre-existing pathology (arterial hypertension, diabetes, chronic nephritis). The cases in these different classes were then divided into two definite groups according to the need for therapy: the first group was treated by rest and hydrallazine as a single therapeutic agent. In the second group multiple agents were needed because of the arterial hypertension, and one was a beta-blocker. Complications were found particularly in the second group of true toxaemias of pregnancy where unfortunately 5 fetal deaths occurred that were attributable to the severity of the hypertension more than to the beta-blockers, which were administered for longer and in higher doses without major complications in recurrent toxaemias and pre-existing arterial hypertension cases.


Local xenogeneic graft-vs-host reaction: a practical assessment of T cell function among cancer patients. The local xenogeneic graft-vs-host reaction (XGVHR) was used as a practical bioassay to assess T lymphocyte function and immunocompetence among cancer patients. Positive XGVHR was found in 99.5% of normal donors, 70% of cancer patients with early stage disease, and 30% of cancer patients with metastatic disease (p less than 0.001). A minimum of 4.5 x 10(6) immunocompetent T lymphocytes are necessary in order to elicit a positive XGVHR. Negative reactions among cancer patients are characterized by the lack of edema fluid accumulation and the appearance of the most basophils at the test site. This suggests that insufficient amounts of lymphokines are being released by the incompetent T lymphocytes, whereas the host is capable of mounting a rejection reaction as evidenced by the appearance of the basophils. Preliminary evidence suggests that the immunologic defect detected by the XGVHR cannot be corrected by monocyte depletion. The identification of putative suppressor T cell subsets may bear immunotherapeutic implications in the future.


[A study of the comparative efficiency of ifenprodil and ritodrine in the treatment of threatened premature labour (author's transl)]. The study compares a sequential test of a beta mimetic drug (ritodrine) and an alpha inhibitor (ifenoprodil) to show that the beta mimetic drug is superior in treating threatened premature labour, because it has a success rate of 90 per cent whereas the second has a success rate of 45 per cent. Ritodrine produces secondary effects in 69 per cent of cases.


Rabbit B spleen lymphocytes and T helper cells. I. Responsiveness to mitogens of B cell subpopulations of different sedimentation velocities and subpopulations bearing or lacking Fcgamma receptors. The response to anti-allotype (anti-Ab4), Nocardia Water Soluble Mitogen (NWSM), pneumococcal polysaccharide type III (SSS III), and human Fc fragments of various purified and unfractionated rabbit spleen cell populations was determined in terms of 3H-thymidine up-take. B cells were isolated either from untreated suspensions of spleen cells or from suspensions from which adherent and phagocytic cells were removed. The purification factor was greater than the enhancement of 3H-thymidine uptake by anti-Ab4, NWSM, and SSS III as compared with the response of unfractionated spleen cells. It thus appears that a helper cell was involved: the mitogen response of purified B cells was enhanced by the addition of T cells. B subpopulations were separated by sedimentation or by rosetting, which allowed us to separate Fcgamma receptor-bearing cells from cells that did not possess this receptor. There were differences between cells responding to B mitogens not only in sedimentation velocity but also in the absolute number of cells. B cells bearing the Fcgamma receptor were less responsive to anti-Ab4 and more responsive to SSS III, NWSM, and human Fc than were B cells lacking the Fcgamma receptor.


Inheritance of lysozyme deficiency in rabbits. The phenotypes of 273 offspring of 56 matings of various genotypes of lysozyme-deficient and normal New Zealand white rabbits were analyzed. It was determined that lysozyme deficiency of rabbits is inherited as an autosomal recessive with complete penetrance. The symbol ld is suggested for the gene for lysozyme deficiency. Further studies on the nature of the condition revealed that the lysozyme deficiency was not due to a lysozyme that was cold labile, or to a lysozyme that was so tightly bound it was unavailable for enzymatic assay, or to a lysozyme with a variant pH optimum. It was demonstrated, however, that the pH profile of the lysozyme remaining in the ld/ld rabbits was different from that found in normal rabbits.


Purification and properties of human B cell-activating factor. Human monocytes, when appropriately stimulated in vitro, release into the culture medium a factor (BAF) that stimulates the IgM response of T-depleted murine splenocytes to heterologous erythrocytes. The behavior of this factor on gel filtration, isoelectric focusing, ion exchange chromatography, and isopycnic centrifugation was studied. BAF appears to be a molecule of 15,000 daltons, pI 6.5, 1.33 g/ml with low solubility at low ionic strength. It is stable to acid, mild heating, and long-term storage. Activity is lost in alkali or by boiling. Papain may reduce BAF activity slightly, whereas trypsin and chymotrypsin have no significant effect. These properties are similar to those of other monokines reported to have a similar m.w.


Acyl-CoA reductase specificity and synthesis of wax esters in mouse preputial gland tumors. Long-chain alcohols are synthesized in the mouse preputial gland tumor (ESR-586) by NADPH:acyl-CoA oxidoreductase. In this study, a series of labeled acids was tested as substrates for the oxidoreductase in a cell-free system from the tumor, and the distribution of label into alcohols, waxes, and other products was determined. The system contained the labeled acid, an acyl-CoA-generating system, an NADPH-generating system, and tumor homogenate. The highest rates of alcohol synthesis were obtained with palmitic (16:0), heptadecanoic (17:0), stearic (18:0), myristic (14:0), elaidic (18:1 trans), and linoleic (18:2) acids, which yielded, respectively, 151, 124, 102, 76, 65, and 35 pmol alcohol/min per mg protein. Decanoic (10:0), lauric (12:0), oleic (18:1 cis), linolenic (18:3), arachidonic (20:4), and behenic (22:0) acids all gave lower activities. Acyl-CoA formation did not appear to be rate limiting with any of the substrates tested except behenic acid. In addition to the fatty alcohol product, a small amount of fatty aldehyde was formed in the system. Incorporation of the labeled fatty acids into wax esters was examined and the distribution of label between the alcohol and acid components of the waxes was determined. Incubation of [1-(14)C]palmitic acid yielded 3.4% free alcohol, 8.3% alcohol esterified in waxes, and 7.7% palmitoyl groups esterified into waxes, whereas, at the other extreme, [1-(14)C]linolenic acid yielded 0.8%, 0.6%, and 38%, respectively, into the homologous components.-Wykle, R. L., B. Malone, and F. Snyder. Acyl-CoA reductase specificity and synthesis of wax esters in mouse preputial gland tumors.


Factors influencing mouse heart creatine kinase efflux and ultrastructure. An isolated mouse heart model has been developed to study the extracellular factors which influence the loss of myocardial enzymes. When added to a tris-HCl buffer/salt mixture at 25 degrees C, glucose, phosphate ion, increased osmolarity, oxygen exclusion and calcium reduced enzyme leakage. Of these, calcium effects on enzyme leakage and ultrastructure were assessed in detail. Concentrations less than or equal to 10(-4) M had no significant effect on enzyme efflux over a 5 hour period. At higher concentrations (10(-3) and 10(-2) M Ca2+), creatine kinase (CK) efflux was significantly altered in a time dependent fashion. In the first hour, 10(-3) and 10(-2) M Ca2+ reduced CK leakage to 33% and 25% of the control values, respectively; and to about 50% of the control values in the second and third hours. This protective effect was lost between the third and fifth hours, when an enzyme efflux 80% greater than control was observed. These studies indicated that CK leakage from mouse heart can be retarded for up to 3 hours by appropriate Ca2+ concentrations. The initial ultrastructural change, in the absence of Ca2+, was a dilatation of the transverse tubules, which gradually enlarged by coalescence. This was followed by a gradual disintegration and ultimate condensation of the myofibrils leaving altered mitochondria floating freely in an apparently intact sarcolemmal bag. These changes appeared to be delayed by Ca2+ for 3 hours, after which no protective effect was evident. Thus, CK leakage is a measure of myocardial autolysis, and numerous simple measures can retard this autolysis for several hours. This raises the possibility of prolonging the preservation of the normal heart in vitro.


