Methodological simplifications in radioimmunoassay of urinary aldosterone. Simplification of radioimmunoassay procedures of urinary aldosterone-18-glucuronide was attempted, taking into consideration the aspects implied by the hydrolysis of urine and the assay itself. The procedure standardized for the hydrolysis step (samples diluted with a two-fold volume of 0.2 N HCl and incubated at 30 degrees C for 16-24 h) proved suitable in terms of practicability and accuracy. Aldosterone antisera, raised in the rabbit against an aldosterone-3-bovine albumin conjugate, were selected according to their specificity towards competing steroids. Depending on the characteristics of the antisera used, an assay of extracts, or even direct measurements of hydrolyzed urines excluding any extraction, were found to yield reliable results. In the case of a high-quality antiserum, evidence for the adequacy of assay on non-hydrolyzed urine extracts for the measurement of the excretion of unconjugated aldosterone was provided by some preliminary data. The results of the experiments, directed at the methodological and clinical validation of the simplified procedures, are reported and discussed in this paper.


[Studies on the activities of peptidases from human leucocytes, and human and guinea pig alveolar macrophages (author's transl)]. Peptidases activities were compared in human leucocytes, guinea pig and human alveolar macrophages. Seversl endo- and exopeptides were characterized; some of them were active at acid pH and others at neutral and alkaline pH. Leucocytes and alveolar macrophages had proteolygic activity for hemoglobin, fibrinogen, collagen and elastin. Using synthetic substrates, several enzymes were characterized: arylamidase, aminopeptidase, carboxypeptidases A and B and cathepsins A and C. The enzymatic activities were much higher in alveolar macrophages than in leucocytes.


Experience with a simple method for estrogen receptor assay in breast cancer. A simple procedure for the assay of specific estrogen receptors in breast cancer tissue is described. Estrogen receptors were detected in 74% of primary tumors, 71% of skin metastases and 63% of lymph node metastases. Postmenopausal patients and younger oophorectomized women had estrogen receptor-containing tumors more frequently, and at higher levels, than uncastrated, premenopausal, patients. The stability of estrogen receptors was not affected by the transportation of samples from distant hospitals, providing that they were kept frozen in Tris buffer, pH 8.0, at all times.


Immunological detection of isoferritins in normal human serum and tissue. A method is described which permits the detection of isoferritins in normal human serum and tissues. The technique makes use of 125I-labelled monospecific anti-human-liver-ferritin antibody to demonstrate the isoferritins after isoelectric focussing of the purified ferritin in polyacrylamide gels. The organ-specific variation in tissue isoferritin profile previously reported in normal subjects has been confirmed by this technique using only 50 ng of each ferritin sample. Serum ferritin from normal healthy subjects was also shown to exhibit a microheterogeneity on isoelectric focussing; six clearly defined isoferritin peaks were detected in the pH range of 5.04 to 5.62. This isoferritin profile of normal serum contained isoferritins over the whole range of the various tissue isoferritins suggesting that a number of organs may contribute to the normal serum ferritin pool.


Acceleration of autoimmunity in NZB/NZW F1 mice by graft-versus-host disease. Chronic graft-versus-host (GVH) disease was induced in NZB/NZW F1 (B/W) hybrid female mice by the weekly injection of parental NZB spleen cells. Control mice received injections of syngeneic spleen cells only. The mice were assayed for antibodies to [3H]DNA and [3H]polyadenylic-polyuridylic acid by a cellulose ester filter radioimmunoassay, and for antibody to thymocytes by a cytotoxicity method. GVH disease accelerated the development of all three antibodies in B/W mice. In addition, sucrose density gradient ultracentrifugation of pooled sera suggested that an accelerated switch from 19S to 7S anti-DNA production may be an early effect of GVH. The mechanism of acceleration is discussed in terms of immunological and viral factors generated by the GVH reaction.


Lymphocyte subpopulations. Human red blood cell rosettes. Human red blood cells (HRBC) even without prior neuraminidase treatment, could form rosettes with human peripheral blood lymphocytes in vitro. The optimum conditions for forming these rosettes were a pH of 7-0 and a medium with 5% bovine serum albumin (BSA). Rosette proportions became much less at a different pH or using lower concentrations of BSA, or replacing BSA with foetal calf sera (FCS) or human sera. Rosette formation was also promoted by prior treatment of HRBC or lymphocytes with neuraminidase. Mixed rosettes of HRBC and sheep red blood cells (SRBC) showed that HRBC receptors were detectable only on lymphocytes that possessed SRBC receptors, suggesting that HRBC rosette-forming cells were probably thymus-derived (T) cells. Next, the properties of human red blood cell (HRBC) and sheep red blood cell (SRBC) rosette-forming cells were investigated by comparing the ability of human peripheral blood lymphocytes to form these two types of rosettes after treatment with various inhibitory reagents. HRBC rosettes were relatively more resistant to inhibition with: (1) proteolytic agents, such as trypsin, alpha-chymotrypsin and pronase; (2) anti-thymocyte serum (ATS); (3) metabolic inhibitors, such as sodium azide and 2,4-dinitrophenol (DNP); (4) cytochalasin B. On further incubation after trypsinization, the lymphocytes recovered some ability to form SRBC rosettes, but continued to lose more of their capability to form HRBC rosettes. All these results were regarded as circumstantial evidence that the HRBC rosettes might represent a subpopulation of human T lymphocytes.


Acetate intolerance during hemodialysis. Acetate is frequently substituted for bicarbonate in hemodialysis solutions. Plasma acetate and bicarbonate concentrations were measured in nine patients with chronic renal failure undergoing hemodialysis with dialyzate containing acetate. In three patients (2 children and 1 adult) plasma acetate concentrations exceeded 15 mEq/liter during the dialysis. The mechanisms leading to acetate intolerance are probably multiple. It cannot be assumed that dialysis with acetate containing solutions will restore the buffer anion deficit characteristic of chronic renal failure.


Effect of liver failure on the ventilatory response to hypoxia in man and the goat. 1. The ventilatory responses to transient and steady-state hypoxia were measured in ten patients with hepatic cirrhosis and in ten healthy control subjects. Successive measurements of these responses were also obtained in six goats before and after the experimental production of liver failure. Changes in the effect of steady-state hypoxia on the ventilatory response to hypercapnia were evaluated by successive studies in another goat. 2. In spite of a respiratory alkalosis during liver failure, the response to transient hypoxia was greater in the patients than in the control subjects. This response was increased after the onset of liver failure in all the goats. 3. In healthy humans and goats the responses to transient and steady-state hypoxia were similar in magnitude. During liver failure there was a disparity between the size of these responses, since the ventilatory increment evoked by steady-state hypoxia was unchanged in spite of the increase in response to transient hypoxia. Steady-state hypoxia consistently enhanced the ventilatory response to hypercapnia in a healthy goat, but frequently depressed the response to hypercapnia during liver failure. 4. The findings suggest that liver failure heightens the sensitivity of the peripheral chemoreceptors to the hypoxic stimulus, but may increase the tendency of the medullary centres to become depressed in hypoxia.


Ventilatory response to carbon dioxide in tetanus. 1. Ventilatory response to carbon dioxide was measured by the rebreathing technique in seven patients with mild tetanus during the disease state and after clinical recovery. 2. The ventilatory response to carbon dioxide was found to be decreased in the tetanus patients during the disease state with normal response after full clinical recovery. It is postulated that the restrictive ventilatory defect was responsible for the decreased ventilatory response to carbon dioxide.


[Treatment of severe hypnotic poisoning with extracorporeal haemoperfusion (author's transl)]. Four patients with severe hypnotic intoxication, twice after suicidal intake of barbital, once of barbital, methaqualone and carbromal, and once of carbromal, were treated with six activated charcoal haemoperfusions. Three patients showed rapid improvement in the level of consciousness followed by complete recovery. One female patient died in cerebral coma due to complete acute cerebromalacia following hypoxia. Serious complications due to the haemoperfusion did not occur. Correct use of activated charcoal haemoperfusion enriches the therapeutic spectrum of severe exogenous intoxications.


Failure to monoaminergic and cholinergic receptor blockers to prevent prostaglandin E2-induced luteinizing hormone release. Receptor blocking drugs were used to determine whether adrenergic, dopaminergic, serotoninergic, or cholinergic synapses are involved in mediating the LH release induced by intraventricularly injected PGE2. Prostaglandin E2 (5mug) was injected into the 3rd ventricle (3rd V) of ovariectomized rats, and plasma LH concentrations before and after treatment were determined by radioimmunoassay. Phentolamine, 20 or 30 mug, or pronethalol, 20 mug (alpha and beta adrenergic receptor blockers, respectively) injected into the 3rd V failed to alter the elevation of plasma LH evoked by PGE2 injected into the ventricle 10 min later. Likewise, LH release following PGE2 was not changed when a dopaminergic blocker, pimozide (0.63 mg/kg, SC), was injected 2 h prior to PGE2. Two antagonists of serotonin, methysergide maleate (3 mg/kg ip) or cinanserin HC1 (1 mg/kg iv) given 2 h or 45 min before PGE2, respectively, failed to alter the action of PGE2. Atropine (100 or 250 mug) injected into the 3rd V 10 min prior to PGE2 was also ineffective in blocking the increase in plasma LH following PGE2. The results of this study indicate that the effect of PGE2 on LH release is not mediated by adrenergic, dopaminergic, serotoninergic, or cholinergic receptors. They also suggest that PGE2 is not acting trans-synaptically but probably directly on the LHRH neuron to induce the discharge of LHRH into the hypophysial portal vessels which then evokes release of LH from the adenohypophysis.


Neurotransmitter regulation of growth hormone and ACTH in the rhesus monkey: effects of biogenic amines. In an attempt to clarify the role of central neurotransmitters in GH and ACTH regulation, chair-adapted unanesthetized adult male rhesus monkeys and chronic indwelling intratrial cannulae were given 30 min infusions of various agonists known to affect central amines, and plasma samples were withdrawn for GH and cortisol determinations. Infusion of acid-saline vehicle alone had no significant effect on plasma GH or cortisol (P less than 0.05). L-Dihydroxyphenylalanine (L-Dopa) (4.5 and 45 mg/kg), but not apomorphine (7 mug/kg), a specific dopaminergic agonist, produced significant elevations of GH. Both noradrenergic (clonidine HCl, 1.5, 15, and 150 mug/kg, and D,L-threodihydroxyphenylserine (D,L-threodops) 90 mg/kg) and serotoninergic (5-hydroxy-L-tryptophan (5-HTP), 45 mg/kg) agonists induced significant GH responses. These findings suggest that GH is regulated in the rhesus monkey by noradrenergic and serotoninergic neurons, whereas participation of dopaminergic neurons has not been established. Significant cortisol responses were only observed following infusion of 5HTP (45 mg/kg). Dopaminergic and noradrenergic agonists not only failed to alter resting cortisol levels but also did not affect the cortisol response to 5-HTP. In the rhesus monkey serotoninergic mechanisms appear to be responsible for the regulation of resting cortisol levels. A catecholamine inhibitory mechanism was not demonstrated in this species.


