A pharmacokinetic model for enterohepatic recirculation in the rat: phenolphthalein, a model drug. A classical compartmental pharmacokinetic model was developed to describe the systemic blood concentration-time profile of phenolphthalein and its glucuronide conjugate (total 3H) following a single intravenous bolus injection of [3H]phenolphthalein. The model incorporates a biliary transport system, including a finite lag time for the biliary phenophthalein glucuronide to be hydrolyzed in the intestine before absorption. Data obtained from bile duct-cannulated animals were fit to the same model excluding any component for intestinal absorption. Agreement between the rate constants obtained for both fits indicates that the model is internally consistent. The model was then used to simulate a 24-hour time-course of phenolphthalein-equivalent blood concentrations which indicates that the long apparent half-lives calculated during this period are artifacts of recirculation.


Pharmacokinetics and analgesic effect of pethidine (meperidine) and its metabolites in the rat. The pharmacokinetics of pethidine (meperidine) and norpethidine (normeperidine) have been investigated after iv administration of pethidine in the rat. The plasma concentration-time curve for pethidine could be described by a triexponential function. The calculated half-lives were 6.0, 18.5, and 64.5 min. Norpethidine, metabolically formed from pethidine, reached maximum plasma concentrations after 30 min and declined biexponentially with half-lives of 66.8 and 301 min. The time course of analgesia after iv administration of pethidine, norpethidine, and p-hydroxypethidine has also been evaluated. When the pharmacokinetic data were compared with the time course of analgesia, the plasma levels of pethidine could be correlated with the analgesic effect after the first rapid distribution phase. The pharmacokinetic constants for pethidine and norpethidine were used to stimulate the plasma levels of these compounds after multiple doses of pethidine. Accumulation of norpethidine was demonstrated to occur, which may be of importance when toxic and analgesic effects of pethidine are evaluated.


Effect of ethanol on the renal excretion and metabolism of choline in the isolated perfused rat kidney. The isolated perfused rat kidney was used to investigate the effect of ethanol on the renal excretion and metabolism of choline. Choline at an initial perfusate concentration of 2.8 mM, with tracer amounts of [methyl-14C]choline, was recirculated through kidneys and radioactivity measured in perfusate, urine, and kidney. 14C-Choline and its metabolites were identified by chromatographic and electrophoretic procedures. Tubular excretion of choline was demonstrated and a transport maximum (Tm) of 1.6 mumol/kidney/min was reached at a choline perfusate concentration of 1.2 mM. Addition of 50 mM ethanol resulted in a 56% increase in the choline Tm and 100 mM ethanol decreased the choline Tm by 25%. The rate of loss of 14C-choline from the perfusate was increased by the lower ethanol concentration and decreased by the higher ethanol concentration. Ethanol at both concentrations diminished the amount of 14C remaining in the kidney. 14C-Betaine was the major choline metabolite and the only 14C-metabolite present in perfusate or urine. Addition of either 50 or 100 mM ethanol increased both glomerular filtration rate and urine volume.


The metabolism of N-hydroxyphentermine by rat liver microsomes. N-Hydroxyphentermine is oxidized to 2-methyl-2-nitro-1-phenylpropane by rat liver microsome preparations. This oxidation accounts for differences noted in levels of N-hydroxyphentermine formed from phentermine in washed and unwashed microsome preparations. The reaction is inhibited by hemoglobin and catalase, whose presence in unwashed microsomes could terminate the N-oxidation of phentermine at the hydroxylamine level. The hydroxylamine-nitro-oxidation appears to be dependent on cytochrome P-450, as the reaction was induced by phenobarbital pretreatment and inhibited by carbon monoxide and 2,4-dichloro-6-phenylphenoxyethylamine.


Biotransformation of 4'-ethynl-2-fluorobiphenyl in the rat. In vitro and in vivo studies. The absorption, metabolism, and plasma pharmacokinetics of a novel anti-inflammatory agent, 4'-ethynyl-2-fluorobiphenyl, was studied in the rat. 4'-[1-14C]Ethynyl-2-fluorobiphenyl was quantitatively absorbed from the gastrointestinal tract. Excretion of radiocarbon into urine was greater than excretion into bile. Appreciate amounts of radiocarbon remained in the carcass 24 hr after dosing. The only metabolite in plasma was (2-fluoro-4'-biphenylyl)acetic acid, which also possessed anti-inflammatory activity. Unchanged 4'-ethynyl-2-fluorobiphenyl was present after administration of higher doses. Peak plasma concentrations of (5-fluoro-4'-biphenylyl)acetic acid were observed within 1 hr of administration. The apparent plasma half-life of this acidic metabolite was 4 hr. The major eliminated metabolite was (4-hydroxy-2-fluoro-4'-biphenylyl)acetic acid. In vivo and in vitro metabolism studies suggest that the major metabolic pathway involves microsomal hydroxylation of the C-H bond of the ethynyl moiety to yield, after rearrangement, a highly reactive intermediate metabolite, 2-fluoro-4'-biphenylylketene.