The relation between dicarbocyanine dye fluorescence and the membrane potential of human red blood cells set at varying Donnan equilibria. The fluorescence, F, of two dicarbocyanine dyes, diS-C3(5) and diI-C3(5), depends both on the membrane potential, E, and on the intracellular pH, pHc, or human red blood cells. Compositions of isotonic media have been devised in which the equilibrium Donnan potential, E, varies at constant pHc and in which pHc varies at constant E. Dye fluorescence measurements in these suspensions yield calibrations of +1.7 % delta F/mV for diS-C3(5) and +0.6 % delta F/mV for diI-C3 (5). While pHo does not affect F of either dye, changes in pHc of 0.1 unit at constant E cause changes of F equivalent to those induced by 2--3mV. Based on these results, a method is given for estimating changes in E from dye fluorescence in experiments in which E and pHc co-vary. The relation of F to E also depends in a complex way on the type and concentration of cells and dye, and the wavelengths employed. The equilibrium calibration of dye fluorescence, when applied to diffusion potentials induced by 1 microM valinomycin, yields a value for the permeability ratio, PK.VAL/PCl, of 20 +/- 5, in agreement with previous estimates by other methods. The calibration of F is identical both for diffusion potentials and for equilibrium potentials, implying that diC-C3(5) responds to changes in voltage independently of ionic fluxes across the red cell membrane. Changes in the absorption spectra of dye in the presence of red cells in response to changes in E show that formation of nonfluorescent dimers contributes to fluorescence quenching of diS-C3(5). In contrast, only a hydrophobic interaction of dye monomers need be considered for diI-C3(5), indicating the occurrence of a simpler mechanism of fluorescence quenching.


Electrical properties of an excitable epithelium. The exumbrellar epithelium of the hydromedusa, Euphysa japonica, is composed of a single layer of broad (70 micrometers), thin (1--2 micrometers) cells which are joined by gap junctions and simple appositions. Although the epithelium lacks nerves, it is excitable; electrically stimulating the epithelium initiates a propagated action potential. The average resting potential of the epithelial cells is -46 mV. The action potential, recorded with an intracellular electrode, is an all-or-nothing, positive, overshooting spike. The epithelial cells are electrically coupled. The passive electrical properties of the epithelium were determined from the decrement in membrane hyperpolarization with distance from an intracellular, positive current source. The two-dimensional space constant of the epithelium is 1.3 mm, the internal longitudinal resistivity of the cytoplasm and intercellular junctions is 196 omega cm, and the resistivity of both apical and basal cell membranes is greater than 23 k omega cm2. Although the membrane resistivity is high, the transverse resistivity of the epithelium is quite low (7.5 omega cm2), indicating that the epithelium is leaky with a large, transverse, paracellular shunt.


On the possible mode of action of serotonin in neurotransmission and brain dysfunction. Criteria necessary to classify a substance as neurotransmitter are used to determine whether disturbances in neurotransmitter function are involved in brain disorders. Six rather stringent criteria for a neurotransmitter are satisfied by the biogenic amine 5-hydroxytryptamine (5-HT): 1. the availability of 5-HT within the CNS is proved by its synthesis and transport, 2. it is stored within the presynaptic bouton, 3. presynaptic stimulation releases 5-HT, 4. it reacts with receptors of the synaptic membranes. 5. a functional equivalence of presynaptic stimulation and subsynaptic 5-HT action can be demonstrated, 6. the transmission is terminated by a retrieval mechanism.--On the basis of this transmitter concept brain dysfunctions as described in the literature are discussed in the light of results obtained using in vitro techniques with isolated synaptic structures. In renal and hepatic insufficiency the transport of the 5-HT precursor tryptophan is affected at the site of the blood-brain barrier. Tryptophan-5-hydroxylase, the rate-limiting enzyme in 5-HT synthesis, is inhibited in hypoxia. The antiparkinson drug 1-aminoadamantane and its 3.5-dimethyl derivative, D 145, inhibit 5-HT catabolism as well as 5-HT storage in synaptic vesicles and reuptake into isolated nerve endings. Since 1-aminoadamantanes enhance the electrically stimulated 5-HT and DA release from nerve endings spatial shifts of transmitter substances within the synaptic area as well as direct receptor stimulation by the drugs may be the cause for the ameliorating effect.


Brain monoamines in hepatic encephalopathy and other types of metabolic coma. Tyrosine (Tyr), tyrosine hydroxylase (TH), tryptophan (Trp), serotonin (5-HT), and 5-hydroxyindole acetic acid (5-HIAA) were assayed spectrofluorometrically and radioenzymatically in various regions of post-mortem brains of human patients with hepatic, uremic, and diabetic coma, liver cirrhosis without coma, and hepatic coma treated with parenteral administration of L-valine, a branched-chain amino acid. The results were as follows: In both hepatic and diabetic coma Tyr was increased as compared to non-comatose cirrhosis and controls, while TH acitivity was within normal limits, indicating sufficient oxygen supply of the brain in both types of coma. Brain DA showed a mild decrease in all types of metabolic coma. Brain Trp was not considerably changed in non-comatose cases of liver cirrhosis and after L-valine treatment of hepatic encephalopathy, but was significantly increased in hepatic coma, with highest elevation in the brainstem tegmentum. Both 5-HT and 5-HIAA were not significantly changed in non-comatose cirrhosis, while a general increase with prevalence for the brainstem was obvious in all types of metabolic coma. After L-valine treatment of hepatic coma, 5-HT levels were usually decreased below control values, while 5-HIAA levels were at or below controls. These results in human post-mortem brains confirm previous CSF and brain findings in experimental and human hepatic and uremic encephalopathies, indicating derangement of brain monoamine neurotransmitter metabolism which is attributed to imbalance of aromatic and branched-chain amino acids in plasma and brain. Increased cerebral 5-HT turnover, particularly in the ascending serotonergic brainstem systems, due to derangement of brain uptake of Trp is suggested to represent an important biochemical substrate of disorders of consciousness in hepatic failure and other types of metabolic encephalopathies. Clinical improvement of hepatic encephalopathy and of the underlying neurotransmitter derangements by administration of L-valine and the possible role of this competitive amino acid on intermediary metabolism and ammonia detoxification are discussed.


CNS Modulation of adrenal tyrosine hydroxylase in Parkinson's disease and metabolic encephalopathies. Tyrosine hydroxylase (TH) activity was assayed radioenzymatically in various regions of post-mortem brains of human individuals without neurologic disorders (controls), with Parkinson's disease, senile dementia, hypertensive encephalopathy, hepatic and diabetic coma, liver cirrhosis without coma, and hepatic coma treated with parenteral administration of L-valine. In addition TH activity of the post-mortem adrenal medulla was assayed in controls, in Parkinson's disease, senile dementia and hypertensive encephalopathy. In Parkinson's disease TH activity was significantly decreased in the nigrostriatal system, and less severe in other brainstem areas, while the raphé-reticular formation and limbic system showed normal values. In addition, there was significant decrease in the TH activity of the adrenal medulla, suggesting that Parkinson's disease is a generalized disorder not limited to distinct CNS areas, and that impairment of the dopaminergic niggro-striatal system may involve the TH activity in the adrenal medulla, thus inducing disorders of the peripheral sympathetic system. Senile brain atrophy showed no definite changes in brain, except the striatum, and adrenomedullary TH, while in one case of hypertensive encephalopathy due to long-term corticosteroid treatment normal TH activity in the adrenal medulla was opposed by decreased striatal TH activity, probably due to cerebral ischemia. TH activity in the caudate nucleus of individuals with both hepatic and diabetic coma were within normal ranges, suggesting a sufficient energy supply of the brain during such metabolic catastrophes, while reduced brain TH activity in patients with hepatic coma who died of acute gastrointestinal bleeding is probably due to severe final cerebral ischemia. No correlative data on brain and adrenomedullary TH activities in metabolic encephalopathies are available so far.


Alterations of putative neurotransmitters and enzymes during ischemia in gerbil cerebral cortex. Bilateral occlusion of common carotid arteries in Mongolian gerbils was produced for the periods (up to 15 min) which were shown to be totally reversible. There was an initial increase of cyclic AMP and GABA levels and enhanced activities of adenylate cyclase and glutamate decarboxylase, as well as the reduction of norepinephrine level and decreased activities of monoamine oxidase, GABA-transaminase and Na+-K+-ATPase. Following these changes, decreased concentration of dopamine, serotinin and glutamate were found. The activities of total protein kinase and acetylcholinesterase were found to be reduced after longer periods of short-term ischemia. The data are consistent with the concept of increased non-controled release of putative neurotransmitters in ischemia.


Changes of some putative neurotransmitters in human cerebral infarction. Dopamine (DA), serotonin (5-HT), tryptophan (TRP), 5-hydroxyindole acetic acid (5-HIAA), and GABA were assayed spectrofluorometrically in various regions of 16 human post-mortem brains with acute and old cerebral infarction. In both recent and older strokes a total depletion of DA and 5-HT in the necrotic tissue was associated with mild reduction of these compounds in remote non-ischemic areas of the injured, and less of the contralateral cerebral hemispheres. 5-HIAA was significantly reduced in acute ischemic necrosis, while the perifocal edema zone showed considerable accumulation of both 5-HT and 5-HIAA. Marked elevation of the 5-HT precursor TRP and of GABA was present in both the necrotic center and perifocal edema of acute infarcts, which also showed a mild reduction of total proteins. The degradation zone surrounding old infarcts showed a mild decrease of both 5-HT and 5-HIAA with normal TRP levels, indicating normalization of the previously increased 5-HT metabolism and turnover after decrease of acute cerebral edema. These data which confirm previous studies in experimental cerebral ischemia and stroke indicate that disorders in the metabolism of brain monoamines and other putative neurotransmitters contribute to the development of postischemic brain damage and the complicating cerebral edema. They are also in keeping with the concept that unilateral focal ischemia produces bilateral effects on brain monoamines.