Adrenergic binding sites and enzyme activities in the heart of hyperthyroid rats. In present study interactions of some adrenergic drugs with the binding of 3H-norepinephrine (NE) and response of some enzymatic systems in the heart of rats with pharmacological hyperthyroidism have been investigated. Binding of NE to cardiac particles was inhibited by isoproterenol, propranolol and in lower concentrations by another beta-blocking drug trimepranol both in control and hyperthyroid hearts in the same degree. However, after addition of nonradioactive  norepinephrine (10(-3) M) the degree of displacement was lower in hyperthyroid than in euthyroid group. Activity of adenylate cyclase was lower in hyperthyroid cardiac particles. This difference remained preserved after stimulation by norepinephrine or NaF. The activities of hormone-sensitive lipase and lipoprotein lipase were increased in preparation of hyperthyroid hearts. The phosphorylase "a" activity was also increased in hyperthyroid cardiac particles. There was no change in cardiac adrenergic binding sites properties in hyperthyroidism with the exception of less displacement of NE by nonlabelled hormone. The results indicate that the increased lipolytic and phosphorylase "a" activities in hyperthyroid hearts are not necessarily linked to elevated activity of adenylate cyclase.


Determination of total insulin (TIRI) in plasma of insulin-treated diabetics and newborn infants of insulin-treated diabetic mothers. Plasma of insulin-treated diabetics and of newborn infants of insulin-treated diabetic mothers contains insulin antibodies which invalidates the radioimmunoassay of insulin. Therefore, the endogenous insulin antibody complex must be splitted at a pH lower than 5 and the total IRI (TIRI) is separated by ethanol extraction. It was investigated the recovery rate in dependence upon plasma volume used for extraction. By reduction of used plasma volume from 500 to 200 mul per extraction the recovery rate was increased from 65.1 +/- 8.4 to 88.3 +/- 4.2% (mean +/- SEM). The low plasma volume of 200 mul for TIRI extraction made it possible to determine TIRI during glucose loads of newborn infants. To eliminate different conditions of incubation for standard and unknown plasma samples the TIRI levels were computed by means of so-called "extracted" standard curve, obtained with extracted insulin from standard insulin dilution in insulin-free pooled human plasma. Using the described method a temporary regeneration of insulin secretion of a newly diagnosed juvenile diabetic after insulin treatment could be shown. In contrast to newborn infants of healthy mothers a biphasic/insulin release was found during the intravenous glucose loads in newborn infants of insulin-treated diabetic mothers.


Glutamine synthetase, glutaminase and phosphodiesterase activities in brain under hypoxia: in vitro effect of cortisol, GABA and serotonin on glutamine synthetase. The effect of hypobaric hypoxia on the activities of glutamine synthetase, glutaminase and cyclic 3'5' AMP phosphodiesterase in rat brain was studied after exposure to 25,000' for 6 h. Glutamine synthetase activity was increased in all the regions of brain studied, and addition of gamma amino butyric acid, serotonin and cortisol in vitro produced a differential response. Glutaminase activity decreased in the whole brain. Cyclic 3'5' AMP phosphodiesterase activity decreased in cerebellum, medulla, hypothalamus and pituitary showing an accumulation of cyclic 3'5' AMP in these regions. The results suggest that glutamine synthesis and degradation are regulated in the central nervous system by cyclic AMP and cortisol: Gamma aminoburyric acid and other compounds can modulate the activity of glutamine synthetase and glutaminase.


Intermediates in the interconversion of acid and alkaline ferrihemoglobin. Structural and kinetic aspects. The acid-alkaline pH-jump in suspension of crystalline sheep hemoglobin has been studied in the range of 5.95 to 8.94. Crystals suspended in 3.8 M Cs2SO4 show a rapid optical transition of half-time equal to or less than 2 ms. As the ammonia concentration is increased in the Cs2SO4-suspended crystals, a second optical transition is observed as a pseudo-first-order reaction, with a rate constant of between 10 and 15 s-1. The alkaline-acid pH-jump proceeds through a very rapid shift of the alkaline-acid equilibrium and is followed by a first-order dissociation constant between 9 and 12 s-1. The dissociation of the ammonia is biphasic, and the ratio between the fast and slow phases is 9.


The reaction kinetics of fluoride and ammonia with acid and alkaline ferrimyoglobin in a crystalline state. Differences between the reactivity of amorphous and crystalline myoglobin have been studied by the rapid-flow method combined with dual-wavelength spectrophotometry. The binding of ammonia to the hydroxide compound has a half-time of 55 ms. The reverse reaction has a half-time of 70 ms. At pH 7.0 the relative half-times of combination and dissociation with fluoride are 10 min for crystalline and 1.8 min for amorphous materials. Reactivity of the crystals to fluoride at pH 6.0 greatly increased as compared with pH 8.7. Half-time at pH 8.7 is 10 min, while at pH 6.0 the half-time is 2.5 s for the crystalline material and 1.4 s for the amorphous material. The exchange of fluoride by azide at pH 6.0 is 3.1-fold faster in amorphous material than in crystalline material.


Circular-dichroism and absorption spectroscopic studies on specific aromatic residues involved in the different modes of aggregation of tobacco-mosaic-virus protein. Conformational changes accompanying the different modes of aggregation of tobacco mosaic virus protein (TMV-protein) were investigated using circular dichroism (CD) and absorption difference spectra in the range of aromatic absorption. Comparing wild-type protein and mutant Ni 2068 (Tyr-139 leads to Cys-139) a tentative localization of aromatic amino acids in the three-dimensional structure is rendered possible. In all modes of aggregation the CD spectra are determined by intrasubunit interactions between aromatic residues, in particular Trp-17 and Trp-52 as well as Tyr-70, Tyr-72 and Tyr-139. The Trp-17-Trp-52 interaction was found to be highly sensitive towards changes of the quaternary structure especially with respect to helical aggregates. This suggests that the environment of the two tryptophan residues is of crucial importance in the three-dimensional structure of the subunit; in the course of aggregation intersubunit interactions compete with the specific intrasubunit Trp-17--Trp52 interactions. It is suggested that Try-70 and Tyr-72 form hydrogen bonds in a strongly hydrophobic environment. Formation of the double disc decreases the rotatory strength, pointing to an increase in conformational flexibility. Spectroscopic and chemical evidence prove that Tyr-70, Tyr-72 and Tyr-139 are in close neighbourhood. Double disc formation by lowering the pH (pH 8 LEADS TO 6.9, I = 0.1 M) or increasing the ionic strength (pH 8, I = 0.1 LEADS TO 0.6 M) is reflected by identical spectral effects in the environment of Tyr-70 - Tyr-72. However the interaction between Trp-17 and Trp-52 indicates significant differences in the conformation which may be important for the formation of higher aggregates, i.e. 'lockwashers', helices, and 'stacked discs'.


Characterization of the oxygenase activity in a mutant of Chlamydomonas reinhardi exhibiting altered ribulosebisphosphate carboxylase. A previously described Mendelian mutant of Chlamydomonas reinhardi, ac i72, exhibiting altered ribulosebisphosphate carboxylase activity and unable to grow on minimal medium is examined for changes in ribulosebisphosphate oxygenase activity. The ribulosebisphosphate oxygenase activity of the enzyme purified from both wild type and ac i72 is compared over a pH range from 7.0 to 9.5. Both enzymes exhibit maximum activity at pH 9.0. However, the ac i72 enzyme is twice as active as the wild type enzyme at a physiological pH of 7.0. The studies in vivo of the products of CO2 fixation of ac i72 and wild type cells in the presence of high and low O2 concentration shows that due to a lower level of carboxylation, the ac i72 cells fix CO2 at half the rate of wild type cells. In ac i72, 24% of the photosynthetically fixed 14C is channelled into the water-soluble fraction as opposed to 6% in wild type. Thin-layer chromatography of the water-soluble fraction showed extensive accumulation of components of the glycolate pathway in ac i72 as compared to wild type. This indicates that the oxygenase activity of the enzyme prevails in ac i72 in vivo. Since a high concentration of glycolate is toxic to cells of C. reinhardi, the high oxygenase activity of ac i72 provides an explanation for the inability of ac i72 to grow phototrophically even though its rate of CO2 fixation is half that of wild type. This toxicity to glycolate is overcome by growth under amber illumination or low O2 concentration.


An intracellular endonuclease of Bacillus subtilis specific for single-stranded DNA. We have fractionated from extracts of Bacillus subtilis the DNase activity specific for single-stranded DNA; the activity separates in two main fractions on Sephadex G-200, a larger one (Mr greater than 400 000) and a smaller one (Mr approximately 30 000). We have purified the smaller, more abundant fraction nearly 3000-fold. The purified enzyme has a pH optimum close to 8, is activated by Ca2+, and is inhibited by EDTA; the enzyme hydrolyses single-stranded DNA at a rate approximately 40 times greater than double-stranded DNA. The mode of action is endonucleolytic on both substrates, but the possiblility that the two activities may reside on different molecules is not ruled out. The products have 5'-P and 3'-OH ends. The enzyme is different from those purified from the culture media of the same organism in several respects; the latter are all extracellular enzymes, they are not specific for single-stranded DNA (except one) and have all an exonucleolytic mode of action.


Guanylate cyclase: assay and properties of the particulate and supernatant enzymes in mouse parotid. A new, very sensitive, rapid and reliable assay for guanylate cyclase has been established based on conversion of [32P]GTP to [32P]guanosine 3':5'-monophosphate and its separation on Dowex 50 and aluminium oxide columns. The optimum conditions for the assay of mouse parotid guanylate cyclase have been established and using this procedure the properties of the enzyme have been investigated. The enzyme was found in both the particulate and supernatant fractions. The particulate enzyme was activated 12-fold by Triton X-100 and the supernatant enzyme activity increased 2-fold. In the presence of detergent guanylate cyclase activity was distributed 85% in the particulate and 15% in the supernatant fractions, respectively. The particulate activity was localised in a plasma membrane fraction. Guanylate cyclase activity was also assayed in a wide variety of other tissues. In all cases enzymatic activity was found in both the particulate and supernatant fractions. The distribution varied with the tissue but only the intestinal mucosa had a greater proportion of total guanylate cyclase activity in the particulate fraction than the parotid. The two enzymes showed some similar properties. Their pH optima were pH 7.4, both enzymes were inhibited by ATP, dATP, dGTP and ITP, required Mn2+ for activity and plots of activity versus Mn2+ concentration were sigmoidal. However, in many properties the enzymes were dissimilar. The ratios of Mn2+ to GTP for optimum activity were 4 and 1.5 for the supernatant and plasma-bound enzymes, respectively. The slope of Hill plots for the supernatant enzyme with varying Mn2+ was 2. The particulate enzyme plots also had a slope of 2 at low Mn2+ concentration but at higher concentrations (above 0.7 mM) the Hill coefficient shifted abruptly to 4. Calcium ions reduced sigmoidicity of the kinetics lowering the Hill coefficient, activated the enzyme at all Mn2+ concentrations but had no effect on the Mn2+:GTP ratio with the supernatant enzyme while with the plasma membrane enzyme Ca2+ had no effect on the sigmoid form of the kinetics at low Mn2+ but prevented the shift to a greater Hill coefficient at higher Mn2+, inhibited the activity at low Mn2+ and shifted the Mn2+:GTP optimum ratio to 4. For the particulate enzyme plots of activity versus GTP concentration were sigmoid (n = 1.3), while the supernatant enzyme exhibited hyperbolic kinetics.