Disposition of flunisolide in the rat, mouse, dog, rhesus monkey, and cynomolgus monkey. Flunisolide (6 alpha-fluoro-11 beta, 16 alpha, 17 alpha, 21-tetrahydroxypregna-1,4-diene-3,20-dione 16,17-acetonide) administered as a single iv or oral dose to rats, mice, dogs, rhesus monkeys, and cynomolgus monkeys had a plasma t 1/2 of 1-3.5 hr and was eliminated mainly via the bile. After iv administration of 14C-labeled flunisolide, radioactivity was widely distributed into tissues and organs. The apparent volume of distribution of flunisolide in these five species was 3.0-8.0 liters/kg. A major metabolite isolated from rhesus monkey urine was shown to be 6 beta, 11 beta, 16 alpha, 17 alpha, 21-pentahydroxypregna-1,4-diene-3,20-dione 16,17-acetonide (6 beta-OH metabolite). Free 6 beta-OH metabolite was a major radioactive entity detected in urine of all species given radiolabeled flunisolide, whereas flunisolide conjugated with glucuronic acid and/or sulfate was a major metabolite detected in the bile of rats, dogs, and cynomolgus monkeys. Following the oral administration of radiolabeled flunisolide, radioactivity was rapidly and efficiently absorbed in all species, but in the rhesus and cynomolgus monkeys most of the plasma radioactivity was due to the 6 beta-OH metabolite and to water-soluble conjugates, suggesting extensive first-pass metabolism of flunisolide.


Metabolism and disposition by the rat of 35S-sulfadiazine alone and in the presence of trimethoprim. The tissue distribution and metabolism of 35S-sulfadiazine (I, SDZ) alone and in the presence of trimethoprim (TMP) was studied in the male rat. In the 72-hr period following a single oral dose (30 mg/kg) of 35S-SDZ/TMP (5/1, w/w), 87% of the radioactivity was recovered in the urine and 15% of the radioactivity was recovered in the feces. The concentrations of drug-related material in the plasma or tissues after 72 hr were less than 0.1 ppm with the exception of the liver (0.13 ppm). Aside from intact drug, the two major urinary metabolites (greater than 5% of the radioactivity in urine) were N4-acetylsulfadiazine (II) and sulfadiazine N4-glucuronide (VI). Three minor urinary metabolites (less than 5%) were identified as N4-acetyl-2-sulfanilamido-4-hydroxypyrimidine (IV), 2-sulfanilamido-4-hydroxypyrimidine (III) and 2-sulfanilamido-5-hydroxypyrimidine (V). Metabolites IV and V are novel metabolites of SDZ and have not been reported previously for any species. The relative amounts of sulfadiazine and its metabolites excreted in the urine and feces as well as the distribution of intact drug and 35S in rat tissues were determined. The metabolites were screened for antibacterial activity; the N4-acetylated metabolites II and IV were inactive, whereas the hydroxypyrimidine metabolites III and V were active against a few organisms but in general much less active than I.


Retention of cytosine arabinoside in mouse lung following intravenous administration in liposomes of different size. An extrusion technique was used to obtain multilamellar lipid vesicles (MLV, liposomes)  of different size distribution. The larger MLV ranged in diameter from 0.1 to 2.6 mu and the smaller from 0.1 to 1.5 mu, and both were composed of phosphatidylserine/phosphatidylcholine/cholesterol in the molar ratio 1:4:5. After intravenous injection of large and small MLV containing encapsulated [3H]cytosine arabinoside (ara-C), their distribution in various organs showed that the fraction of the dose associated with lung was greater for large MLV relative to small MLV by factors of 3.6--10 after 1 hr, 5.3--14 after 4 hr, and 17--23 after 24 hr. For large MLV more than 50% of drug remaining in vivo after 24 hr was associated with the lung, compared with 2.5% for small MLV. Almost all of the 3H associated with lung at all times for both large and small MLV could be accounted for by unchanged ara-C. Differences in 3H levels between small and large MLV in other tissues were much less dramatic or were not significant. The apparent in vivo stability of the liposomes was not affected by size. The data are consistent with an initial trapping of large MLV during first passage in the lung, with subsequent binding and retention. Release of ara-C from large or small MLV in the lung is apparently slow relative to meatbolism.


Biotransformation of mazindol. II. Absorption and excretion in the dog and man. On administration of tritium-labeled mazindol (l) to man and the dog, absorption was slow but complete. Excretion was slow in both dog and man, but the dog did excrete the drug and/or metabolites faster than man. Blood levels of l in man after a single oral dose of a 2-mg tablet reached a peak value of 2.5 ng/ml, which remained essentially unchanged for at least 6 hr. After multiple doses of a 2-mg tablet t.i.d. for 4 days to subjects who had been receiving nonlabeled l, 2 mg t.i.d. for 6 months, the blood level reached a maximum concentration of 10.8 ng/ml, measured 2 hr after the last dose on day 4. This value is somewhat higher than predicted by the single-dose data, indicating that in man, mazindol does not induce its own metabolism.