When--and why--should nutritional state control neurotransmitter synthesis? The rates at which neurons synthesize such Group I neurotransmitters as serotonin, acetylcholine, and the catecholamines norepinephrine and dopamine depend physiologicall on the availability to them of the circulating precursors for these compounds (tryptophan, choline and tyrosine, respectively). The concentrations of precursor in the circulation and in neurons change rapidly after food consumption, depending upon what is eaten. Nutrient intake thus normally influences the synthesis of these neurotransmitters. Neurons that emit signals by releasing serotonin, acetylcholine, dopamine, or norepinephrine participate in the control of a number of bodily functions and behaviors (e.g., hunger, food choice, sleep, alertness, sensitivity to environmental stimuli and disease states). Dietary manipulations (or the consumption of individual nutrients) can thus be used as tools for the experimental analysis of functions mediated by monoaminergic or cholinergic neurons, and as adjuncts in the treatment of some diseases of these neurons. It is unclear "why" the evolutionary process should have "allowed" the neurotransmission mediated by acetylcholine or the monoamine transmitters to be influenced by the vagaries of food choice. One possible benefit that might accrue to the organism as a result of this dependency would be the use of cholinergic or monoaminergic neurons as "sensors", providing the brain with information about peripheral metabolic state. Thus carbohydrate consumption, which--by altering plasma amino acid levels accelerates brain serotonin synthesis--enhances the release of a transmitter (serotonin) that tends to diminish the animal's desire to consume carbohydrates.


Postnatal rat sympathetic neurons in culture. I. A comparison with embryonic neurons. 1. A morphological and physiological comparison was made between embryonically and postnatally derived superior cervical ganglion neurons (SCGN) grown in dissociated cell culture. It was found that while morphologically distinct, the physiological properties of the postnatal neurons were the same as their embryonic counterparts. 2. Intracellular injection of horseradish peroxidase (HPR) demonstrated that SCGN from any age of animal elaborated two basic types of processes, although the pattern of process ramification was unique for each neuron. The two types of proceses were 1) the large, smooth, rapidly tapering; and 2) the thin, nontapering variety, which often contained varicosities along its length. It is suggested that the former are dendritic in function, while the latter act as axons. 3. A difference was noted in somal size and the number of primary processes extended by the embryonic and postnatal neurons, with the latter more closely resembling the in vivo morphology. 4. Resting potentials and action-potential amplitudes of postnatal SCGN were comparable to those found previously for embryonic SCGN in vitro. 5. Iontophoretic application of putative neurotransmitter substances revealed the presence of acetylcholine receptors (AChR) on both embryonic and postnatal SCGN. Picrotoxin-sensitive depolarizing responses to iontophoresed gamma-aminobutyric acid (GABA) was seen on a few embryonic neurons, but not on the older cells. No responses were detected when norepinephrine (NE), glutamate, cAMP, substance P, or dopamine were applied to the SCGN of either age group. 6. Synatpic interaction between postnatal SCGN were found at an earlier in vitro age (12 days) than was the case for embryonic neurons (20 days). 7. Synaptic transmission was found to be chemical in nature. This was shown by 1) a dependence on external Ca2+ concentrations; 2) steplike fluctuations in synpatic potential amplitude, and 3) a variation in potential amplitude with changes in membrane potential. 8. It is concluded that the postnatal SCGN are able to survive in culture even when taken from animals up to 12.5 wk old. The elaboration of processes is in many ways strikingly similar to sympathetic neurons in the animal, and they are able to form functional synaptic interactions.


Gastroesophageal reflux causing respiratory distress and apnea in newborn infants. Respiratory distress, apnea, and chronic pulmonary disease since birth were identified in 14 infants who also had symptomatic gastroesophageal reflux. Birth weights varied from 760 to 4,540 gm. All infants had radiographic changes similar to those in bronchopulmonary dysplasia. Cessation of apnea and improvement of pulmonary disease occurred only after medical (8) or surgical (6) control of gastroesophageal reflux. Simultaneous tracings of esophageal pH, heart rate, impedance pneumography, and nasal air flow in five infants demonstrated that reflux preceded apnea. Apnea could be induced by instillation of dilute acid, but not water or formula, into the esophagus. Prolonged monitoring of esophageal pH more than two hours after feeding in 14 other infants less than 6 weeks of age (birth weight 780 to 3,350 gm) without a history of recent vomiting indicated that reflux was not greater than in normal older children.


Aarskog syndrome: significance for the surgeon. A familial syndrome of short stature associated with facial dysplasia and congenital anomalies was reported by Aarskog in 1970. Subsequently, at least 53 patients in 13 affected families have been reported. The family to be described came under investigation following referral of one of the nine siblings to the surgical clinic at Childrens Hospital of Los Angeles for the evaluation of a right inguinal hernia. Recognition of the syndrome and further evaluation of the remaining siblings led to the diagnosis of several genital anomalies requiring surgical correction.


The evaluation of the force to expel oily injection vehicles from syringes. Measurement of the force needed to expel oily injection vehicles from all-glass syringes through hypodermic needles has shown that the force required to maintain various rates of expulsion is in agreement with a modified form of the Poiseuille equation. The flow from disposable plastic syringes with rubber plunger tips fits this equation only if a correction is made to take account of the resistance to movement of the plunger (which is effectively zero for all-glass syringes). This resistance or binding force was shown to be increased by exposure to vegetable oils, and its magnitude was inversely dependent on vehicle viscosity. This increase was caused by swelling of the rubber plunger tips due to uptake of the oily vehicles by the rubber. No such increase in binding force was observed with a viscous aqueous vehicle.


Evaluation of changes in drug particle size during tableting by measurement of dissolution of disintegrated tablets. Three poorly soluble drugs (chloramphenicol, phenacetin and prednisolone) were compressed into tablets of 10% drug content on a physical testing instrument at three different compression pressures. The dissolution profiles were determined by a modification of the U.S.P. method for drug suspensions, granules before compression, disintegrated and intact tablets. By comparison of the dissolution rates for disintegrated tablets with those for granules before compression, or suspensions, it is possible to separate the change in particle size during compression from the pressure-dependent dissolution behaviour of intact tablets. A comparative measurement of dissolution for disintegrated tablets with that for granules provides a useful method for elucidating the particle bonding or cleavage within the tablet during compression.


Effect of drug content and drug particle size on the change in particle size during tablet compression. Three size fractions for each of three poorly soluble drugs were compressed into 10 mm diameter tablets of four different dilution ratios. The compression was carried out on a physical testing instrument at four compression levels of 49.0, 98.1, 196.2 and 294.3 MN m-2. The effect of drug content and drug particle size on the change in particle size during tableting was examined by the determination of the dissolution rate for disintegrated tablets. A linear relation was obtained when plotting 1n(T80%) versus drug content. There was a critical particle size where the phenomena of cleavage and bonding during tableting balanced each other, but this varied with drug content.


Catalytic tritiation of drugs and analysis of the tritium distribution by 3H n.m.r. spectroscopy. Propranolol, phenobartitone, diphenylhydantoin, amphetamine, imipramine and propoxyphene have been tritiated using tritiated water and platinum catalyst (from the dioxide and sodium borohydride), and the pattern of labelling has been ascertained by 3H n.m.r. spectroscopy. The results show that this exchange procedure can lead to satisfactory incorporation of tritium at 'stable' aromatic positions. For imipramine where incoroporation of tritium was low, an alternative acid-catalysed procedure was satisfactory.


A high performance liquid chromatographic assay of cis- and trans- isomers of tricyclic neuroleptic drugs. Tricyclic neuroleptics based upon the thioxanthene nucleus exhibit geometrical isomerism and the major pharmacological activity resides in the Z-component. An h.p.l.c. procedure which enables the separation detection and quantification of these isomers is described. The method is applicable to the analysis of flupenthixol, clopenthixol, chlorprothixene, doxepin and dothiepin. Measurement of the isomer-ratios in various samples of flupenthixol has shown that small batch to batch variations are apparent. The determination of the isomer-ratio in formulations has been shown to rely upon the complete extraction of the medicament. This is due to the differential release of the components from the tablet matrix with the cis-isomer being favoured. There is little difference observed between the adsorption isotherms of the two components (onto charcoal) but in competition experiments differential adsorption may be demonstrated. This has clear implications for the pharmacokinetics of these drugs.