Influence of unsaturated fatty acids in chloroplasts. Shift of the pH optimum of electron flow and relations to deltapH, thylakoid internal pH and proton uptake. Linolenic acid (C18:3) is the main endogenous unsaturated fatty acid of thylakoid membrane lipids, and seems in its free form to exert significant effects on the structure and function of photosynthetic membranes. In this investigation the effect of linolenic acid was studied at various pH values on the electron flow rate in isolated spinach chloroplasts and related to deltapH, the proton pump and the pH of the inner thylakoid space (pHi). The deltapH and pHi were estimated from the extent of the fluorescence quenching of 9-aminoacridine. Linolenic acid caused a shift (approximately one unit) of the pH optimum for electron flow toward acidity in the following systems: (a) photosystems II + I (from H2O to NADP+ or to 2,6-dichlorophenolindophenol) coupled or non-coupled; (b) photosystem II (from H2O to 2,6-dichlorophenolindophenol in the presence of dibromothymoquinone). In photosystem I conditions (phenazine methosulphate), the deltapH of the control increased as a function of external pHo with a maximum around pH 8.8. When linolenic acid was added, the deltapH dropped, but its optimum was shifted toward more acidic pHo. The same phenomena were also observed in photosytems II + I (from H2O to ferricyanide) and in photosystem II conditions (from H2O to ferricyanide in the presence of dibromothymoquinone). However, the deltapH was smaller and the sensitivity of the proton gradient toward linolenic acid was eventually higher than for photosystem I electron flow activity. The proton pump which might be considered as a measure of the internal buffering capacity of thylakoids was optimum at pHo, 6.7 in the controls. An addition of linolenic acid diminished the proton pump and shifted its optimum toward higher pHo. As a consequence, pHi increased when pHo was raised. At the optimal pHo 8.6 to 9, pHi were 5 to 5.5. Additions of increasing concentrations of linolenic acid displaced the curves toward higher pHi. A decrease of pHo was therefore required to maintain the pHi in the range of 5-5.5 for maximum electron flow. In conclusion, the electron flow activity seems to be delicately controlled by the proton pump (buffer capacity), deltapH, pHi and pHo. Fatty acids damage the membrane integrity in such a way that the subtile equilibrium between the factors is disturbed.


Uronic acid dehydrogenase from Pseudomonas syringae. Purification and properties. 1. Uronic acid dehydrogenase was purified to homogeneity. After a 338-fold purification a yield of 16% was achieved with a specific activity of 81 mumol NADH formed min-1 mg protein-1. 2. The purity of the enzyme was controlled by disc electrophoresis, sodium dodecylsulfate electrophoresis and ultracentrifugation. 3. A molecular weight of 60 000 was determined by gel chromatography and by ultracentrifugation. 4. The native enzyme is composed of two subunits, their molecular weight being 30 000 as estimated by sodium dodecylsulfate electrophoresis. The subunits as such are inactive. 5. The absorption spectrum with a maximum at 278 nm shows no evidence for a prosthetic group. 6. For catalytic activity no SH groups and no metals seem to be necessary. 7. The Michaelis constants determined with the pure enzyme are for glucuronic acid Km = 0.37 mM, galacturonic acid Km = 54 muM and NAD+ (with glucuronic acid) Km = 80 muM. 8. A weak reverse reaction could be observed with glucaric acid lactones at acidic pH. 9. NADH is competitive with NAD+. The inhibitor constant is Ki = 60 muM. 10. The NAD+ binding site seems to be of lower specificity than the uronic acid binding site.


The purification and properties of the trimethoprim-resistant dihydrofolate reductase mediated by the R-factor, R388. The R-factor R388 mediates the production of a trimethoprim-resistant dihydrofolate reductase. This enzyme has a different molecular weight and pH profile to the trimethoprim-sensitive enzyme of the Escherichia coli host. The R-factor mediated enzyme was separated completely from the host E. coli enzyme by DEAE-cellulose ion-exchange chromatography. The purified R-factor enzyme was about 20 000 times less susceptible to trimethoprim than the E. coli enzyme and although it was inhibited competitively by trimethoprim, its inhibitor constant (Ki) was 20 000 times greater than that of the host enzyme. The R388 and E. coli enzymes also differed in their substrate specificity requirements. In addition, the R388 enzyme suprisingly conferred high level resistance to the broad spectrum dihydrofolate reductase inhibitor, amethopterin. The possible origins of the R388 enzyme are discussed.


Glycosaminoglycan biosynthesis in arterial wall. Hexosaminyltransferase and glucuronyltransferase in cell membranes of aortic media-intima. Hexosaminyltransferase and glucuronyltransferase are well known for their role in the biosynthesis of proteoglycans. These two enzymes are characterized in rough and smooth membranes, obtained following subcellular fractionation of aortic media-intima. They require the presence of Mn2+ or Mg2+ for activity. The optimum concentration for these two cations is 5 mM. The optimum pH for hexosaminyltransferase and glucuronyltransferase is approximately 6.8 in Tris buffer, and their optimum temperature is 30 degrees C. Hexosaminyltransferase has an apparent Km of 0.27 nM. Glucuronyltransferase has an apparent Km of 0.21 nM. Uptake of labeled sugars by endogenous proteoglycans is inhibited by puromycin. Hexosaminyltransferase and glucuronyltransferase are present in both rough and smooth submicrosomal fractions. The different endogenous glycosaminoglycans are labeled during our incubation experiments. The greatest incorporation is noted for hyaluronic acid and heparan sulfate; the least is seen for chondroitin sulfate. The results obtained in vitro for incorporation of labeled precursors into endogenous proteoglycans are consistent with those observed during the study in vivo of the turnover of these macromolecules.


A study of the lysyl residues in the basic pancreatic trypsin inhibitor using 1H nuclear magnetic resonance at 360 Mhz. Fourier transform 1H nuclear magnetic resonance (NMR) experiments at 360 MHz using convolution difference techniques to improve the spectral resolution were employed to investigate the resonances of the lysyl residues in bovine pancreatic trypsin inhibitor. The observations in both native protein and in chemically modified protein containing Nepsilon-dimethyllsysine show that three of the four lysines extend predominantly freely into the solvent, whereas lysine-41 is involved in an intramolecular interaction with tyrosine-10. Since in the single crystal structure tyrosine-10 is involved in an intermolecular interaction with arginine-42 of the neighboring protein molecule, the NMR data thus reveal a local conformation difference for bovine pancreatic trypsin inhibitor in solution and in the crystalline form which appears to result primarily from intermolecular interaction in the crystal lattice.


Effect of triiodothyronine on rat liver chromatin protein kinase. 1. Injection of triiodothyronine to rats stimulates protein kinase activity in liver chromatin nonhistone proteins. A significant increase was found after two daily injections. A 4-fold increase was observed with the purified enzyme after eight daily injections of the hormone. No variations were observed in cytosol protein kinase activity. Electrophoretic pattern, effect of heat denaturation, effect of p-hydroxymercuribenzoate seem to indicate that the enzyme present in treated rats is not identical to the enzyme in control animals, which suggests that thyroid hormone has induced nuclear protein kinase. Diiodothyronine, 3, 3', 5'-triiodothyronine have no effect on protein kinase. 2. Chromatin non-histone proteins isolated from rats injected with triiodothyronine incorporated more 32P when incubated with [gamma-32P]ATP than the chromatin proteins from untreated rats. Thyroidectomy reduced the in vitro 32P incorporation. It is suggested that some of the biological activity of thyroid hormone could be mediated through its effect on chromatin non-histone proteins.


Subcellular distribution of histone-degrading enzyme activities from rat liver. Chromatin prepared from liver tissue contains a histone-degrading enzyme activity with a pH optimum of 7.5-8.0, whereas chromatin isolated from purified nuclei is devoid of it. The histone-degrading enzyme activity was assayed with radioactively labelled total histones from Ehrlich ascites tumor cells. Among the different subcellular fractions assayed, only lysosomes and mitochondria exhibited histone-degrading enzymes. A pH optimum around 4.0-5.0 was found for the lysosomal fraction, whereas 7.5-8.0 has been found for mitochondria. Binding studies of frozen and thawed lysosomes or mitochondria to proteinase-free chromatin demonstrate that the proteinase associated with chromatin isolated from frozen tissue originates from damaged mitochondria. The protein degradation patterns obtained after acrylamide gel electrophoresis are similar for the chromatin-associated and the mitochondrial proteinase and different from that obtained after incubation with lysosomes. The chromatin-associated proteinase as well as the mitochondrial proteinase are strongly inhibited by 1.0 mM phenylmethanesulfonyl fluoride. Weak inhibition is found for lysosomal proteinases at pH 5. Kallikrein-trypsin inhibitor, however, inhibits lysosomal proteinase activity and has no effect on either chromatin-associated or mitochondrial proteinases. The higher template activity of chromatin isolated from a total homogenate compared to chromatin prepared from nuclei may be due to the presence of this histone-degrading enzyme activity.


Anion and amine uptake and uncoupling in submitochondrial particles. 1. Unlike chloroplasts, submitochondrial particles are not uncoupled by nigericin + KCl or NH4Cl. Also the uncoupling effect of lipophilic anions is largely independent of the addition of weak bases. 2. Low concentrations of permeant anions cause a shift of the steady-state energy level rather than a cycle of energy utilization. The degree of inhibition of ATP synthesis by tetraphenylboron is larger than required for the uptake of the anion. 3. Lipophilic anions such as bromthymolblue, bromcresolpurple, and 8-anilino-1-napthalene sulphonate cause a pH-independent, 50% uncoupling in submitochondrial particles at concentrations of 3, 30 and 30 muM, respectively. The passive interaction of bromthymolblue and bromcresolpurple appears as a pH-dependent distribution between two pHases. ATP causes a pH-independent slight shift in the anion distribution, with negligible anion accumulation. 4. Addition of amines to energized submitochondrial particles results in two types of effects; uptake of amines and uncoupling. While in chloroplasts amine uptake and uncoupling are closely associated, this is not the case in submitochondrial particles. The uncoupling effect is observed only with lipophilic and not with hydrophilic amines, and the degree of uncoupling increases with the lipophilicity of the amines. The amine uptake, on the other hand, is accompanied by negligible uncoupling. 5. While the uptake of amines is dependent on the presence of non-permeant anions, such as Cl-, the uncoupling effect is independent of Cl-. Furthermore the amine uncoupling is markedly enhanced by lipophilic anions. 6. The view is discussed that the uncoupling effect of lipophilic anions and lipophilic amines in submitochondrial particles is due to a catalytic energy dissipation rather than to a stoichiometry energy utilization. The molecular mechanism of uncoupling presumably involves a cycling of charges after a perturbation of the membrane structure.