Characterization of mephenytoin metabolites in human urine by gas chromatography and mass spectrometry. Metabolites of mephenytoin (5-ethyl-3-methyl-5-phenylhydantoin) were characterized in human urine following chromatography on XAD-2 resin, permethylation, and combined gas chromatography and mass spectrometry. Four glucuronide metabolites previously unidentified in man were characterized as their permethylated derivatives by chemical-ionization and electron-impact mass spectrometry. These metabolites included 5-ethyl-5-(hydroxyphenyl)-3-methylhydantoin O-glucuronide; 5-hydroxyethyl-3-methyl-5-phenyl-hydantoin O-glucuronide; 5-ethyl-5-(hydroxymethoxyphenyl)-3-methylhydantoin O-glucuronide; and a metabolite tentatively identified as 5-ethyl-5-phenylhydantoin N3-glucuronide in which both N-demethylation and glucuronide conjugation of the hydantoin ring have occurred. Mephenytoin, N-demethylmephenytoin, 5-ethyl-5-(hydroxyphenyl)-3-methylhydantoin, and 5-ethyl-5-(hydroxymethoxyphenyl)-3-methylhydantoin were characterized in extracts of enzymatically hydrolyzed urine.


The metabolism of DL-[14C]verapamil in man. The metabolism of DL-verapamil in man was studied after oral administration of an aqueous solution of DL-[14C]verapamil. Between 67 and 71% of the 14C administered was excreted in the urine within 5 days. Verapamil was extensively metabolized; only 3--4% of the dose was excreted in the urine as unchanged drug. Cleavage of the C--N--C bond by N-'dealkylation, preferentially at the C-atom belonging to the shorter side chain, was the main metabolic step. Verapamil and its N-dealkylated metabolites were further metabolized by O-demethylation. Inasmuch as approximately 10 times more of the metabolites formed from the tertiary amine verapamil are secondary amines than are primary amines, it would seem that N-dealkylation of this tertiary amine proceeds at a much higher rate than the N-dealkylation of the secondary amine metabolites to primary amine metabolites.


The disposition of l-3-[(dimethylamino)-(m-dioxan-5-yl)methyl]pyridine in man. l-3-[(Dimethylamino)-(m-dioxan-5-yl)methyl]pyridine hydrochloride (LY 108380) is being evaluated in man as a potentially useful, nonaddicting analgesic agent. This substituted dioxane is structurally different from any currently known analgesic. Following im administration of the 14C-labeled compound to healthy volunteers, the drug was absorbed rapidly (t1/2(abs) = 2--20 min). Pharmacokinetic analyses suggested that LY 108380 was widely distributed and extensively bound in tissues. The drug was not bound to plasma proteins in vitro or in vivo. In the blood, radioactivity was distributed in both red cells and plasma; a cell/plasma radioactivity ratio of 0.5 was maintained for about 1 hr. The t1/2 for elimination of LY 108380-14C from plasma was about 1.3 hr, although radioactivity persisted in plasma for over 100 hr. At the time of peak radioactivity, the parent compound was the major constituent in plasma; quaternary N-glucuronide and N-desmethylated metabolites were also detected in plasma. Levels of radioactivity in saliva were 2--5 times higher than those in plasma shortly after drug administration. About 82% of the radioactivity was eliminated in the urine, 6% in expired air (as 14CO2), and 1% in feces. The major metabolite of LY 108380 (55% of the dose) was a quaternary amine formed by glucuronidation at the pyridine nitrogen. Less than 10% of the dose was N-demethylated to secondary and primary amines, and about 2% was excreted unchanged.


Chemical synthesis and biological occurrence of carboxylic acid metabolites of delta1(6)-tetrahydrocannabinol. Potential metabolites of (-)-3,4-trans-tetrahydrocannabinol (THC) with a carboxylic acid function in the side chain were synthesized as their methyl esters in the delta1(6)-series. The chromatographic and mass-spectrometric properties of the five side-chain homologues were examined and utilized to facilitate the isolation and the identification of the corresponding monocarboxylic acid metabolites of delta1(6)-THC in the mouse, guinea pig, and rabbit formed after ip administration. The metabolites were identified by gas chromatography and mass fragmentography. In mouse liver delta1(6)-THC-7-oic acid and 3'',4'',5''-trisnor delta1(6)-THC-2''-oic acid were identified as metabolites. In guinea pig liver these acids occurred together with 4'',5''-bisnor-delta1(6)-THC-3''-oic acid and 5''-nor-delta1(6)-THC-4''-oic acid. Rabbit liver contained delta1(6)-THC-7-oic acid, 2'',3'',4'',5''-tetranor-delta1(6)-THC-1''-oic acid and 4'',5''-bisnor-delta1(6)-THC-3''-oic acid. Further, the structure of three dicarboxylic acid metabolites of delta1(6)-THC in rabbit kidney were determined by mass spectrometry and proton  magnetic resonance spectroscopy.