Biological effectiveness, in goldfish, of some p-substituted acetanilides alone and in the presence of poloxamers. The death times of goldfish have been measured in aqueous solutions containing different concentrations of p-substituted acetanilides alone or in the presence of poloxamers. Plots of reciprocal death time versus concentration were linear with a positive concentration intercept, the minimum effective concentration (MEC). The MEC values were directly related to the pi value (hydrophobic-lipophilic constant) of the functional group on the acetanilide indicating that activity is directly related to lipophilicity. Slopes of reciprocal death time versus drug concentration were linearly related to pi values but there was no direct dependence. The presence of poloxamers in aqueous acetanilide solutions reduced the goldfish death time. The effect of the poloxamers is believed to be one of rendering the goldfish membrane more permeable to drugs.


Elimination of prostacyclin (PGI2) and 6-oxo-PGF1 alpha in anaesthetized dogs. The plasma concentration of 6-oxo-PGF1 alpha was measured by radioimmunoassay after constant rate infusion of 6-oxo-PGF1 alpha or prostacyclin (PGI2) into anaesthetized dogs. A steady-state plasma concentration was rapidly attained with both compounds. After termination of the infusions, the concentration of 6-oxo-PGF1 alpha declined according to a bi-exponential process. The steady-state plasma concentrations of 6-oxo-PGF1 alpha obtained after infusion of 6-oxo-PGF1 alpha and PGI2 were approximately 10 times higher than the corresponding steady-state level of PGF2 alpha measured after infusion of PGF2 alph into the same dogs. The data presented suggest that PGI2 and 6-oxo-PGF1 alpha are eliminated more slowly than PGF2 alpha, probably because they are not taken up and metabolized in the lungs as rapidly as PGF2 alpha.


Effect of taurine on release of 3H-GABA by depolarizing stimuli from superfused slices of rat brain cerebral cortex in vitro. The effect of taurine on potassium (30 mM), veratrine (10 micrograms ml-1) and ouabain (100 microM) induced release of 3H-GABA from superfused slices of rat brain cerebral cortex had been studied. Taurine (10--100 mM) potentiated K+-induced release of 3H-GABA but was more potent in potentiating veratrine- and ouabain-induced release, having significant effects at 5 mM and 1 mM respectively. The potentiation of stimulus induced release does not appear to be due to inhibition of reuptake and the effect is consistent with both a 'modulatory' role and anticonvulsant action for taurine.


Effect of ethanol on adrenaline-stimulated glucose uptake in rat white adipose tissue. The effect of ethanol on adrenaline-stimulated glucose uptake by rat white adipose tissue has been examined in vitro. Ethanol (3%) inhibited the stimulatory effect of adrenaline on glucose uptake whereas it failed to inhibit the effect of adrenaline on free fatty acid production. Addition of calcium (12.5 mM) to the incubation medium restored adrenaline's effect on glucose uptake. Addition of propranolol also restored the effect of adrenaline inhibited by ethanol. Ethanol did not inhibit insulin-stimulated glucose uptake. These results suggest that ethanol modifies the coupling of the adrenoceptor to the glucose transport system in adipose tissue that is stimulated by adrenaline.


The disposition of [14C]metronidazole in rats following vaginal and oral administration. The absorption, tissue distribution and excretion of [17C]metronidazole (14C-MTZ) were compared during the first 4 h after administration of 10 mg kg-1 of 14C-MTZ either orally or intravaginally (i.v.g.) to rats. Peak 14C blood concentrations were reached at 1 h in both groups. Blood samples collected at 0.5, 3 and 4 h had a higher 14C concentration in orally dosed rats (P less than 0.05) than in i.v.g.-treated animals. About 3% of the i.v.g. applied dose remained in the vagina at 4 h. After 4 h, the plasma, liver, kidney, brain, lung and uterus concentrations of 14C were similar in both groups, whereas the blood, skeletal muscle and fat 14C values were significantly greater in the orally dosed rats. The total recoveries of 14C in the urine and faeces did not differ (ca 38% over 4 h) between the two groups. These results suggest that the kinetics of metronidazole are similar after the administration of equal amounts of this drug by either route.


Beta adrenoceptor blocking properties of MK-761. MK-761 is a new class of compound which has beta adrenoceptor antagonist and vasodilating properties in a single molecule. The compound has in vitro beta adrenoceptor blocking activity in the isolated cat heart papillary muscle and isolated rat atria. Unlike propranolol, it did not depress contactile force. Intrinsic sympathomimetic activity was not observed in vitro, (isolated cat papillary muscle, isolated atria) but the compound has some intrinsic sympathomimetric activity because it slightly increased heart rate in reserpinized rats. MK-761 was found to be approximately as potent as timolol and pindolol in blocking cardiac and vascular beta adrenergic receptors in anesthetized dogs. The drug was not, however, cardioselective. Oral beta adrenoceptor blocking activity was observed in rats at doses similar to those which decreased blood pressure.


Measurement of distortions in fixed partial dentures resulting from degassing. 1. During the degassing phase, fixed partial dentures removed immediately when the temperature reached 1,900 degrees F (1,038 degrees C) with soldered connectors do not distort more than those with cast connectors. While not tested, heat-soaking could be expected to cause a greater amount of distortion, especially for fixed and supported partial dentures having soldered connectors. 2. Use of custom-made sagger trays giving support to long-span fixed partial dentures at the ends and within the span is desirable for both degassing and porcelain firing, regardless of whether or not the connectors are cast or soldered. 3. The accumulative amount of possible distortion resulting from soldering, degassing, and porcelain firing may be clinically significant if precautions are not affected.


The accuracy of soldering investment.

The accuracy of soldering investment. Testing soldering investments of different thermal expansions established that an acceptable dimensional change in a three-unit fixed partial denture occurs with an investment which expands at 0.70% to 0.72%.


Acid and neutral hydrolases in Trypanosoma cruzi. Characterization and assay. Twelve acid hydrolases, 4 near-neutral hydrolases, and alkaline phosphatase were demonstrated in 0.34 M sucrose homogenates of Trypanosoma cruzi strain Y: p-nitrophenylphosphatase and alpha-naphthylphosphatase, with optimum pH at approximately 6.0; alpha=ga;actpsodase. beta=ga;actpsodase. beta=g;icpsodase, N-acetyl-beta-glucosaminidase, cathepsin A and peptidase I and III, with optimum pH between 5.0 and 6.0; and arylsulfatase, cathepsin D, alpha-arabinase and alpha-mannosidase with optimum pH at approximately 4.0. alpha-Glucosidase, glucose-6-phosphatase and peptidase II had optimum pH at approximately 7.0. beta-Glycerophosphatase had a broad pH-activity curve from 4,0 to 7.4, with maximum activity at pH 7.0. The main kinetic characteristics of these enzymes and their quantitative assay methods were studied. No activity was detected for alpha-fucosidase, beta-xylosidase, beta-glucuronidase, elaidate esterase, acid lipase, and alkaline phosphodiesterase.


[Arteriography in polyarteritis nodosa. Diagnostic findings and value of repeated examinations (author's transl)]. The results of 18 abdominal arteriographies (general aortography, selective renal, superior mesenteric, and coeliac arteries) in 11 patients with polyarteritis nodosa (P.A.S.), are reported and analyzed. In all tissues, the frequency of the lesions was 66% for micro-anéurisms, 100% for stenoses, and 83% for thromboses. These are seen mainly in the kidney, but isolated lesions may occur in the liver and gastro-intestinal tract. As they vary with time and the area affected, selective contrast studies of the main stems of the abdominal aorta and, in some cases, repeated arteriographic examinations, are necessary.


Nicotinamide adenine dinucleotide phosphate-decanaldehyde adduct as an inhibitor of beef brain NADP-linked aldehyde reductase. The synthesis and characterization of the addition product of NADP and decanaldehyde are described. This adduct is an inhibitor of beef brain NADP-linked aldehyde reductase (Ki = 2.3 x 10(-6) M) along with the NADP adducts of butyraldehyde, phenylpropionaldehyde, and phenylacetaldehyde.