Oral antipyretic therapy: evaluation of benorylate, an ester of acetylsalicylic acid and paracetamol. The capacity of benorylate, an ester of acetylsalicylic acid and paracetamol, to reduce fever in children was compared with that of the components as such or as a combination. The series of cases studied consisted of 66 patients between the ages of 4 months and 12 years with rectal temperatures above 38.5 degrees C. Temperatures were recorded at 15 and 20 min and 1, 2, 4 and 6 hrs after the administration of the drug. The antipyretic effect of combined acetylsalicylic acid (11 mg/kg) and paracetamol (14 mg/kg) was superior to the effect of benorylate with a dose of 25 mg/kg and even of 50 mg/kg as well as better than the effect of either drug alone. Acetylsalicylic acid (10 mg/kg) and paracetamol (12.5 mg/kg) alone produced a significantly greater antipyretic effect than benorylate with a dose of 25 mg/kg. Given in a dose of 35--40 mg/kg, benorylate seems to have a significant antipyretic effect. However, this effect is clearly smaller than that of either of its components, acetylsalicylic acid or paracetamol. Therefore benorylate is probably not suitable to be used as a general antipyretic agent in children.


Effect of ischemic anoxia on electrical and mechanical activity of the totally isolated porcine stomach. Electrical and mechanical activity were recorded from totally isolated whole porcine stomachs perfused with homologous blood. Stagnant, complete ischemia was then produced by closing the gastric artery and vein for various periods of time up to 3 h. After a given period of ischemia, blood was recirculated for 10 min and then pentagastrin was injected into the gastric artery. The vagus nerve was also electrically stimulated at various stages of anoxia and recirculation. After up to 90 min of ischemia, stomachs were able to demonstrate electrical control activity of low frequency after 10 min of blood recirculation. After injection of pentagastrin, electrical control activity rapidly became normal. When ischemia lasted 120 min or longer, recirculation of blood did not induce reappearance of electrical control activity, but pentagastrin injection produced a normal response. After ischemia of 240 min duration or longer, recirculation and pentagastrin had no effect. In previously ischemic stomachs the response to vagal stimulation occurred only in the stomachs responding to pentagastrin.


Combined azaperone and metomidate anaesthesia in liver transplantation in the pig. Combined azaperone and metomidate anaesthesia has been used in 86 surgical procedures on 84 piglets, either as such or deepened and prolonged. 51 animals were sacrificed at the end of the procedure as planned before. The anesthesia allowed the performance of various short and long operations. Out of the 33 remaining pigs, submitted to 35 operations, 4 died during the procedure of a technical fault; 2 did not recover from a deep hypothermia (below 10 degrees C); 1 died from the hepatic coma induced through the operative procedure. The other 26 awoke and recovered spontaneous breathing within 1-4 h following the type of anaesthesia and operating procedure they had submitted, which dured from 15 to 330 min.


Cardiovascular and beta-adrenergic blocking effects of timolol. The haemodynamic effects of timolol and its inhibiting action on the cardiovascular and bronchial effects of isoproterenol have been studied. Splanchnic nerve activity was recorded. The antiarrhythmic action of timolol was studied on guinea pig isolated atria, using arrhythmias induced by epinephrine, ouabain or coronary ligation in the dog. Timolol is a very potent beta-adrenoceptor blocking agent, without specificity on beta1- or beta2-receptors. No intrinsic beta-stimulating or depressant effects were found. Timolol reduced splanchnic discharges. The antiarrhythmic effect of timolol was limited to epinephrine-induced arrhythmias.


Effects on rectal temperature in rats of gamma-aminobutyric acid; possible mediation through putative transmitters. The rectal temperature of male rats was measured in a thermoneutral environment (25 degrees C) and at ambient temperatures of 15 and 35 degrees C. Unless otherwise specified all drugs were administered intracerebroventricularly (i.c.v.) and all results are reported for the thermoneutral environment. Exposure to 15 degrees C did not affect the rectal temperature but exposure to 35 degrees C produced hyperthermia. At 15 and 25 degrees C, 20 mug GABA produced hyperthermia which was longer lasting at the former ambient temperature. GABA (20 mug) prevented the hyperthermic effect of exposure to 35 degrees C and produced hypothermia in animals maintained at this temperature for 1 hr. A low dose (1 mug) of NA produced hyperthermia and a higher dose (mug) hypothermia. In rats pretreated with sodium salicylate (i.p.), 20 mug GABA and 1 mug NA produced hypothermia instead of hyperthermia, suggesting the release of PGE in mediating hyperthermia. The hypothermic effect of 10 mug NA and of GABA observed at 35 degrees C was blocked by phentolamine, an indication of the possibility of alpha-adrenoceptor mediation.


Analysis of cardiac chronotropic responses to diazepam and bromazepam in conscious trained dogs. In conscious trained dogs, administration of bromazepam (0.3 mg/kg p.o.) or diazepam (0.3 and 1.0 mg/kg p.o.) had no influence on heart rate. A higher dose (10 mg/kg p.o.) of two benzodiazepines elicited a positive chronotropic effect which was rapid in onset and of long duration. The beta-adrenoceptor blocking agent practolol (2.5 mg/kg i.v.) did not revert heart rate after the benzodiazepines to the same level as in controls, indicating that the tachycardia was not produced by an increase in sympathetic outflow to the heart. For diazepam, a sympathetic--parasympathetic interaction cannot be excluded. However, diazepam and bromazepam significantly reduced the tachycardia which is normally observed after administration of methylatropine (0.5 mg/kg i.v.) alone or in combination with practolol. In anaesthetized dogs, bromazepam failed to modify the heart rate responses to electrical stimulation of cardiac vagal or sympathetic nerves, excluding an action on this compound on ganglionic transmission and cardiac cholinoceptors and adrenoceptors. It is concluded that high doses of diazepam and bromazepam influence the heart rate of conscious dogs in a biphasic way. Firstly, they cause a central reduction of vagal tone to the heart resulting in tachycardia. Secondly, the two drugs decrease the cardiac pacemaker rate directly. Since the overall effect is tachycardia, the central action is more pronounced.


The amphetamine-induced inhibition of dopamine biosynthesis in rat striatum. The dopamine biosynthetic machinery of intact synaptosomes of rat striatum showed a 5-fold increase in development from 3-day-old neonates to adults, and it was fully developed between 2-3 weeks after birth. Concurring with this development was the appearance 2 weeks after birth of a regulatory mechanism(s) through which amphetamine in vivo induced an inhibition of dopamine biosynthesis. The inhibition was not appreciably reversed when haloperidol, in addition to amphetamine, was administered.


Toxicological aspects of dimethyl-ether. The authors report the results of a series of investigations on the toxic effects produced in mice and rabbits by inhalation of Dimethyl-ether. Median lethal concentration (LC50) and Median lethal time (LT50) were determined in the mouse. Also the effects of DME inhalation on some physiological parameters (blood pressure, heart rate, blood gas and pH data) were evaluated in the rabbit.


[Gastric irrigation and secretion in the ligatured or fistuled pylorus rat upon influence of intra-duodenal olive oil (author's transl)]. Pylorus ligature hides the inhibitory effects of endogenous cholecystokinine-pancreozyme (CCK-PZ) on gastric mucosal secretion and irrigation, whereas the juice collected through transduodenal pyloric fistula makes this phenomenon obvious. It appears that the pyloric fistula encourages inhibition of gastrinic secretion, so that the CCK-PZ can achieve its effects.


Properties of glutamine aminohydrolases in subcellular fractions of liver of tumour bearing mice. Glutamine aminohydrolase is found to be present in microsomal and soluble supernatant in liver of EAC-bearing mice. Enzymes obtained from these two sources were characterized and found to behave differently from the mitochondrial glutaminase of both normal and tumour-bearing mice.


[Ability of adrenergic nerves to accumulate exogenous noradrenaline under some pharmacological influences]. By employing spectrofluorometry and quantitative fluorescent histochemistry the norepinephrine (NE) content in the tissue and in the adrenergic nerves of Vas deferens of the rat after depletion of the transmitter's reserves with tyramine and their subsequent replenishment through incubation of the tissue with exogenous NE was measured. The object of investigations was also the influence of antidepressants (phthoracizine and imipramine) and of the cholinolytic spasmolytin (diphacyl) on processes of the NE accumulation. Phthoracizine, impramine and spasmolytin were found to capable of inhibiting accumulation of the exogenous NE in the tissue and in the adrenergic nerves of Vas deferens, provided the mediator is present in the extraneuronal medium in a concentration of 0.5 gamma/ml. When the neurotransmitter was introduced into the medium in higher concentrations (10 gamma/ml) the antidepressents and cholinolytic did not produce the said effect. A correlation of spectrofluorometric and quantitative fluorescent-histochemical findings was observed in cases when the tissue contained less than 50% (i.e. less than 9 gamma/ml) of the total NE reserves.


[Possible participation of potassium ions in the regulation of local cerebral blood flow]. The microinjections of mock spinal fluid with various potassium concentrations into the cat and monkey brain tissue were made in acute and chronic experiments. The vascular reactions as studied with the microphotography and H-clearance method were found to be linearly related to the potassium concentrations within the range of 0--12 mEq/1, being constrictory below 5 mEq/1 and dilatory above that. An interaction between MSF potassium and pH seems to be weak if any, since the slope of the potassium vascular activity diagram remains unchanged within 6.8--7.8 pH change. The data obtained suggest that potassium ions take part in rCBF control since potassium is known to escape from stimulated nerve cells and thus substantially increase an extracellular potassium concentration.


The alternate complement pathway in inflammatory bowel disease. Quantitation of the C3 proactivator (factor B) protein. A component of the complement system's alternate pathway was investigated in ulcerative colitis and Crohn's disease. The mean C3PA (Factor B) titer in normals was 74 +/- 15%; in ulcerative colitis, 92 +/- 18%; and in Crohn's disease, 119+/- 24%. Significance was at the P less than 0.001 level when the mean values for the ulcerative colitis and the Crohn's disease groups were compared to normal subjects. Titers did not change significantly with exacerbation or amelioration of the diseases or when patient groups were analyzed according to the mode of treatment received.