Isolation and identification of urinary metabolites of oxycodone in rabbits. Metabolism of oxycodone was studied in the rabbit and found to proceed through five metabolic pathways: O-demethylation, N-demethylation, N-oxidation, 6-keto reduction, and glucuronidation. Six urinary metabolites were isolated and identified in the unconjugated form: 14-hydroxydihydromorphinone, 14-hydroxydihydrocodeine, 14-hydroxydihydrocodeinone N-oxide (oxycodone N-oxide), 14-hydroxydihydroisocodeine, 14-hydroxydihydrocodeine N-oxide, and noroxycodone, together with unchanged oxycodone. Identification was made by means of various chromatographic and spectral comparisons with authentic samples. Oxycodone, 14-hydroxydihydromorphinone, 14-hydroxydihydrocodeine, 14-hydroxydihydroisocodeine, and noroxycodone were also identified as aglycons of conjugated metabolites.


Formation of two metyrapone N-oxides by rat liver microsomes. In the presence of phenobarbital-pretreated rat liver microsomes and under oxidative conditions, metyrapone is transformed in vitro into reduced metyrapone and two other metabolites. In an effort to further characterize those metabolites, large-scale incubations of metyrapone were performed. Untransformed substrate and metabolites were extracted into chloroform under alkaline conditions and separated by thin-layer chromatography. The nature of the metabolites as N-oxides located on either pyridine ring was established by physical methodologies, mainly electron-impact and chemical-ionization mass spectrometry, and also by chemical reactions with titanous chloride. The formation of both N-oxides was increased in microsomes from phenobarbital-, but not from 3-methylcholanthrene-pretreated animals. N-Oxide formation during metyrapone metabolism might be an important step in its inhibitory action on the cytochrome P-450-mediated drug metabolism.


Metabolic reduction of 1-nitrosoadamantane by rabbit liver microsomes. Properties of a C-nitroso reductase system. Washed microsomes from rabbit liver reduced 1-nitrosoadamantane to N-hydroxy-1-aminoadamantane in the presence of a cofactor solution under aerobic conditions; no further reduction of the hydroxylamino metabolite to 1-aminoadamantane (amantadine) occurred. Reduced pyridine nucleotide cofactors are needed for the metabolic reduction. The rate of formation of N-hydroxy-1-aminoadamantane depended upon the microsomal protein content, the time of incubation and the concentration of 1-nitrosoadamantane incubated. The metabolic reduction occurred in air as well as under nitrogen or carbon monoxide. Cupric chloride, mercuric chloride, cysteamine, FAD, and FMN decreased significantly the C-nitroso reductase. The properties of the C-nitroso reductase differed from those of other microsomal reductive pathways.


Enhancement of reductive metabolism of p-nitrobenzoate and nitrazepam in isolated perfused rat liver by ethanol. Reductive metabolism of p-nitrobenzoate (2 mM) was studied in the isolated perfused rat liver, after acute ethanol dosing, with use of a hemoglobin-free perfusion medium. Formation of reduced metabolites under control conditions (0.3 mumol per g of liver per hr) was enhanced fivefold (1.4 mumol/g/hr) in the presence of ethanol (38 mM), thus reaching hepatic reductase activities occurring under anaerobic conditions (1.4 mumol/g/hr). Ethanol failed to increase hepatic nitro reduction when alcohol dehydrogenase was inhibited by pyrazole. Addition of acetaldehyde led to a marked stimulation of nitroreductase activity. Carbon monoxide did not influence the ethanol-mediated enhancement of nitroreductase activity but almost abolished the enhancement caused by anoxia. Reductive azo cleavage of salazosulfamide was not enhanced by ethanol. When nitrazepam was used as the substrate (1 mM) for the isolated perfused rat liver, addition of ethanol (38 mM) led to an enhanced content of 7-amino derivative in the liver and in the perfusate, whereas the formation of 7-acetylamino derivative remained unchanged. The distribution of nitrazepam in liver and perfusate was not altered by ethanol.