Conformational analogues of dopamine. Synthesis and pharmacological activity of (E)- and (Z)-2-(3,4-dihydroxyphenyl)cyclopropylamine hydrochlorides. (E)- and (Z)-(+/-)-2-(3,4-dihydroxyphenyl)cyclopropylamine hydrochlorides were synthesized as part of a program to assess the importance of conformational isomerism with respect to the various peripheral biological actions of dopamine. Although neither of the compounds possessed dopaminergic activity in the canine renal blood-flow model, both agents were weak alpha-adrenergic agonists and exhibited cardiostimulatory properties similar to dopamine. The E isomer was apprxoximately 5 times more potent than the Z isomer in its alpha-adrenergic activity and approximately 15 times as potent in its cardiac effects. Possible reasons for the lack of renal dopaminergic activity exhibited by the E isomer are presented.


Tripeptide analogues of melanocyte-stimulating hormone release-inhibiting hormone (Pro-Leu-Gly-NH2) as inhibitors of oxotremorine-induced tremor. Fourteen di- and tripeptide analogues of MIF, Pro-Leu-Gly-NH2, have been synthesized and assayed for inhibition of oxotremorine-induced tremor. Replacement of Pro by HCO-Pro or cyclopentanecarboxylic acid gave inactive analogues, while some peptides of the general structure less than Glu-Leu-Gly-NR1R2 were highly active. Thus, R1 = C3H8 and R2 = H gave 4 times the activity of MIF, R1 = I-C3H8 and R2 = H gave 13 times the activity of MIF, and R1 = R2 = CH3 gave 29 times the activity of MIF. cyclo(-Pro-Leu-), Pro-Lys-Gly-NH2, and Pro-Arg-Gly-NH2 had no activity. Apparently, small modifications in the structure of MIF can yield highly active analogues with potential clinical value, e.g., in the treatment of Parkinson's disease or mental depression.


Absolute configuration of glycerol derivatives. 7. Enantiomers of 2-[[[2-(2,6-dimethoxyphenoxy)ethyl]amino]methyl]-1,4-benzodioxane (WB-4101), a potent competitive alpha-adrenergic antagonist. The enantiomers of 2-[[[2-(2,6-dimethoxyphenoxy)ethyl]amino]methyl]-1,4-benzodioxane (4) were prepared from the chiral 2-[(tosyloxy)methyl]-1,4-benzodioxanes [(2S)- and (2R)-5]. The corresponding (2R)- and (2S)-2-(aminoethyl)-1,4-benzodioxanes [2R)- and (2S)-7] were prepared by a modified Gabriel synthesis and converted to the enantiomers of 4 by condensation with 2,6-dimethoxyphenoxyacetaldehyde (8) and reduction of the intermediate imine with NaBH4. The enantiomer (2S)-4 was 40--50 times as potent as the enantiomer (2R)-4 in antagonizing the alpha-adrenergic response of methoxamine-induced contraction of rabbit aortic strips, showing a pA2 = 9.0. This result is consistent with the previous observation that S enantiomers of 2-[(alkylamino)methyl]benzodioxanes are more potent antagonists at a alpha-adrenergic receptors than the R enantiomers.


A cooperative education program for nurse practitioners/physician's assistants. Traditionally, nurse practitioners and physicians' assistants have been trained separately. Despite similarities in curriculum and graduate practice, there has been little or no articulation between their training programs or professional organizations. In 1977 the Family Nurse Practitioner Program at the School of Medicine, University of California, Davis, and the Primary Care Associate Program at Stanford University Medical Center merged clinical curricula. In this article the authors describe the cooperative educational venture and evaluate its first year.


Evidence for a role of phosphatidyl ethanolamine as a modulator of membrane-membrane contact. Phosphatidyl ethanolamine (PE) is shown to be effective in producing membrane aggregation. The aggregation of PE and PE/PC (phosphatidyl choline) mixed vesicles was studied as a function of pH and cation composition of the medium. The kinetics and equilibria were studied in stopped-flow rapid mixing experiments, in which PE vesicles prepared at pH 9.2 were "jumped" to pH 7. H+ ions protonate PE- and promote vesicle aggregation in a cooperative fashion. Vesicles containing PC have a decreased tendency to aggregate compared to pure PE vesicles. The apparent rate constant for aggregation was about two orders of magnitude below that for diffusion controlled aggregation and was virtually the same for PE and PE/PC mixed vesicles. A theoretical description of equilibrium for vesicle aggregation is developed in terms of three parameters: the equilibrium constant for the protonation of PE (KA), the equilibrium constant for aggregation (Keq) and the number of PE molecules in an effective area that the two vesicles must interact in order to aggregate (Neff). These parameters are compared with values and trends expected for electrostatic calculations based on dipolar repulsion and short-range binding, to which hydrogen bonding may contribute. The results are interpreted in a self-consistent fashion to indicate: (i) that PE and PC mix randomly, (ii) that head-to-tail binding occurs between PE(PC) molecules on apposing vesicles, (iii) that electrostatic screening accounts for the decrease in KA as a function of the molar fraction of PC per vesicle, (iv) that the PE must be 90% protonated before aggregation can occur, and (v) that for all the lipid systems we considered, the point at which the extent of dimerization is half maximal is close to the physiological pH, indicating that PE may have a regulatory effect in the aggregation of biological systems.


On two-dimensional passive random walk in lipid bilayers and fluid pathways in biomembranes. The lateral mobility of pyrene,  pyrene decanoic acid, and 1-palmitoyl-2-pyrene decanoyl-phosphatidyl choline (pyrene lecithin) in lipid bilayers is determined by the excimer formation technique. This method is applied to vesicles of lecithins differing in chain length and in the degree of saturation of the hydrocarbon chains. These values are compared with results in cephalins of different chain length and in dipalmitoyl phosphatidic acid at variable pH. The influence of cholesterol is investigated. The results are analyzed in terms of the Montroll model of two-dimensional random walk. The jump frequency of the probe molecule within the lipid lattice is obtained. The advantage of this measure of transport in lipid layers is that it does not involve lipid lattice parameters. The main results of the present work are: (i) The lateral mobility of a given solute molecule in lamellae of saturated lecithins is independent of hydrocarbon chain length and rather a universal function of temperature. (ii) In unsaturated dioleyl lecithin the amphiphatic molecules have lateral mobilities of the same size as in saturated lipids. The jump frequency of pyrene, however, is by a factor of two larger in the unsaturated lecithin. (iii) The jump frequencies in phosphatidyl ethanolamines are about equal to those in lecithins. (iv) In phosphatidic acid layers the hopping frequencies depend on the charges of the head groups of both the lipids and the probes. (v) Cholesterol strongly reduces the jump frequency in fluid layers. (vi) The lateral mobility in biological membranes is comparable to that in artificial lipid bilayers. The experimental results are discussed in terms of the free volume model of diffusion in fluids. Good agreement with the predictions made from this model is found. A striking result is the observation of a tilt in dioleyl-lecithin bilayer membranes from the hopping frequencies of pyrene and pyrene lecithin. A tilt angle of phi = 17 degrees is estimated.


Effect of pH on the calcium metabolism of isolated rat kidney cells. The effects of metabolic and respiratory acidosis and alkalosis on cellular calcium metabolism were studied in rat kidney cells dispersed with collagenase. In both types of acidosis, the intracellular pH, total cell calcium, and the cell relative radioactivity after 60 min of labeling are significantly depressed. Kinetic analysis of 45-ca desaturation curves shows that acidosis decreases all three cellular calcium pools and depresses calcium fluxes between the superficial and cytosolic pools and between the cytosolic and mitochondrial pools. In alkalosis the intracellular pH, the total cell calcium, and the cell relative radioactivity are significantly increased. Kinetic studies show that in alkalosis, only the mitochondrial pool is consistently increased. Calcium exchange between the mitochondrial and cytosolic pool is increased in metabolic alkalosis only. These results suggest that hydrogen ion is an important modulator of calcium metabolism, and that the intracellular pH rather than extracellular pH is the critical factor in determining the calcium status of cells during altered acid-base conditions.


Behavioral treatment parameters with primary dysmenorrhea. Fourteen women with primary dysmenorrhea were administered four sessions of systematic desensitization (SD) by either a male or a female therapist. The following measures were taken during the flow periods before and after treatment and at a 6-month follow-up: menstrual symptom checklist, medication usage, invalid hours, and menstrual attitudes. At pretreatment, menstrually distressed women had significantly higher scores on all measures compared to a normative group and an explicitly nondistressed group. At posttreatment, treated women's scores on the dependent variables were significantly reduced. All indices were reduced to a "nondistressed level" at posttreatment and at 6-month follow-up. Type of dysmenorrhea (congestive vs. spasmodic), trait anxiety level, and therepist sex did not predict differential responsiveness to SD. SD did not affect frontailis EMG, peripheral blood flow, or pain threshold. A Retrospective Symptom Scale of menstrual distress was found to be highly reliable, valid, and sensitive.