Measurement of gastric functions during digestion of ordinary solid meals in man. A method of measuring gastric secretions and emptying rates after ingestion of an ordinary (solid-liquid) meal has been developed and validated. The technique quantifies movements of volume across the pylorus using constant duodenal perfusion with a nonabsorbable marker, polyethylene glycol (PEG), which, in turn, quantifies emptying into the duodenum of another marker, [14C]PEG, incorporated in the meal. Acid and pepsin outputs can be determined without manipulation of the intragastric pH. Employing this method, we have simultaneously quantified acid, pepsin, and total secretory outputs; rates of gastric emptying of meal and secretions; and serum gastrin levels during digestion. These data characterize physiological responses to ordinary food in health.


A dopamine receptor in esophageal smooth muscle of the opossum. We examined the possibility that dopamine may play a role in nerve-mediated "off" responses of esophageal body (EB) and relaxations of lower esophageal spincter (LES) smooth muscle. The effects of dopamine, epinine, and dopamine antagonists on EB and LES smooth muscle were studied on these responses. Dopamine and epinine caused a dose-related fall in basal LES muscle tension and in amplitude of EB muscle "off" responses. Threshold dose for both was about 10(-7) M, and maximal dose was about 10(-4) M. At high concentrations, they also caused repetitive transient contractions of both LES and EB muscle after the period of inhibition. These effects were antagonized by haloperidol, 10(-5) M, and bulbocapnine, 10(-5) M, but were not influenced by propranolol, 10(-5) M, nor by phenoxybenzamine, 10(-5) M. Neither haloperidol nor bulbocapnine influenced responses to electrical field stimulation. Tetrodotoxin 10(-7) M abolished the responses to electrical field stimulation but did not antagonize the effects of dopamine and epinine. EB and LES smooth muscle contain a dopamine receptor. It is unlikely that dopamine is involved in responses to electrical field stimulation.


[Continuous subcutaneous pH monitoring in newborns with abnormal metabolism (author's transl)]. In newborns with postpartum acidosis the subcutaneous interstitiel space pH was monitored continuously with the special electrode of Stamm and Moeller. This was compared with intermittent determinations of the pH in the capillary blood from the heel of the infant. The severity of the metabolic disturbance can be recognized in all test cases with both methods. The correction of the acidosis develops in a concordant manner, in a concordant magnitude and at a largely synchronous rate. The observations show that it is possible to monitor the postpartum adaptation phase by continuous pH monitoring in the interstitial space of the subcutaneous tissue and to observe the efficiency of the treatment of acidosis, administration of oxygen, or a buffer infusion.


[The significance of gestational age in evaluating the antepartal cardiotokogram (author's transl)]. The influence of gestational age on the probable meaning of features in the antepartal CTG which are not clearly pathological was investigated in 232 births in 1973/74. The umbilical artery pH was significantly lower among premature infants exhibiting abnormal, than among those exhibiting normal CTG patterns. No similar difference was found in term or overdue births. The clinical diagnosis of neonatal condition using the Apgar scheme confirmed biochemical findings: The frequency of depressed babies is in turn clearly higher in cases with abnormal antepartal CTG patterns. It is assumed that the reason for this is the higher sensitivity of premature babies to haemodynamic disturbances. Thus, it follows that premature babies exhibiting antepartal patterns which are not completely normal require close observation.


Characteristics of lipopolysaccharides of Salmonella typhi isolated from carriers and patients suffering from typhoid fever. Lipopolysaccharides (LPS) of Salmonella typhi strains, isolated from carriers and patients suffering from typhoid fever, were characterised according to their biochemical properties, morphological structure and degree of aggregation of complexes. All preparations of LPS, regardless of their origin, were morphologically heterogeneous. Free electrophoresis and immunoelectrophoresis demonstrated that LPS preparations were composed of components possessing different mobilities in electric fields. LPS of bacterial strains isolated from both carriers and patients, split upon reaction in immunoelectrophoresis with specific antiserum 73, rabbit antiserum to Salmonella typhi Vi Bhatnagar and 0-901 split into anodic and cathodic fractions. The anodic fraction reacted similarly as Vi antigen. LPS from Salmonella typhi Ty-2 yielded only the cathodic fraction, typical for O antigen. LPS from strains which were passaged twice in nutritional medium possessed identical properties as LPS from fresh cultures of Salmonella typhi. Electron microscopy revealed that LPS appears as long bands, rods, ellipsoid forms and amorphous material. Contrary to amorphous material, the bands, rods and ellipsoid forms possessed three-layer structure.


Reduction of blood platelet monoamine oxidase activity in schizophrenic patients on phenothiazines. A newly developed assay for monoamine oxidase (MAO) activity in blood platelets (serotonin used as substrate) was applied for the measurement of the enzyme activity in 76 schizophrenic patients. No significant reduction was found in the blood platelet MAO activity in a group of 33 untreated schizophrenic patients, as compared to that in the normal controls. Male patients revealed to have lower enzyme activity than females in the schizophrenic group, as we described previously in the normal subjects. Treatment with phenothiazines caused significant reduction of blood platelet MAO activity, while platelet serotonin content and platelet count appeared to be not affected by the drug treatment. The authors suggest that blood platelet MAO activity may be related to hormonal factors but not to psychiatric diagnosis of schizophrenia or constitution liable to schizophrenic illnesses.


[Inflammatory cerebro-vascular disease: angiographic findings and distribution patterns (author's transl)]. Although cerebral angiography should be approached with caution in the diagnosis of inflammatory cerebro-vascular disease there are some characteristic angiographic findings which may be helpful for classification and differential diagnosis. The proximal cerebral arteries are favourably affected by basal meningitis and thrombangiitis obliterans with resulting stenoses and occlusions. Whereas those inflammations originating from neighbouring skull structures mostly involve the intracavernous parts of the carotid artery, the tuberculous and mycotic arteritis prefer the supraclinoid carotid siphon. Peripheral vascular changes are found in luetic endangiitis, necrotizing and toxic angiitis and in collagenoses. Simultaneous involvement of the temporal arteries is of great diagnostic importance demonstrating the systemic character of the inflammatory process; in Horton's arteritis it can be a pathognomonic finding. Infectious endocarditis, some mycoses and malaria may lead to embolic occlusion of cerebral vessels. Mycotic aneurysms mostly have a broad base or a fusiform shape and do not prefer the localizations of congenital aneurysms. Angiographically, abscesses, tuberculomas and viral encephalitis may result in circumscribed hypervascularized areas. The characteristic angiographic findings are exemplified and discussed on the basis of 8 cases of inflammatory cerebro-vascular disease (tuberculosis, pneumococcal and unspecific bacterial meningitis, syphilis, mycosis, Takayasu-syndrome, panarteritis nodosa, temporal arteritis).


Oxidative deamination of biogenic amines by intestinal amine oxidases: histamine is specifically inactivated by diamine oxidase. The ability of the gut to inactivate various amines by oxidative deamination was tested with a 130-fold purified amine oxidase preparation from dog small intestine. Of 34 amines tested, putrescine, benzylamine, cadaverine, and serotonin were the most favourable substrates. Histamine was inactivated rapidly by this enzyme preparation, too. Histamine derivatives methylated at the imidazole nucleus were also deaminated, whereas Nalpha-methylhistamine was only a poor substrate and Nalpha, Nalpha-dimethylhistamine was not a substrate at all. Using a second procedure for the purification of amine oxidases from gut, the separation of a soluble monoamine oxidase from diamine oxidase was achieved by gel filtration on Sephadex G-200. The diamine oxidase deaminated putrescine (Km = 1.3 x 10(-4)M) and histamine (Km = 6.6 x 10(-5)M), but not serotonin, and was inhibited by aminoguanidine, but not by pargyline. The soluble monoamine oxidase inactivated serotonin (Km = 4.5 x 10(-4)M), but not histamine and putrescine and was inhibited by pargyline, but not by aminoguanidine. It was concluded that in dog small intestine (as well as in rabbit small intestine) only diamine oxidase was capable of inactivating histamine by oxidative deamination.


Comparative toxicology in vitreous humor and blood. Drug and toxic substances were detected in blood and vitreous humor in fifty-six cases, in which causes of death were both from an overdose of the particular substances and from other unrelated causes. Five instances are reported in which two drug substances were detected in blood and vitreous humor from the same subject. Patients having long survival times, as well as those dying from unrelated causes, reveal drug values to approach unity, when the blood and vitreous concentrations are compared. The ratios reached at equilibrium probably depend on solubility of the drug in vitreous humor, lipid solubility and the percentage protein-bound in the blood. The vitreous humor provides another parameter of testing and may be useful in studies of survival time.


Electrodecantation of serum proteins.

Electrodecantation of serum proteins. The sedimentation of albumin under the action of the electric and gravitational fields was determined as a function of time in discontinuous experiments in a rectangular cell, using serum with the albumin fraction stained blue. It was shown that even under the influence of strong electric fields, the upper boundary of the albumin layer fell no further than the mid-point of the cell. In continuous single-stage separation of gamma-globulin from other serum proteins, only about half the gamma-globulins can be obtained from the solution because it remains homogeneously distributed throughout the solution and is only free from albumin and other proteins in the upper half of the cell. In experiments with continuously operated triangular cells, the process was optimized to give gamma-globulin of 97.5% purity in a yield of 80%, at serum flow-through rates of up to 0.5 l/h in a block composed of 40 cells.


Recent studies on cytochrome P-450-linked functions in isolated rat liver cells. In rat liver cells isolated by perfusion in the perfusion in the presence of collagenase, the major portion of cytochrome P-450 is present in the oxidized, nonsubstrate-bound, low spin state. Drug addition to a suspension of liver cells results in the rapid formation of the cytochrome P-450 (Fe3+)-substrate complex which in turn is followed by the appearance of other species with different spectral characteristics before steady state drug monooxygenation is achieved. Cytochrome P-450-linked metabolism of various tested drugs and carcinogenic polycyclic hydrocarbons by isolated rat liver cells is as fast, or faster, as with rat liver microsomes supplemented with a NADPH generating system. Both experimental models respond similarily to phenobarbital or 3-methylcholanthrene pretreatment of the animals and to various of the wellknown inhibitors of drug metabolism. Except with liver cells isolated from fasted, phenobarbital-treated rats, generation of cytosolic NADPH seems sufficient to support optimal drug metabolism even in the absence of added substrates of intermediary metabolism. In isolated liver cells oxidized drug metabolites undergo subsequent metabolic conversion, most often to form the corresponding glucuronides and sulphates. These are readily excreted, whereas non-conjugated products, e.g. free phenols, tend to accumulate intracellularly. Cellular glucuronide formation is strongly inhibited by ethanol-presumably due to an unfavorable effect of the increased NADH/NAD+ ratio on the synthesis of uridine-5'-diphosphoglucuronic acid (UDPGA). In contrast, low concentrations of ethanol have no, or only a slight stimulatory effect on the cytochrome P-450-linked step of drug metabolism and there are indications that the oxidation of low concentrations of ethanol is in fact stimulated by a facilitated reoxidation of cytosolic NADH occuring during drug monooxygenation.