The effect of ammonium sulfate on the metabolism of dimethylnitrosamine and other xenobiotics by rat hepatic microsomes. The addition of 125--1000 mM (NH4)2SO4 to rat hepatic washed microsomal preparations was found to stimulate markedly the rate of in vitro metabolism of the hepatocarcinogen dimethylnitrosamine. Solute treatment also stimulated the activities of NADPH-cytochrome c reductase, NADPH oxidase, the N-oxidation of N,N-dimethylaniline, and the fluorescent interaction of 8-anilino-1-naphthalenesulfonic acid (ANS) with hepatic microsomes. (NH4)2SO4 had a varied effect on the activities of a number of mixed-function oxidase (MFO) enzyme activities. Whereas the activities of aniline 4-hydroxylase and 4-nitrobenzoic acid nitroreductase were enhanced at all solute concentrations, several other MFO enzyme activities were either progressively inhibited or stimulated at low and inhibited at high (NH4)2SO4 concentrations. Solute treatment had no effect on microsomal cytochrome P-450 content but inhibited the activities of glucose 6-phosphatase and UDP-glucuronyltransferase. All of the observed changes in enzyme activities and ANS-microsome fluorescence interaction were found to be reversible when the solute was removed by centrifugation. These findings suggest that (NH4)2SO4 and certain other solutes can reversibly modify the conformation of microsomal membranes in such a manner as to affect microsomal enzyme activities.


Atenolol: a review of its pharmacological properties and therapeutic efficacy in angina pectoris and hypertension. Atenolol is a beta-selective (cardioselective) adrenoceptor blocking drug without partial agonist or membrane stabilising activity. Its profile of action most closely resembles that of metoprolol which differs only in that it has some membrane stabilising activity. Atenolol has been well studied and is effective in the treatment of hypertension and in the prophylactic management of angina. Its narrow dose response range obviates the need for highly individualised dose titration. In patients with angina its long duration of beta-blocking activity allows once daily dosage, whereas other beta-blockers, unless in sustained release dosage forms, need to be given in divided doses. Other beta-blockers can be given once daily in hypertension, but at presnt the evidence for effective control with a once daily regimen is more convincing with atenolol. Further studies are need to clarify any important differences in blood pressure control between the various beta-blocking drugs, both in conventional or sustained release dosage forms. As with metoprolol, atenolol is preferable to non-selective beta-blockers in patients with asthma or diabetes mellitus. Atenolol has been well tolerated in most patients, its profile of adverse reactions generally resembling that of other beta-blocking drugs, although its low lipid solubility and limited penetration into the brain results in a lower incidence of central nervous system effects than seen with propranolol. Atenolol is eliminated virtually entirely as unchanged drug in the urine and dosage needs to be reduced in patients with moderate to severely impaired renal function (glomerular filtration rate less than 30 ml/min). There is no need for modification of dosage of atenolol in liver disease.


[Pharmacological activity of N-basic substituted derivatives of 1,3-dimethylxanthine]. The authors carried out studies on cats, guinea pigs, rats and mice, using isolated smooth muscle organs (bronchi, ileum of guinea pig), and examined bronchodilatating, diuretic, antihistaminic action and acute toxiticy of three N-basic substituted derivatives of 1,3-dimethiylxanthine (theophyline) namely: 7-bis-(2-hydroximethyl)-amino-ethyl theophyline under the form of a base (compound G111, tartarate (G112) and nicotinate (G113). The studies were performed in comparison with theophyline. It was found that the substance G113 had a considerable bronchodilatating effect, surpassing in strength and velocity that of theophyline. The examined substances had antihistaminic effect of G113 as the antihistaminic effect of G113 surpassed that of theophyline under experimental conditions in vivo. It was established that all three compounds manifested diuretic activity close to that of theophyline. The derivatives of theophyline, examined by us, had considerably lower toxicity (7,17, resp. 8 times) than that of theophyline. On the basis of the experimental results and some advantages of the examined compounds (good solubility, low toxicity) perspectives are seen for their eventual clinical trial as bronchodilatating and diuretic drugs.


[Inhibition in neurophysiology].

[Inhibition in neurophysiology]. In Neurophysiology, inhibition is the effect of one neuron upon another tending to prevent it from initiating impulses. At cellular level, there are 3 modes of inhibition: postsynaptic inhibition, presynaptic inhibition and electrical inhibition. The formers are mediated by chemical transmitter. Examples of functional inhibition are presented. They explain its role in the coordination of movement and posture, in the integration of the sensation and in the behaviour.


[Models of inhibition in animal behavior]. The concept of inhibition in psychopharmacology is discussed by presenting different models of behavioral inhibition in animals. The inhibitions induced by fear or anxiety appear as a result of punishment introduction into an unknown situation, frustration... These inhibitions are particularly sensitive to minor tranquilizers. The inhibitions induced by "fatigue" appear when avoidance situations continue for 24 hours or when a situation demands a marked increase of animal activity to obtain the same reward (food). These inhibitions are particularly sensitive to psychoanaleptics. The inhibition induced by placing a animal in an inescapable situation is particularly sensitive to antidepressants.