Setting generality of blood pressure reductions and the psychological treatment of reactive hypertension. The setting generality of treatment effects was examined for a client with 3 years' history of essential hypertension. Self-control progressive relaxation training led to durable normotensive blood pressures (BP) in the natural environment and psychology clinic, although medical setting BP remained elevated. Further assessment indicated that this latter problem was a function of conditioned anxiety to medical setting stimuli. Systematic desensitization led to reductions in medical setting BP to normotensive levels, and laboratory psychophysiological assessments confirmed the elimination of the anxiety response. The present case study raises the issue of to what extent such reactive hypertension is a problem for other individuals similarly diagnosed and demonstrates the importance of the assessment of generality of treatment effects for essential hypertension.


Inhibition of glucose-6-phosphate dehydrogenase activity by scorpion Leiurus quinquestriatus (H and E) venom. Crystalline glucose-6-phosphate dehydrogenase was significantly inhibited by L-quinquestriatus venom. Neither boiling nor dialysis could abolish this inhibition completely. Raising the concentration of either G6P or NADP in the reaction mixture reversed the inhibition, indicating that the latter was competitive with respect to the substrate and the coenzyme.


Acute febrile systemic reaction to polyvalent pneumococcal vaccine. An acute, severe febrile reaction with leukocytosis followed vaccination with polyvalent pneumococcal polysaccharide vaccine in a splenectomized patient with myeloproliferative disease. Recovery required five days.


Polymicrobial bacteremia.

Polymicrobial bacteremia. Of 26,961 blood cultures taken during an 18-month period at the Cincinnati General Hospital, 1,715 (6%) were positive. Ninety-four patients had blood cultures containing more than one organism. Although aerobic and anaerobic streptococci were the most frequently isolated bacteria, a variety of microorganisms, including Staphylococcus aureus and the Klebsiella-Enterobacter-Serratia group, was isolated in different combinations depending on the underlying disease. Neurological illness, malignant neoplasms, burns, and decubitus ulcers were among the most common underlying conditions found. The overall mortality was 54%, but only 58% of these deaths were specifically related to an episode of polymicrobial bacteremia. Patient survival was significantly related to appropriate antimicrobial therapy.


Intracellular pH measurement in frog muscle by means of 31P-nuclear magnetic resonance. The 31P-NMR technique was used for the monitoring of intracellular pH and studying its heterogeneity in the femoral biceps muscle of Rana catesbiana under anaerobic conditions. The value of intracellular pH of fresh muscle calculated from the chemical shift of intracellular inorganic phosphate (P1) was 7.3 on average and the line width of P1 was about 0.2 ppm. As the line width determined by the relaxation mechanism was 0.099 ppm, the P1 signal in fresh muscle was concluded to consist of overlapped narrow components, which indicated the heterogeneity of muscular pH (about 0.2 pH unit). Living muscle showed gradual acidification due to glycolysis and the decrease in heterogeneity. When glycolysis was suppressed by iodoacetic acid, slight alkalization due to the breakdown of creatine phosphate was observed. When the Lohmann reaction was suppressed by 2, 4-dinitro-1-fluorobenzene, rapid acidification accompanied by the appearance of a new acidic component was observed with the onset of ATP decrease. This new component was not detected in the muscle pretreated with glycerol to disrupt the transverse tubules. Therefore, it is likely that this new acidic component originates in the intracellular compartment, and not in the cellular difference.


[Stimulating effects of alanine and glycine on guinea-pig taenia coli (author's transl)]. Effects of alanine and glycine on the mechanical and electrical activities of guinea-pig taenia coli were investigated. Alanine (0.1--10 mM) and glycine (0.5--10 mM) produced a dose dependent contraction of guinea-pig taenia coli. The stimulating effects of alanine and glycine were not inhibited in the presence of atropine (5 muM), tetrodotoxin (0.1 micrograms/ml), diphenhydramine (4 muM), methysergide (3 muM), strychnine (10 muM), and were not influenced by treatment with indomethacin (3 muM). However, these effects were inhibited in the presence of a Ca antagonist, verapamil (10 muM). When the electrical activities of the taenia coli were recorded by the single sucrose-gap method, alanine and glycine (5--10 mM) produced a reduction of membrane potential and increased spike heights and frequencies of action potential. In LiCl or Choline-Cl and in Na-isethionate solutions, the stimulating effect of alanine was not abolished, but was completely inhibited in KCl-depolarized preparation. From these results, it is considered that both alanine and glycine may directly produce contraction by a depolarization of the cell membrane of guinea-pig taenia coli.


[Effect of blockaders of cardiac beta-adrenergic structures on the central hemodynamics and contractile function of the myocardium in acute experimental coronary insufficiency]. The indices of central hemodynamics and myocardial contractile function were studied in 35 dogs before and in different periods after the administration of drugs which block beta-receptors: propranolol, pindolol, and talinolol. The drugs blocking the beta-adrenergic receptors were administered against the background of an intact myocardium to 15 dogs and against the background of acute coronary insufficiency to another 15; acute ischemia was induced in 5 dogs to which the drugs were not given. It was established that beta-adrenergic blocking agents have a beneficial effect in the acute stage of myocardial ischemia; they exert a marked influence on the consumption of oxygen by the myocardium, intramyocardial tension, and the contractile capacity and rhythm of the heart. Talinolol produced the most favourable effect.


[pH changes and the K+ and Na+ concentration in the blood of the coronary vein in experimental myocardial infarct complicated and not complicated by ventricular fibrillation]. It was found that following occlusion of the coronary artery in dogs, the rate of increase in K+ concentration in blood plasma draining directly from the focus of ischemia is greater in cases complicated by ventricular fibrillation. Fibrillation always occurs against the background of a decrease in pH and an increase in the K+ level in blood plasma draining from the focus of ischemia. It is suggested that inhibition of the development of disorders of acid-base and ion equilibrium in the myocardium would be an effective means of preventing ventricular fibrillation in the acute stage of myocardial infarction.


[High dose betablocker treatment in experimental renal hypertension (author's transl)]. Rats with experimental renovascular hypertension were treated with high doses of beta-blocking agents. Pindolol 10 mg/kg per day increased Goldblatt-type hypertension, whereas Propranolol 100 mg/kg per day showed an antihypertensive effect. A linear correlation between the weight of the left-heart ventricle and the systolic blood pressure was found in animals treated with Pindolol as well as in untreated Goldblatt rats. On the contrary, the weights of the left-heart ventricle were significantly higher in those animals treated with Propranolol than in the other groups. The results show that Pindolol in high doses, possibly on account of its sympathomimetic activity, leads to an increase in Goldblatt hypertension, but does not influence the pressure-dependent progression of left-heart hypertrophy. On the other hand, Propranolol, possibly on account of a cardiodepressory effect, leads to a decrease in blood pressure.


[Chufa (Cyperus esculentus) as a source of vegetable fats in a sealed life-support system]. The cultivation schemes, productivity, total biochemical, lipid in particular, composition of nodules of chufa (Cyperus esculentus) were studied, using a phytotron. Upon continuous illumination chufa yielded a high total productivity and a satisfactory coefficient of economic effectiveness (not less than 50%). Chufa nodules have an optimal ratio of proteins, carbohydrates and fats containing essential fatty acids. To meet man's requirements for vegetable oils and essential fatty acids, it is necessary to produce daily 150--200 g dry chufa nodules which are quite acceptable as a dietary ingredient.


[Cardia function after proximal-gastric vagotomy (author's transl)]. In 36 patients with chronic duodenal ulcer disease LES-function before and after proximal-gastric vagotomy was examined manometrically and by determining pH-values in a prospective study. In addition, esophagus biopsies were taken 5 cm proximal of the cardia. 12 healthy volunteers served as controls. A direct effect of proximal-gastric vagotomy on LES-function could not be shown. On the contrary, in 38,9% of the patients examined, preoperatively proven esophagitis disappears nearly completely 12 months following proximal gastric vagotomy. This positive effect of vagotomy is to be interpreted as a beneficial effect on regurgitated intestinal juice, more so than an effect on cardia function itself.


[Influence of pyloroplasty and pyloric stenosis on motoric and secretory function of the stomach after selective proximal vagotomy--an experimental study (author's transl)]. In conscious fullgrown minipigs simple SPV alone, SPV and pyloric stenosis and SPV and pyloroplasty were performed. After a liquid test meal the motoric and secretory function of the stomach were examined simultaneously by a modified method of intestinal perfusion and aspiration. After simple SPV initially a marked decrease of gastric volume and normal emptying into the duodenum were found. With additional pyloric stenosis no significant change was found. The pyloroplasty lead to an increase of gastric volume and delayed emptying. The acid secretion after feeding reduced by SPV was not changed significantly neither by pyloroplasty nor by pyloric stenosis. The baseline values of serum gastrin were elevated after SPV as well as after SPV in combination with pyloric stenosis or pyloroplasty. After food stimulation there was a delayed increase of gastrin after SPV which differed from that after SPV with pyloric stenosis or pyloroplasty only during the first hour. These results show that after SPV no further improvement of the motoric and secretory function can be achieved by an additional pyloroplasty. Furthermore these findings permit the conclusion that even after SPV with additional artificial pyloric stenosis no delayed gastric emptying occurs and that there is no negative effect postoperatively on the acid secretion and gastrin production.