D-glucose dehydrogenase from Bacillus megaterium M 1286: purification, properties and structure. 1) Glucose dehydrogenase from Bacillus megaterium has been purified to a specific activity of 550 U per mg protein. The homogeneity of the purified enzyme was demonstrated by gel electrophoresis and isoelectric focusing. 2) The amino acid composition has been determined. 3) The molecular weight of the native enzyme was found to be 116000 by gel permeation chromatography, in good agreement with the values of 120000 and 118000, which were ascertained electrophoretically according to the method of Hedrick and Smith and by density gradient centrifugation, respectively. 4) In the presence of 0.1% sodium dodecylsulfate and 8M urea, the enzyme dissociates into subunits with a molecular weight of 30000 as determined by dodecylsulfate gel electrophoresis. These values indicate that the native enzyme is composed of four polypeptide chains, each probably possessing one coenzyme binding site, which can be concluded from fluorescent titration of the NADH binding sites. 5) In polyacrylamide disc electrophoresis, samples of the purified enzyme exhibit three bands of activity, which present the native (tetrameric) form of glucose dehydrogenase and two monomeric forms (molecular weight 30000), arising under the conditions of pH and ionic strength of this method. 6) The enzyme shows a sharp pH optimum at pH 8.0 in Tris/HCl buffer, and a shift of the pH optimum to pH 9.0 in acetate/borate buffer. The limiting Michaelis constant at pH 9.0 for NAD is 4.5 mM and 47.5 mM for glucose. The dissociation constant for NAD is 0.69 mM. 7) D-Glucose dehydrogenase is highly specific for beta-D-glucose and is capable of using either NAD or NADP. The enzyme is insensitive to sulfhydryl group inhibitors, heavy metal ions and chelating agents.


[Enzyme induction in Streptomyces hydrogenans. V. Characterization of testosterone-17 beta-dehydrogenase and its induction by steroids]. Testosterone 17beta-dehydrogenase can be enriched from Streptomyces hydrogenans. The enzyme dehydrogenizes testosterone with Km=13muM and estradiol-17beta with Km=21muM to the corresponding 17-ketoderivatives. NAD forms NADH with Km=125muM. The enzyme is strongly inhibited by androstandione and 17alpha-methyltestosterone. The Ki for 17alpha-methyltestosterone is 18muM. The enzyme activity increases with increasing pH up to alkali-mediated denaturation at about pH 10. The optimum temperature is at 45 degrees C. If Streptomyces hydrogenans is cultivated in the absence of steroids, the specific activity of testosterone 17beta-dehydrogenase in the cytosol of the microorganisms amounts to 10 mU/mg protein, and increases up to 10-fold if the cells are cultivated in the presence of certain steroids. Testosterone, alpha-dihydrotestosterone, beta-dihydrotestosterone, estradiol-17beta, and 17alpha-methyltestosterone are very effective inducers. Thus, for the first time, the ability of estradiol-17beta to induce an enzyme synthesis in a microorganism is shown. The steroid-dependent induction is inhibited by testosterone acetate and rifamycin SV. Cyproterone, however, does not decrease the testosterone-dependent enzyme induction of testosterone 17beta-dehydrogenase.


Boar acrosin, II: Amino acid composition, amino terminal residue and molecular weight estimations by ultracentrifugation. The molecular weight of boar acrosin in neutral solution was estimated to be 41000 +/- 1000 by high-speed sedimentation equilibrium analysis. This result is in good agreement with the value found earlier[1] by sodium dodecylsulfate polyacrylamide gel electrophoresis. The sedimentation coefficeint of acrosin obtained by active enzyme centrifugation of partly purified preparations is in accordance with the sedimentation coefficient of the pure preparation estimated by conventional sedimentation velocity analysis. The sedimentation coefficient of acrosin is considerably decreased in slightly acidic solution (pH 4), indicating that changes in the tertiary structure occur upon acidification. The amino acid composition of the acrosin preparation homogeneous by electrophoretic and chromatographic criteria and in sedimentation studies was determined. Valine was found as the unique N-terminal amino acid. However, in microheterogeneous forms of acrosin, alanine and methionine were also detected in end group analysis.


Characterization of protein kinases from Blepharisma intermedium. Three protein kinases (EC 2.7.1.37) were detected in Blepharisma and partially purified. The enzymes were most active with histone as substrate protein. The stability of the bond between phosphate and protein acceptor showed the characteristics of seryl- or threonylphosphate. Protein kinase I was solubilized by ultrasonication or freezing and thawing, while the enzymes II and III were readily solubilized by mild homogenization. Protein II and III were noticeably activated by cAMP and cGMP, while protein kinase I was inhibited by cAMP. Associated with protein kinase II and III activity was the ability to bind labeled cAMP. The following molecular weights were determined: 90000 for enzyme I, 280000 for enzyme II, and 95000 for enzyme III. Various apparent Michaelis constants were estimated.


[Solubilization and purification of a 3alpha-hydroxysteroid dehydrogenase in rat liver microsomes (author's transl)]. The microsomal 3-hydroxysteroid dehydrogenases were solubilized with lubrol, a non-ionic detergent. A 3alpha-hydroxysteroid dehydrogenase is purified about 100-fold by double affinity chromatography on 5alpha-dihydrotestosterone-Sepharose. This enzyme can use both NADH and NADPH as coenzymes.


The dissociation of insect embryos for cell culture. Procedures and solutions were developed for dissociating embryos of Blattella germanica in preparation for primary cell culture. Trypsin solutions were maximally effective at 0.01% for germ bands but higher concentrations, 0.05 to 0.1% were needed for embryos in later stages.


Effect of sodium butyrate on mammalian cells in culture: a review. Sodium butyrate produces reversible changes in morphology, growth rate, and enzyme activities of several mammalian cell types in culture. Some of these changes are similar to those produced by agents which increase the intracellular level of adenosine 3',5'-cyclic monophosphate (cAMP) or by analogs of cAMP. Sodium butyrate increases the intracellular level of cAMP by about two fold in neuroblastoma cells; therefore, some of the effects of sodium butyrate on these cells may in part be mediated by cAMP. Sodium butyrate appears to have properties of a good chemotherapeutic agent for neuroblastoma tumors because the treatment of neuroblastoma cells in culture causes cell death and "differentiation"; however, it is either innocuous or produces reversible morphological and biochemical alterations in other cell types.


On the nature of the presumed receptor for IgE on mast cells. III. Kinetics of the blocking of the PCA reaction by cell-free particulate preparations from rat peritoneal mast cells and effect of pH and calcium concentration on the reaction. The 'binding' of IgE to particulate preparations derived from sonicated purified rat mast cells was measured by the blocking of PCA titrations of the supernatant solutions from incubations with such preparations. It was found that the PCA blocking reaction was inhibited by the addition of calcium ion to the incubations. The blocking reaction was strongly dependent on the pH of the incubations, being maximal at pH values lower than 5-0. The blocking reaction proceeded in a linear manner for at least 3 h provided that no more than 70 percent of the amount of IgE initially supplied had been removed by the particulate fraction. Only mast cell-derived preparations were capable of effecting PCA blocking.


Effect of glucose and sucrose on survival in batch culture of Streptococcus mutans C67-1 and a noncariogenic mutant, C67-25. The growth and survival of two strains of Streptococcus mutans in 5% (wt/vol) glucose or sucrose broth was investigated. S. mutans strain C67-1 showed little loss of viability after 30 h of incubation in batch culture in the presence of either sugar. S. mutans strain C67-25, a noncariogenic mutant of C67-1 that has lost the ability of the latter to produce sticky, insoluble extracellular polysaccharide when grown in sucrose broth, showed a dramatic loss of viability after 30 h of incubation in either glucose or sucrose broth, the effect being most marked in the presence of glucose. The loss of viability was shown to be due to acid production. Insoluble extracellular polysaccharide production appears to be a phenomenon favoring the survival of organisms subjected to high sucrose levels. Other factors must be involved, however, since there are differences between the two strains as regards their survival in glucose broth.


Microcapsule of type III strains of group B Streptococcus: production and morphology. The yield of purified type III polysaccharide of group B Streptococcus was significantly improved by modification of the growth medium. Culture of organisms in standard Todd-Hewitt broth resulted in acid accumulation during the exponential phase of growth and poor yield of type III polysaccharide when extracted from cells by washing with neutral buffer solution. By increasing the buffering capacity of the broth medium, acid accumulation was prevented, and the number of viable cells was increased at the stationary phase of growth. Further, by increasing the concentration of glucose in the buffered medium, the yield of type III polysaccharide was increased two to three times. Electron microscopic investigations of cells grown in the modified broth medium demonstrated a thicker microcapsule than was found in organisms grown in standard broth.


Central regulation of blood eosinophilia by the beta-adrenergic system in rats. The effect of isoprenaline and three different beta-adrenergic blocking agents on blood eosinophilia was evaluated in the rat. Eosinopenic effect of isoprenaline was antagonized by intraperitoneal pretreatment with propranolol and by high but not low doses of practolol. Sotalol, a beta-blocker which in contrast to propranolol does not penetrate the blood-brain barrier, did not affect the eosinophil count when injected intraperitoneally, but produced a marked increase in number of circulating eosinophils following intracerebroventricular injection. Intraperitoneal administration of isoprenaline and propranolol as well as intracerebroventricular injection of sotalol did not affect eosinophil count in adrenalectomized or hypophysectomized rats. These results suggest that beta-adrenergic agents regulate the number of circulating eosinophils through a central mechanism and that presence of both hypophysis and adrenal glands is required for this action.


Comparison between two antibody populations in the EBV system: anti-MA versus neutralizing antibody activity. EBV-neutralizing antibody titers were determined in 11 sera derived from African Burkitt lymphoma or nasopharyngeal carcinoma patients and in the corresponding serum fractions retained above XM 100 Diaflo membranes after low pH treatment, and after recombination of the retained and passed fractions by neutralization of the acidified samples. They were compared with the corresponding antimembrane antigen (MA) titers in seven of the same sera. While all sera tested showed substantial increase of the anti-MA activity in the retained fraction, resulting in a significantly increased mean titer, EBV neutralizing activity did not change at all after identical treatment or changed only in a random fashion, resulting in stable mean titers. It is suggested that anti-MA and neutralizing antibodies are directed against at least partly different antigens on the virus.