[Inhibition and psychotropic agents. Therapeutic disinhibitory agents]. The concept of disinhibitory agent is heterogeneous. If the disinhibitory property refers to inhibition in its clinical meaning, all psychotropic drugs can answer the denomination of disinhibitory agent when they are prescribed in some pathological entities. Traditionally this denomination refers to a few major tranquillizers, the antipsychotic action of which is linked to the improvement of the affective indifference and of the decrease of the psychomotor activity. However the pharmacokinetic data reveal that some potent sedative neuroleptics can become disinhibitors at high doses and some disinhibitory agents can become sedative substances at higher doses.


[Neuroleptic disinhibitory agents].

[Neuroleptic disinhibitory agents]. The concept of "disinhibition" appeared in 1956 (Broussolle and Dubor) with a study of the activity of prochlorpémazine in chronic schizophrenia. Each chemical group of neuroleptics includes at least one or two disinhibitory compounds. This activity intervenes upon the affective and intellectual life without necessarily the usual extra-pyramidal akathisia side-effects. Psycho- and sociotherapy are strongly adviced with disinhibitory effect.


[Anxiolytic drugs and inhibition].

[Anxiolytic drugs and inhibition]. The term "désinhibition" has a different meaning for the psychopharmacologist and for the psychiatrist. The action of anti anxiety agents on animal behavior is described with a special interest for the passive and active avoidance tests. The mode of action and the site of action of benzodiazepines is discussed, focussed on the existence of an endogeneous ligand displacing the binding of diazepam. The differences between low and high doses of anti-anxiety agents are stressed in patients and normal volunteers, and the treatment of inhibition by benzodiazepines is also discussed.


Conformational changes in bovine-liver glutamate dehydrogenase: a spin-label study. A spin-labelled analogue of p-chloromercuribenzoate reacts specifically with glutamate dehydrogenase. The most marked change in the properties of the spin-labelled enzyme is a fivefold decrease in the rate of reduction of the coenzyme by L-glutamate and no change in the rate of oxidation by 2-oxoglutarate. The electron spin resonance spectrum is a sensitive probe for the conformational state of the enzyme. Spin-labelled glutamate dehydrogenase in the presence of saturating concentrations of NADPH and 2-oxoglutarate or L-glutamate shows a complete conformational change while in the presence of NADP+ and 2-oxoglutarate only half of the protomers have changed conformation. The conformational change upon addition of NADPH to the spin-labelled glutamate dehydrogenase in the presence of 2-oxoglutarate happens in a concerted way between 20 and 80% saturation with NADPH. One of the conformations is favoured by the activator ADP while the other is favoured by the inhibitor GTP.


Magnetic-resonance studies of the geometry of bound substrate, coenzyme and activator on bovine-liver glutamate dehydrogenase. ADP and ATP with a spin-label linked to the terminal phosphate are activators of glutamate dehydrogenase and bind to the same site as the activator ADP. There is hardly any interaction with the coenzyme site. Glutamate dehydrogenase can be modified with a ketone spin-label at a site in the active centre[Andree and Zantema, (1978) Biochemistry, 17, 778--783]. The spin-labelled activators interact with ketone spin-labelled glutamate dehydrogenase in the same way as with native glutamate dehydrogenase relative to the activator site, but show a stronger binding to the coenzyme site. Upon binding to the coenzyme site a spin-spin interaction between the ketone spin-label and the spin-labelled activators is observed. Nuclear magnetic resonance studies of the linewidth of 2-oxoglutarate and NADP+ bound to their functional sites on glutamate dehydrogenase without and with spin-labels result in distances between the ligand nuclei and the spin-labels. The results show that NADP+ binds in an open conformation consistent with the conformation in other dehydrogenases. The activator ADP binds in the neighbourhood of the active centre, but with very little or no overlap with the coenzyme site.


Amino acid sequence studies of horseradish peroxidase. Amino and carboxyl termini, cyanogen bromide and tryptic fragments, the complete sequence, and some structural characteristics of horseradish peroxidase C. Horseradish peroxidase C dominates quantitatively among the isoperoxidases of horseradish root and has an isoelectric point close to 9. It consists of a hemin prosthetic group, 2 Ca2+ and 308 amino acid residues, including 4 disulfide bridges, in a single polypeptide chain that carries 8 neutral carbohydrate side-chains. The molecular weight of the polypeptide chain is 33890. Assuming an average carbohydrate composition of (GlcNAc)2, Man3, Fuc, Xyl for each carbohydrate chain, the molecular weight of native horseradish peroxidase C is close to 44 000. Cyanogen bromide fragments of reduced and carboxymethylated apo-peroxidase were purified by a combination of gel filtration and isoelectric focusing in urea, and cystine-containing tryptic fragments of apo-peroxidase were purified by gel filtration followed by disulfide cleavage and rechromatography at the initial conditions. The present paper discusses (a) isoelectric points and charge distribution within the native protein, the apoprotein and the cyanogen bromide fragments, (b) a buried pyrrolidonecarboxylyl amino terminus, (c) heterogeneity at the carboxyl terminus, and (d) a possible domain structure, likely from partial tryptic digestion.