Control of pulmonary ventilation during exercise and mediators in the blood: CO2 and hydrogen ion. In reviewing the blood borne "mediators" to ventilation during exercise, our emphasis is on VCO2 and [H+]. We consider that exercise ventilation is more closely linked to VCO2 than to VO2. Lactic acid has a dual effect on ventilation, as it results in an increased CO2 output as well as increased [H+], an independent ventilatory stimulus. Examples from acid-base disturbances hypoxia and training are used to illustrate the relationship of VE to VCO2 and [H+].


Peripheral chemoreceptors and exercise hyperpnea. The carotid bodies appear to be the only peripheral chemoreceptors mediating ventilatory control during exercise in man. While little is known about the mechanism of stimulation, much is known about the effects of carotid body stimulation upon pulmonary ventilation (VE). These effects have been produced by hypercapnia, hypoxia, metabolic acidosis, arterial blood pressure, temperature, and catecholamines. A signal from CO2 flow is attractive because of the strong correlation between CO2 output and VE during exercise. The carotid body's role in the hyperpnea depends on the intensity of exercise. During heavy exercise, pH falls and hyperventilation ensues. The carotid bodies appear to be the exclusive mediators of the ventilatory compensation for the acidosis at this exercise intensity. For moderate exercise, mean arterial PCO2 does not change. Therefore, how is the CO2 signal transmitted to the respiratory center? Two current theories are: (1) since arterial PCO2 and pH oscillate with each breath, the amplitude and period of these oscillations may change during exercise and may be of sufficient magnitude to stimulate the carotid bodies, and (2) there exists a disequilibrium between hydrogen ion activity within the red blood cell and in the plasma because carbonic anhydrase is found in the former but not the latter. This theory assumes that the enzyme is not accessible to the plasma.


The brain's role in exercise hyperpnea.

The brain's role in exercise hyperpnea. Three aspects concerning the role of the central nervous system in the control of exercise hyperpnea are reviewed. First, the integration of sensory input stresses the concept of multiple sites of integration--with the end result that both adequate gas exchange and near-optimal mechanical response of the lung and chest wall are achieved during exercise. Secondly, the potential role of the "central" [H+] chemoreceptor is discussed--in terms of the mechanisms available for the protection of brain ECF [H+] and the stimulus-response characteristics of this important chemical sensor and a critical analysis of how it may be evaluated. Finally, the question of forebrain influences on exercise hyperpnea and the "sensation" of ventilatory effect is discussed, with particular emphasis on the multi-purpose regulation of breathing in athletic endeavors.


Degloved sole and heel.

Degloved sole and heel. A case of a degloving injury of the sole and heel is reported. A major problem with this rather unusual injury is resurfacing the defect, as it is a weight-bearing area. The management of the injury with a microvascular free flap is presented and discussed.


Pulmonary ascariasis.

Pulmonary ascariasis. A case of pulmonary ascariasis is reported for the first time in Australia. Because of increasing immigration from countries which have a high incidence of ascariasis (especially those of South-East Asia), and increasing travel to Asian countries, the awareness of this infestation as a cause of respiratory disease may be of great importance.


[Bone marrow insufficiency and hemangioendothelioma of the liver after thorotrast application: Late effects of thorotrast (thorium dioxide) in man (author's transl)]. The report details with the late effects following administration of thorotrast in two patients: multicentre metastating hemangioendothelioma of the liver respective bone marrow insufficiency developed after application of thorotrast 34 respective 36 years ago. The clinical and autoptical findings are described and attention is drawn to the relevance of this disease at present time.


Purification and properties of cAMP independent glycogen synthase kinase and phosvitin kinase from human leukocytes. cAMP independent glycogen synthase kinase and phosvitin kinase activity was purified from the 180 000 x g supernatant of human polymorphonuclear leukocytes by ammonium sulphate precipitation and phosphocellulose chromatography. The cAMP independent glycogen synthase kinase eluted from the phosphocellulose at 0.54 M NaCl (peak A) separate from the major phosvitin kinase eluting at 0.68 M NaCl (peak B). The kinase activity of both peaks tended to form aggregates, but in the presence of 0.6 M NaCl, the peak B enzyme had Mr 250 000, 7.2S and the peak A enzyme Mr 38 000, 3.8S. The ratio between synthase kinase and phosvitin kinase activity in peak A was 1:3.2 and in peak B 1:31.4. In addition the kinase activities differed with respect to sensitivity to temperature, ionic strength and CaCl2. It is suggested that the peak A enzyme represents the cAMP independent glycogen synthase kinase of leukocytes, whereas the peak B enzyme is a phosvitin kinase, which is insignificantly contaminated with some synthase kinase (peak A) and contains a separate, second synthase kinase. Synthase kinase had Kmapp 4.2 microM for muscle glycogen synthease I and Kmapp 45 microM for ATP. GTP was a poor substrate. The activity was not influenced by cyclic nucleotides, Ca2+, or glucose-6-P. Synthase I from muscle and leukocytes was phosphorylated to a ratio of independence of less than 0.05.


The temperature and pH-dependent transition of hen lysozyme. Characterization of two temperature-defined domains and of an N-acetylglucosamine (inhibitor)-insensitive form. The previously described temperature and pH-dependent transition in the solid state of hen lysozyme was studied in solution. Experiment concerning the velocity of lysis of M. luteus by lysozyme and its behavior in presence of an inhibitor (GlcNAc) as well as a reinvestigation of the Arrhenius curves over a large range of pH, demonstrated the existence of two temperature-induced domains. An inhibitor-insensitive lysozyme form was characterized at 40 degrees (physiological temperature).


Glucocorticoid induction of tyrosine aminotransferase in cultured cells. For over a decade, tyrosine aminotransferase induction in tissue culture cells has been a useful model system in which to study glucocorticosteroid action. In the 1960s, the establishment in culture of rat hepatomas expressing the inducible enzyme, already known to be induced in liver in vivo, provoked a wide-ranging series of experiments. The data from these experiments have provided considerable information regarding the mechanism of action of steroids. These include the fundamental facts that the steroids act directly on the induced cell in unmetablized form, that removal of steroid results in deinduction, that induction does not require DNA synthesis or massive changes in RNA synthesis, and that cytoplasmic receptor occupancy by active steroids correlates closely with the steroids' ability to affect inductions. Studies in tissue culture cells have led to the analysis of transcriptional and posttranscriptional models attempting to explain enzyme induction. The effects on enzyme induction of nonsteroid hormones and other factors have been studied through the use of tissue culture cells. Finally, cells and clones of cell variants are being used to study enzyme induction, through biochemical analysis and cell genetic approaches, including somatic cell hybridization.


Multihormonal control of tyrosine aminotransferase in isolated liver cells. The regulation of tyrosine aminotransferase activity by glucocorticoids and cyclic AMP was investigated in isolated liver parenchymal cell suspensions. The induction and maintenance of elevated levels of tyrosine aminotransferase activity in liver cells were completely dependent upon the presence of both the synthetic glucocorticoid, dexamethasone, and glucagon of dibutyryl cyclic AMP. No induction was observed when any of these compounds were tested alone. Immunotitration experiments revealed that the 6- to 7-fold increase in tyrosine aminotransferase activity following the addition of dexamethasone and glucagon was accompanied by a parallel increase in the amount of immunologically reactive enzyme protein. Pulse-labeling experiments established that this increase in enzyme protein could be fully accounted for by a corresponding increase in rate of synthesis of tyrosine aminotransferase. Neither hormone had any effect on the rate of degradation of the enzyme. The increase in tyrosine aminotransferase synthesis evoked by the presence of both hormones was blocked by the addition of actinomycin D or cycloheximide to the medium, demonstrating that RNA and protein synthesis were required for the induction of enzyme activity. By varying the time and order of addition of the inducers and inhibitions, evidence was obtained that the hormones act sequentially. The steroid hormone acts first, presumably to increase the level of functional tyrosine aminotransferase mRNA or its precursor. The processing of this precursor to a translatable form or the specific translation of tyrosine aminotransferase mRNA is apparently dependent upon a specific cyclic AMP-controlled process. In vivo experiments demonstrated that both glucocorticoids and cyclic AMP increase the level of functional tyrosine aminotransferase mRNA in the liver. The actions of the steroid hormone and cyclic nucleotide were blocked by alpha amanitin, establishing the requirement for ongoing gene transcription. The protein synthesis inhibitors, cycloheximide, emetine, and puromycin, were as effective as cyclic AMP in increasing tyrosine aminotransferase mRNA activity. The action of these inhibitors is probably related to their ability to elevate hepatic intracellular cyclic AMP levels, thus mimicking cyclic AMP administration. Extension of these in vivo studies to isolated liver cells will provide a valuable system for investigating the regulation of gene expression by glucocorticoids and cyclic AMP.