Gastric acid secretion, serum-gastrin levels and psychomotor function under the influence of placebo, insulin-hypoglycemia, and/or bromazepam. Gastric acid output, blood-glucose, serum-gastrin and psychomotor-performance were measured in four healthy subjects one hour before and two hours after the intravenous injection of (a) 2ml saline, (b) 0.2 U/kg b.w. insulin, (c) 0.1 mg/kg b.w. bromazepam. Each subject underwent one experiment of each type. The study was layed out as a Latin-square and analysed accordingly. Gastric acid secretion was measured by means of intragastric titration and a telemetering capsule; blood-glucose and serum-gastrin levels as well as psychomotor performance as a measure of vigilance were determined in 15-minute-intervals. In the saline series (a), none of the four parameters showed any systematic variation. In series (b), a bimodal response of acid output to insulin, initial inhibition and subsequent stimulation was observed in all subjects. Serum-gastrin levels showed only a slight and transient increase in the first thirty minutes. Psychomotor performance decreased markedly with progressing hypoglycemia, and increased when glucose levels rose again. In the bromazepan series (c), acid output and psychomotor performance decreased and, after the first hour, increased almost parallely, while glucose and gastrin levels remained unchanged. In series (d), an additive effect of insulin and bromazepam occurred: acid output and psychomotor performance were lower than after insulin alone; peak acid secretion, maximal hypoglycemia and peak of serum-gastrin were shifted to the right. It is concluded that the lowered basal as well as insulin-stimulated acid secretion after bromazepam is due to the central effect of the drug, and that this effect is mediated to the gastric glands directly via autonomic nervous pathways without involving a release of endogenous gastrin.


[Correlation between plasma concentration and clinical effect of neuroleptics and antidepressants]. Results of investigations concerning the correlation between plasma concentrations and clinical effect of neuroleptics and antidepressives are summarised. In the case of neuroleptics there is in general no relationship between clinical parameters of activity and plasma concentration. For antidepressives there is, in most cases, a correlation between side-effects and plasma concentrations. Whether the therapeutic effect is dependent on the plasma concentration or not, is however open to some doubt. The practical value of plasma concentration estimations during therapy with the above mentioned drugs is therefore limited. Inadequate efficacy and/or poor tolerance can occasionally be explained by estimating plasma concentrations which alone, however, scarcely allow one to make prophecies. In determination of the optimal individual dosage must continue to be an empirical process based on clinical observations and experience.


EEG sleep studies of insomniacs under flunitrazepam treatment. This study investigates the effect of flunitrazepam, a new benzodiazepine, on the sleep of insomniac patients under chronic treatment. Polygraphic recordings have shown that this drug decreases not only the activity of the wakefulness system, but also the activity of the synchronizing system of slow-wave sleep. The subjective feeling of improved and sounder sleep seems to be related to a decrease of wakefulness pressure as well as to a decrease of body motoricity, but not with the modification of sleep stages themselves. Flunitrazepam appears to possess some regulatory properties on REM sleep, since this stage is enhanced in patients with an initial low amount of REM sleep and decreased in those having a higher initial REM sleep. Flunitrazepam possesses potent and useful hypnogenic properties in man but does not induce physiological sleep.


A histochemical study of the apparent deamination of proteins by sodium hypochlorite. The possible chemical mechanisms by which neutral solutions of sodium hypochlorite containing a high concentration of sodium chloride abolish the acidophilia of proteins in sections of fixed tissue are reviewed. The most probable one is the chlorination of the protein terminal amino groups, followed by the breakdown of the N-chloramine so formed into alpha-ketocarboxylic acid, nitrile or aldehyde groups. Hypochlorite solutions certainly do not deaminate tissue sections as was previously thought. Experimental evidence for the formation of relatively stable N-chloramine groups in situ and their limited conversion to aldehydes is reported. For example, the acidophilia of hypochlorite-treated sections was found to be restored after flooding them with hydriodic acid followed by the extraction of the liberated iodine with an alcohol. The significance of these experimental findings is discussed.


Effects of dbcAMP and theophylline on rat adrenal medulla grown in tissue culture. Explants of rat adrenal medulla were grown in tissue culture. The effects of various doses of dbcAMP ranging from 0.001 mM up to 1 mM and equimolar amounts of theophylline were recorded by phase contrast optics and catecholamine histochemistry (glyoxylic acid method) over six days. There was a dose-dependent inhibition of the normally occurring outgrowth of Schwann cells, "chromaffin" cells and axons from the explants. Maintenance of glyoxylic acid-induced fluorescence in "chromaffin" cells was dose-dependent, too. Since theophylline is known to enhance intracellular levels of cAMP only, these effects are probably due to the action of cAMP. cAMP obviously maintains the degree of differentiation of chromaffin cells. Thus it could be argued that a certain degree of dedifferentiation is a prerequisite for the formation of axons from these cells.


The possibilities and limitations of membrane methods for the histochemical demonstration of cholinesterases. The thiocholine method for the histochemical detection of cholinesterases according to Karnovsky-Roots was adapted for unfixed cryostat sections by addition of the agar solution to the incubation mixture and by using the semipermeable membrane interposed between the section and the incubation medium. The procedure prevents the leakage of the enzyme activity of the section and is suitable for tissues where the cholinesterase activity is low.


Copper catalyzed alkaline autoxidation of selenocystamine. In alkaline medium and in the presence of cupric ions selenocystamine undergoes autoxidation and is entirely transformed into selenohypotaurine. Among the different metal ions tested, Fe, Co, Ni, Cu, Ag, Mg, Mn, only cupric ions are effective in catalyzing the reaction. The reaction shows an optimum around pH 13. In most respects the autoxidation of selenocystamine is similar to the alkaline autoxidation of cystamine. Some data on the paper and ion exchange chromatographic behaviour of selenohypotaurine and selenotaurine are reported, as also details for the synthesis of selenotaurine.


The binding of nucleotides to 3'-nucleotidase from wheat germ. The 3'-mononucleotidase (3'-ribonucleotide phosphohydrolase, EC 3.1.3.6) from wheat germ has been purified 2,000 fold. The enzyme has a molecular weight of approximatley 32,000, as judged by the use of G-100 gel filtration, and does not attack 2'- or 5'- nucleotides. In order to obtain some indications on the structural requirements for binding and reactivity, the purified enzyme has been subjected to kinetic analyses, including initial velocities with several 3'-ribomononucleotides, inhibition by 5'- nucleotides and nucleotide-analogues, and effect of pH and sulphydryl compounds. The data indicate one base binding site at the active site of the enzyme. This site appears to be the same involved in the binding of both substrates and inhibitors, with higher affinity for purine nucleotides than for pyrimidine compounds, in the order guanosine greater than adenosine greater than inosine greater than uridine greater than cytidine nucleotides.


Improved histological localization of GABA-transaminase activity in rat cerebellar cortex after aldehyde fixation. A method for the chemical fixation of the enzyme GABA-transaminase in nervous tissue is described. It is shown that after perfusion with a formaldehyde/glutaraldehyde fixative, activity of the enzyme in cerebellar cortex is demonstrable whilst cellular morphology is preserved. Results from the improved technique have shown new sites of GABA-transaminase activity in cerebellar cortex. In view of these results a special function for glial cells in this area of brain has been suggested.


Effect of plasma [K+] on the DC potential and on ion distributions between CSF and blood. Keeping the arterial pH at 7.4 and PaCO2 at 40 mmHg in eight anesthetized dogs, we acutely raised plasma potassium concentration from 3.4 to 8.2 meq/1, then allowed it to decay back to control levels. The cerebrospinal fluid (CSF)-blood electrical potential difference (pd) increased 13.2 mV per 10-fold increase in plasma [K+]. Again keeping arterial pH at 7.4 and PaCO2 at 40 mmHg, we elevated plasma [K+] in four dogs from 3.3 to 8.0 meq/1 and maintained this level for 6 h. We found 1) that the PD increased from a control value of +1.3 to +8.9mV, showing no tendency to decay over the 6 h; and 2) that the change in PD did not affect the distribution of Na+, K+, H+, Cl-, or HCO3- between blood and CSF over the 6 h. These results suggest that under these conditions the PD between CSF and blood may play no effective role in determining the distributions of these charged species by 6 h. These results are contrasted with recent findings which suggest that H+ and HCO3- are distributed according to passive forces between CSF and blood.


Ontogeny of tracheal fluid, pulmonary surfactant, and plasma corticoids in the fetal lamb. We examined fetal plasma corticoids and flow rate, electrolyte composition, and surfactant content of tracheal fluid in chronic experiments with eight fetal lambs. From 120 to 148 days of gestation the rate of fluid production was 4.5 ml/kg per h, and there was no change in mean fluid sodium (147.8 meq/1), chloride (153.1 meq/1), calcium (2.2 mg/100 ml), and pH (6.23). Tracheal fluid potassium increased from 4.3 meq/1 at 120-130 days to 8.9 meq/1 at term, while plasma sodium, chloride, calcium, pH, and potassium were constant at 146.1 meq/1, 110.0 meq/1, 12.1 mg/100 ml, 7.39, and 4.0 meq/1, respectively. Plasma corticoids were less than 1.5 mug/100 ml total (0.3 mug/100 ml free) until 130 days, when they increased rapidly to 10.5 total (3.2 free) at 148 days. Surfactant was first detected in tracheal fluid between 124 and 133 days and its secretion increased rapidly after 135 days to a value of 125 mug/kg per h at 148 days. A sudden increase in fetal plasma corticoids does not seem to be the stimulus for appearance of surfactant in the lamb, although these hormones may induce the rapid accumulation of surfactant prior to delivery.


Hydrogen ion concentration and oxygen uptake in an isolated canine hindlimb. Oxygen utilization (VO2) and lactate production by an isolated perfused canine hindlimb was evaluated at various hydrogen ion concentrations. A membrane lung perfusion system was established such that blood flow and temperature could be fixed at normal levels. Oxygen, nitrogen, and carbon dioxide (CO2) gas flows to the membrane lung were independently regulated to provide a fixed arterial oxygen content (CaO2). By changing CO2 flow, the pH of the arterial blood was varied between 6.9 and 7.6 at 10-min intervals. The mean O2 delivery (CaO2 X blood flow) was between 16.3 ML O2/min and 20.5 ml O2/min. Standard error of the mean in each dog, however, was less than 0.4 ml O2/min. VO2 was linearly related to the pH of the perfusing blood: VO2% = 100.1 pH - 643 (r = 0.866). Oxygen consumption was inversely related to PCO2: VO2% = -0.62 PCO2 + 124, but the correlation was less good (r = 0.729). Lactate production was linearly related to the pH of the perfusing blood (above a pH of 7.4): lactate produced = 22.5 pH - 162.5 (r = 0.75). At a pH below 7.4, lactate was not produced. Oxygen consumption of skeletal muscle appears critically dependent on extracellular fluid pH. A change in pH of 0.1 alters VO2 almost exactly 10%. Alkalosis is a potent stimulus to lactic acid production by skeletal muscle.