Artifacts imitating aging of glucose-6-phosphate dehydrogenase in human erythrocytes. Data in the literature based on the technique of graded osomotic hemolysis have been re-evaluated. Differences were previously found in the glucose-6-phosphate dehydrogenase/hemoglobin ratio and in the heat stability of the enzyme in hemolysates of 'old' and 'young' cells. These differences were believed to be due to the aging of the enzyme. As the erythrocyte membrane acts as a molecular sieve under hypotonic conditions [cf. Cseke, E., Váradi, A., Szabolcsi, G., and Biszku, E. (1978) FEBS Lett. 96, 15--18], the hemolysate obtained when a fraction is lysed does not properly represent the content of the lysed cells. As hemoglobin is lost from cells which are not yet lysed, the enzyme/hemoglobin ratio is underestimated in 'old' cells and overestimated in 'young' cells. It is further shown that the observed differences in the heat stability of glucose-6-phosphate dehydrogenase in the fractions obtained by graded hemolysis are due to the presence of different concentrations of endogeneous NADP. Therefore the published data obtained by graded osmotic hemolysis do not prove the assumption that the enzyme is aging during the lifetime of the erythrocyte.


Interaction of ADP with skeletal and cardiac myosin and their active fragments observed by proton release. The technique of proton release measurement has been used to explore the binding of ADP to skeletal and cardiac myosins and their active fragments in a variety of conditions. It has proved possible to obtain binding profiles on intact myosin in the filamentous, undissolved form in physiological solvent conditions. Binding constants are given. At higher ionic strength (0.5 M potassium chloride) the binding profile of magnesium-ADP. is compatible with the presence of two types of site, differing from one another both in respect of affinity and the number of protons released per site. Studies with cardiac myosin reveal no such indications of heterogeneity, and are consistent with the presence of a single population of thermodynamically indistinguishable sites. In the absence of divalent cations, in solutions containing potassium ions and EDTA, ADP binds with absorption rather than liberation of protons. The pH profile of proton absorption at saturation can be fitted in terms of an ionising group with an unperturbed pK of 9.4, and at least one of lower pK(5.9). The dissociation constant (pH8 at 5 degrees C) is about 8 microM, and the affinity for uncomplexed ADP is thus only slightly weaker than that for magnesium-ADP


Binding of coenzyme and substrate and coenzyme analogues to 6-phosphogluconate dehydrogenase from sheep liver. An X-ray study at 0.6 nm resolution. The analogues of the coenzyme NADP+, nicotinamide--8-bromo-adenine dinucleotide phosphate (Nbr8ADP+) and 3-iodopyridine--adenine dinucleotide phosphate (io3PdADP+), were prepared. Nbr8ADP+ was found to be active in the hydrogen transfer adn io3PdADP+ is a coenzyme competitive inhibitor for 6-phosphogluconate dehydrogenase. The binding of NADP+, NADPH and NADPH together with 6-phosphogluconate as well as that of both analogues to crystals of the enzyme 6-phosphogluconate dehydrogenase has been investigated at 0.6-nm resolution using difference electron density maps. The molecules bind in a similar position in a cleft in the enzyme subunit distant from the dimer interface. The orientation of the coenzyme in the site has been determined from the io3PdADP+ -NADP+ difference density. The ternary complex difference density extends beyond that of the nicotinamide moiety of the coenzyme and tentatively indicates substrate binding. No clear identification of the bromine atom of Nbr8ADP+ can be made. However, the analogue is bound more deeply in the cleft than is NADP+. The NADPH density is the most clearly defined and has thus been used to fit a molecular model using an interactive graphics system, checking for preferred geometry. A possible conformation is presented which is significantly different from that of NAD+ in the lactate dehydrogenase ternary complex.


Effects of DMSO, pH, stretch and calcium on the thick filaments in an amphibian smooth muscle. Conventional fixation with glutaraldehyde fixatives at pH 7.4 did not preserve/promote thick filaments in Bufo smooth muscle. However, if pH was lowered to 6.0, thick filaments were present and addition of dimethyl sulfoxide (DMSO) led to a greater number of thick filaments. Stretch alone had little effect on the presence of thiber seen. Calcium had little effect on the numbers of thick filaments, but it affected the appearance of the thick filaments. Low calcium (10(-7) M) caused a higher proportion of rod-shaped filaments, while in high calcium (10(-3) M) most thick filaments were ribbons. The great lability of the thick filaments in amphibian smooth muscles makes them ideal for studying factors which affect the appearance of thick filaments in smooth muscle, but it also raises the question of the degree of aggregation of myosin in the living cell.