Posttranscriptional regulation of glucocorticoid-regulated functions. Relying heavily on studies of TAT regulation in cultured rat hepatoma cell lines, we have attempted in this brief review to discuss possible mechanisms for posttranscriptional regulation of glucocorticoid-sensitive enzymes and to chronicle the evidence for and against posttranscriptional mechanisms for specific enzyme induction by glucocorticoids. Initially, mechanisms were considered that would reconcile results showing sensitivity of both induction and deinduction of TAT to inhibitors of RNA synthesis with studies demonstrating first that glucocorticoids regulate the rates of specific enzyme synthesis and, then, that glucocorticoids regulate levels of enzyme-specific mRNA. Such reconciliation proved unnecessary when it was demonstrated that inhibitors of RNA synthesis such as actinomycin D were not specific for RNA synthesis, but also had effects on mRNA turnover and protein metabolism. The bulk of evidence to date establishes that glucocorticoids promote the production of enzyme-specific mRNA for the proteins whose synthesis is regulated by thses steroids. Nevertheless, there is still very little direct evidence that steroids can modulate rates of specific gene transcription. The glucocorticoid stimulation of mouse mammary tumor virus RNA production in cultured cell lines is the only example to date where such a mechanism is supported by RNA-DNA hybridization studies. Posttranscriptional actions of steroids on the turnover, processing, or extranuclear transport of specific mRNA precursors remain potential steps at which glucocorticoids might function. The rapid turnover of some glucocorticoid-regulated enzymes and their mRNAs not only ensures a rapid response to steroid addition or withdrawal, but also subjects these proteins to relatively large fluctuations upon alterations in overall protein or mRNA metabolism. Thus many of the inductions and repressions of hepatic TAT and TO by mediators other than the glucocorticoids may be attributable entirely to nonspecific mechanisms.


Somatic cell fusion in the study of glucocorticoid action. The basic phenomena of cell fusion and hybrid cell formation are briefly described and the potential of somatic cell hybridization in studies on the expression of differentiated cellular functions is discussed. The technique of cell hybridization has been applied to two types of cellular responses to glucocorticoids. The induction of specific proteins has been investigated in hybrids of inducible cells with uninducible cells. Most studies dealt with the liver-specific enzyme tyrosine aminotransferase, whose inducibility was extinguished in the majority of the hybrids between hepatoma and nonliver cells. However, upon chromosome segregation, inducibility reappeared in some of these hybrid cells. The current ideas about cellular control of inducibility are discussed. The other major glucocorticoid-responsive system investigated in cell hybridization studies consists of lymphoid cells which are killed when exposed to the steroid. Such sensitive cells were hybridized with several types of glucocorticoid-resistant lymphoid lines, and sensitivity was found to be dominant over resistence. Hybrids between sensitive and resistant lymphoid cells, however, showed an increase in the frequency at which resistance occurred as compared to the rate observed with the wild-type parental cells. No complementation to steroid sensitivity was found in hybrids between different types of resistant cells with defects in the glucocorticoid-specific receptor system.


Syneristic and antagonistic effects of glucocorticoids on insulin action. HTC cells, an established line of rat hepatoma cells in tissue culture, provide a useful experimental model system for studying the interaction of glucocorticoids and insulin in the regulation of protein metabolism. The actions of insulin and glucocorticoids on amino acid transport and protein degradation are antagonistic in this cell line. In contrast, the actions of these two hormones are additive with regard to the induction of tyrosine aminotransferase. The addition of insulin to HTC cells previously incubated with dexamethasone causes a rapid further doubling in the cellular concentration of this enzyme. The properties of the induction by insulin differ in several respects from the induction by glucocorticoids. The former occurs immediately, without the characteristic lag observed during induction by steroids. Insulin induction of transaminase does not require concomitant RNA synthesis, and does not cause the accumulation of specific mRNA for this enzyme as do glucocorticoids. Using specific immunoprecipitation techniques, we have demonstrated that insulin stimulates a nonselective increase in the rate of total protein synthesis in HTC cells, and a selective decrease in the rate of degradation of tyrosine aminotransferase relative to total protein. Thus the induction of transaminase by insulin involves two distinct actions of the hormone, affecting both synthesis and degradation of protein.


The role of glucocorticoid hormones as biological amplifiers. Recent research in hormone action has been aimed at studying single effects in well-defined systems. As exemplified in several chapters of this book, it has been possible to deduce a general mechanism of action of the glucocorticoids using this approach. Most hormones, and the glucocorticoids in particular, do not act as independent agents in the intact animal. Although the best known example of how glucocorticoids interact with other hormones is the amplification of the effect of those whose action is mediated by cAMP, these steroids also augment the effects of a variety of other hormones and effectors. Such interactions are of interest in clinical medicine as well, since glucocorticoid hormones are used in combination with other drugs in a number of conditions, including the treatment of asthma, allergies, and certain kinds of shock and cancer. Neither the biochemical nor the pharmacologic basis for the effects of the glucocorticoids is known. In some cases the actions of other hormones are not observed unless the tissue has first been exposed to glucocorticoids. In these instances the glucocorticoids are said to exert a "permissive effect," since they allow a process to proceed at a maximal rate even though the steroid itself has no effect on this process. There is no doubt that such examples exist, as documented above: thus the concept of a "permissive effect" does have utility. The term fails to describe the more general role the glucocorticoids play, since in many instances the steroid also has a direct effect on the process itself, or optimizes a process in which the primary effector is not as yet known. Because of these cases, and because the historically more general usage first proposed by INGLE [1] seems to have been forgotten, use of the term "permissive effect" has been avoided in this chapter. An ultimate goal in glucocorticoid hormone research is to identify the mechanisms involved in the amplification effect these hormones exert. Now that the actions of these hormones and of the hormones they interact with are being defined, such work is within the realm of feasibility.


DNA-damaging and mutagenic effects of 1,2-dimethylhydrazine on Bacillus subtilis repair-deficient mutants. Mutagenic, DNA-damaging, and in vivo alteration of DNA have been demonstrated for 1,2-dimethylhydrazine (DMH), a potent inducer of adenocarcinomas of the large intestine and colon of rats. These activities are pH-dependent, with 6.5 giving optimum response. There was no requirement for metabolic activation with rat-liver S9 mix when the appropriate Bacillus subtilis mutant strains were used. The Rec- strains recA8 and mc-1 were greater than 300-fold more sensitive to the DNA-damaging activity of DMH than was their isogenic wild-type parent. The DNA isolated from DMH-treated mc-1 had altered spectroscopic characteristics, and gave a greatly reduced transformation efficiency. Treatment of B. subtilis strain TKJ6321 with DMH at pH 6.5 induced His+, Met+ mutations in substantial numbers at low concentrations of this chemical. The use of B. subtilis mutants in these studies has therefore made it possible to demonstrate mutagenic and DNA-damaging activity in bacteria for this potent carcinogenic chemical.


Fatty acid biosynthesis in Wallerian degeneration of rat sciatic nerve. In-vitro fatty acid biosynthesis was studied in normal rat sciatic nerve and during wallerian degeneration. Normal nerve incorporated 1,3-C14-malonyl-CoA and 1-C14-acetyl-CoA into fatty acids by a de-novo biosynthetic pathway. The reaction product with highest radioactivity was palmitic acid, and the free fatty acids of nerve contained 90% of the total fatty acid label. During wallerian degeneration, there was a rapid increase of fatty acid biosynthesis that reached a peak between 16 and 24 days after nerve section. Values declined to normal levels at approximately 50 days.


[Improvement in the quality of rape seed by combining silage with fodder sugar beets. 1. Quality evaluation of the silages]. Industrial-scale studies showed that the addition of rape-seed flakes to half sugar mangels improves the feeding value and the quality of the silages. Furthermore, it was found that the development of yeasts was inhibited and that the propagation of acidifiers was retarded as the proportion of rape-seed increased. The microbiological analysis indicates that the amount of rape-seed added to the half sugar mangels should not exceed 10%.