Cerebrospinal fluid sampling technique and Astrup pH and PCO2 values. The pH and PCO2 values measured by the Astrup technique were compared in cerebrospinal fluid (CSF) obtained using two different sampling techniques: 1) a direct or in vivo technique and 2) the widely accepted syringe sampling technique. In 65 pairs of measurements in 9 dogs it was found that the pH was always overestimated and the PCO2 always underestimated in the syringe sample when compared to the in vivo sample. The equations describing the relationships are as follows: 1) pH (syringe = 0.995 pH (in vivo) + 0.084 and 2) PCO2 (syringe) = 0.873 PCO2 (in vivo) + 0.2. The amount by which the syringe sample underestimated the true PCO2 value increased with the absolute PCO2 value, consistent with the possibility of there being a diffusional loss of CO2 during the transfer of CSF from the syringe to the pH electrode (PCO2 (in vivo)- PCO2 (syringe) = 2.4, 4.9, 7.5, and 10.0 mmHg at in vivo PCO2's of 20, 40, 60, and 80 mmHg). This study indicates that the technique used for sampling CSF is crucial to the expected accuracy of the results and that the number of transfers of CSF during the sampling and measurement procedures should be minimized in order to obtain reliable results.


Hypoventilation in ponies after carotid body denervation. Seven ponies were subjected to carotid body denervation (CD) and two ponies were sham operated (S). Measurement of arterial blood gases and arterial blood and cerebrospinal fluid (CSF) acid-base balance were made prior to and 1,2,4,9, and 17 wks after surgery in unanesthetized animals. Resting ventilation and ventilatory responsiveness to hypoxia and NaCN infusion were assessed prior to and 2,9, and 17  wks after surgery. Alveolar hypoventilation in the CD ponies was marked 1-2 wk after surgery when VE and VA were reduced 40% and 10%, respectively, from control and PaCO2 was 12-15 mmHg above control. However, the effect was not nearly as great 4, 9, and 17 wk after surgery when the PaCO2 stabilized at approximately 6 mmHg above control PaCO2. Arterial blood pH was normal in the hypercapnic CD ponies, but CSF pH remained acid relative to normal throughout the 17-wk period. Changes in ventilatory responsiveness to hypoxia and NaCN tended to parallel changes in resting ventilation. These findings suggest: 1) the carotid bodies are essential in ponies to maintain normal ventilation: 2) in CD ponies peripheral chemosensitivity is partially regained at some unestablished locus; and 3) pH compensating mechanisms in chronically hypercapnic ponies function relatively better in blood than in CSF.


Total and regional cerebral blood flow during moderate and severe exercise in miniature swine. To determine the influence of exercise on cerebral blood flow, we ran 14 swine at 3-6 mph and at 0-10% grades on a treadmill for 30 min at moderate and severe levels of exercise. Measuring heart rate, cardiac output, and aortic pressure via implanted probes, we injected 15-mum radiolabeled microspheres via the left atrium before and during exercise. We measured their distribution by gamma spectrometry, determining total cerebral blood flow, regional blood flow, and ratio of flow to gray and white matter. Heart rate, cardiac output, and aortic pressure rose progressively with increasing exercise. Total cerebral flow resembled that reported in humans, i.e., it did not change significantly with exercise. Regional flow distribution also failed to change significantly with exercise. The ratio of gray to white matter flow did not change except to the cerebellum where it rose significantly from resting values at both moderate and severe exercise. Gray matter received more flow than white matter during all three conditions of observation. Cerebral blood flow was remarkably constant during even severe exercise.


Method for measuring hepatic uptake of oxygen or other blood-borne substances in situ. A preparation is described by which hepatic arterial blood flow and portal venous blood flow can be accurately and continuously measured while simultaneously providing a method by which multiple blood samples can be taken from the hepatic artery, portal vein, and hepatic vein without disrupting hepatic hemodynamics or causing hemodilution. By this means hepatic uptake or release of blood-borne substances can be measured in situ and correlated with hemodynamic parameters. In 13 splenectomized cats, oxygen uptake by the denervated liver was 4.5 +/- 0.3 ml . min-1. 100 g-1 of tissue, representing 54% of total oxygen removed by the splanchnic bed. The hepatic hemodynamics determined by this method are similar to those reported by others in vivo and the metabolic state of the liver remained stable for at least 2 h during which an average of 29 blood samples were taken. Advantages of this preparation over other methods of obtaining similar data are discussed.


Analysis of pesticide residues by chemical derivatization. II. N-methylcarbamates in natural water and soils. A method for the quantitative determination of several N-methylcarbamates in natural waters and the applicability of the derivative to soil samples using a previously published extraction procedure are described. After extraction of the carbamates from the substrate, the carbamates are hydrolyzed in a 10% methanol-potassium hydroxide solution to form the phenolic hydrolysis products, which are isolated and derivatized with pentafluorobenzyl (PFB) bromide to produce the PFB ether derivatives. The PFB derivatives are cleaned up and fractionated on a silica gel microcolumn and determined by electron capture gas-liquid chromatography (GLC). Eight organophosphate pesticides and 2 phthalate acid esters that hydrolyze to phenols or phthalic acid were evaluated as potential interferences and were found not to interfere with any of the carbamates tested. Quantitative determinations of 0.1 mug carbofuran and 3-ketocarbofuran and 0.5 mug carbaryl, metmercapturon, and Mobam in a 1 L water sample are possible. Propoxur was not determined at levels less than 1 mug/L due to the short GLC retention time of the derivative and interferences from the reagents at the lower levels.


Separation of pesticide residues from lipids prior to gas-liquid chromatographic analysis. Lipids are separated from dieldrin, endrin, and p,p'-DDE residues by saponification in ethanolic sodium hydroxide, acidification with dilute sulfuric acid, and adsorption chromatography on deactivated alumina, using petroleum ether as the eluant. Dieldrin, endrin, and p,p'-DDE are efficiently recovered (95-102%), and p,p'-DDT is converted to p,p'-DDE, which is then recovered with high yield (90-96%). Extremely low lipid carryover (less than 0.3-0.5%) is observed for 0.5-1.0 g samples of chicken fat.


Collaborative study of the Food Chemicals Codex method for the determination of the neutralizing value of sodium aluminum phosphate. Fifteen laboratories participated in a collaborative study to evaluate the Food Chemicals Codex method for the determination of the neutralizing value of sodium aluminum phosphate. The AOAC method for determining the neutralizing value of sodium acid pyrophosphate, sec. 8.010, was also included in the study. The precisions of the Food chemicals Codex method, based on the between-replicate standard deviation and on one collaborator making one determination, are 1.16 and 3.66, respectively. The Food Chemicals Codex method for the determination of the neutralizing value of sodium aluminum phosphate has been adopted as official first action.


Affinity chromatography of trypsin and related enzymes. I. Preparation and characteristics of an affinity adsorbent containing tryptic peptides from protamine as ligands. An absorbent for the affinity chromatography of trypsin [EC 3.4.21.4] (AP Sepharose) was prepared. The ligand was a mixture of oligopeptides (mainly di- and tripeptides) containing L-arginine as carboxyl termini, and was obtained from a tryptic digest of protamine. Trypsin was absorbed at relatively low pH (7-4), but was not absorbed at the optimum pH of catalysis (8.2). This was clearly explained on the basis of the pH dependence of the interaction of trypsin with its products. Inactivated trypsin, trypsinogen, and chymotrypsin were not absorbed. The absorption of active trypsin was interferred with by either benzamidine or urea. From these observations, it is evident that AP Sepharose is an affinity adsorbent. AP Sepharose was useful for purification of commercial bovine trypsin. A preliminary application for the purification of Streptomyces griseus trypsin was also successful.


Effects on tryptophyl absorption of the ionization of the catalytic carboxyls in hen and turkey lysozymes. The difference spectra of hen and turkey egg-white lysozymes [EC 3.2.1.17] produced by acidification were measured. The difference spectra of both lysozymes had peaks at 295 and 301 nm which are characteristic of tryptophyl residues. The pH dependence curves of the extinction differences (delta eplision) at 301 nm and 295 nm for hen lysozyme were identical with the corresponding curves for turkey lysozyme. The pH dependence of delta eplision at 301 nm was analyzed assuming that the extinction at 301 nm is due to Trp 108 only, which interacts with the catalytic carboxyls, Glu 35 and Asp 52.  The macroscopic pK values of Glu 35 and Asp 52 in both lysozymes thus determined were 6.0 and 3.3, respectively. These values were in excellent agreement with those determined by measuring the pH dependence of the circular dichroic band at 305 nm (Kuramitsu et al. (1974) J. Biochem, 76, 671-683; (1975) ibid. 77, 291-301). The pH dependence of delta eplision at 295 nm could not be completely explained in terms of the electrostatic effects of the catalytic groups on Trp 108.


Resonance Raman scattering from hemoproteins. Effects of ligands upon the Raman spectra of various C-type cytochromes. Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome f, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b5) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron uas found around 1540 cm=1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm-1) to alkaline (1533 cm-1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome f, the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm-1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c3, the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome b5. Since two axial ligands of the heme iron in cytochrome c3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm-1 instead of the strongest band at 1313 cm-1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical disortion of heme.


Studies on the microsomal electron-transport system of anaerobically grown yeast. III. Spectral characterization of cytochrome P-450. A carbon monoxide-binding pigment which shows an absorption peak at about 450 nm in the reduced carbon monoxide difference spectrum was purified from the microsomal fraction of yeast grown anaerobically. The spectral characteristics of the pigment were practically identical with those of cytochrome P-450 of hepatic microsomes, especially from polycyclic hydrocarbon-induced animals. The pigment was denatured to P-420, and bound with ethyl isocyanide in the reduced state. Although Type I spectral change was not evident, the pigment showed Type II and modified Type II spectral changes upon binding with some organic compounds, as in the case of hepatic cytochrome P-450. These observations clearly indicate that the carbon monoxide-binding pigment of yeast microsomes may be designated as cytochrome P-450 of yeast.


Polarographic studies on ubiquinone-10 and rhodoquinone bound with chromatophores from Rhodospirillum rubrum. Redox components bound with chromatophores of Rhodospirillum rubrum, and pure samples of ubiquinone-10 and rhodoquinone were studied polarographically at 24 degrees. In a mixture of ethanol and water (4 : 1, v/v) at pH 7, ubiquinone-10 and rhodoquinone had half-wave potentials (E1/2) OF +43 MV and -63 mV, respectively. For both quinones, values of the electron transfer number (n) were 2 , and plots of E1/2 versus pH formed straight lines with slopes of -30 mV/pH in the neutral pH range; thus, values of the proton transfer number (n-a) were estimated to be 1 for both quinones. When bound with chromatophores, ubiquinone-10 and rhodoquinone had E1/2 values of +50 mV (n=2) and -30 mV (n=2), respectively, at pH 7. Values of (n-a) were estimated to be 1 for ubiquinone-10 and 2 for rhodoquinone. A component (POC-170) thought to be one of the active center bacteriochlorophylls (Liac-890) was characterized; it has E1/2 value of -170 mV at pH 7 and its oxidation-reduction is possibly brought about by dehydrogenation-hydrogenation. Conceivably, the oxidation-reduction sites of ubiquinone-10, rhodoquinone and POC-170 partly, if not all, exist on the surface of chromatophore membrane or project outside the membrane, because of their accessibility to the polarographic electrode.