Studies on the absorption, distribution and elimination of 6-o-chlorophenyl-2,4-dihydro-2(N-methyl-piperazin-1-yl)-methylene-8-nitro-1H-imidazo[1,2-a] [1,4]benzodiazepin-1-one methanesulphonate in the male rat and rabbit. The fate of a novel imidazo-benzodiazepine (I) was studied in male rats and rabbits using 14C and 3H-labelled I. In both species the compound was rapidly and widely absorbed after an oral dose of 5 mg/kg to give peak tissue and plasma levels after 1 hour in the rat and 4 hours in the rabbit. The highest concentrations of radioactivity were present in the liver (rat) and liver, kidney and subcutaneous fat (rabbit). Plasma levels of radioactivity fell to 3% of the maximum value in 24 hours in the rat but 48 hours were required for a similar fall in the rabbit. The main route of elimination of radioactivity was via the bile followed by excretion in the faeces. For the rat the rate of biliary elimination was 16.6% of the administered dose/hour; for the rabbit this rate was 5.6%/hour. Recovery of administered radioactivity during 0-24 hours for urine and faeces respectively was 4.8% and 69% for the rat and 23.2% and 10.9% for the rabbit. Up to 97% of the radioactivity administered to rats could be recovered in the excreta in the 7 days following dosing. Up to 90% of the dose administered to rabbits appeared in the excreta during 10 days. No unchanged (I) could be detected in the urine or bile. The radioactive metabolites were polar products, some of which were in the form of glucuronide conjugates.


Metabolites of 3-hydrazino-6-[bis-(2-hydroxyethyl)amino]pyridazine dihydrochloride in rat urine. In 24 hr urine of rats orally given 150 mg/kg of 3-hydrazino-6-[bis-(2-hydroxyethyl)amino]pyridazine dihydrochloride (DL 150), no unchanged compound was detected. Three metabolites, less polar than DL 150, were isolated, their structures assigned by UV, MS, IR and 1H NMR spectroscopies, and confirmed by synthesis. They are: 3-[bis-(2-hydroxyethyl)amino]-6-isopropoxypyridazine (1); 3-[bis-(2-hydroxyethyl)amino]pyridazine (2); 3-methyl-6-[bis-(2-hydroxyethyl)amino]-s-triazolo[4,3-b]pyridazine (3). The metabolism of DL 150 in the rat follows some of the metabolic pathways reported for hydralazine.


Alterations in regional cerebral hemodynamics and metabolism produced by focal cerebral ischemia. Since diffuse changes in blood flow and metabolism occur in the brain following a focal ischemic insult, changes of cerebral blood flow on the side opposite a cerebral infarction were observed. We have examined the course of the cerebral hemodynamic changes occurring in 15 patients with unilateral acute strokes. In 12 of these patients, significant blood flow changes (over 15%) occurred in the nonischemic hemisphere during the period of observation. A similar time course of change was found in the ischemic hemisphere and the flow changes in the two hemispheres appeared parallel to one another (diaschisis). Thus we have demonstrated a significant reduction in flow in the contralateral cortex following a unilateral ischemic insult. This reduction in flow was of the order of 38% from the control value which agrees quite well with the reduction in cortical glucose consumption of the order of 31%. We postulate that these changes are the hemodynamic and metabolic concomitants of diaschisis and may in part be due to a transneuronal depression of function.


Precordial mapping of ST-segment elevation and Q waves following anterior myocardial infarction. The effects of established beta-blocking drugs. The electrocardiographic (ECG) signs of ST-segment elevation and the development of Q was using 72-lead precordial surface mapping, and the release of creatine kinase (CK) activity has been studied in 47 patients with uncomplicated anterior myocardial infarction. These findings were compared with a further nine patients who had acute myocardial infarction but were receiving long-term beta-blocking drugs. It was found that ST-segment elevation and Q waves had rapidly changing and different natural histories and that beta-blocking drugs altered the natural history of ST-segment changes but had no effect on the pattern and time course for the loss of electrically active myocardium. There was a close relationship between the precordial area of ST-segment elevation at 2--3 h and the final development of Q waves in the patients with uncomplicated anterior myocardial infarction. No similar relationship could be found in those on beta-blocking drugs. The pattern of changes in plasma CK and its MB isoenzymes activity were similar for both groups. The relationship between early ST-segment elevation and the final area of Q waves may prove useful in clinical practice. This may not apply where beta-blocking drugs are commenced before the initial recording of ST-segment elevation.


Calcium overload and mechanical function in posthypoxic myocardium: biphasic effect of pH during hypoxia. Mechanical function and calcium accumulation in the myocardium during and after hypoxia were examined in the isolated but arterially perfused interventricular rabbit septum. The pH of the perfusate during hypoxia was varied from 7.4 to 6.6 by increase of the pCO2. All septa were reoxygenated for 30 min at pH 7.4. In the posthypoxic period the recovery of developed tension was greatest and the magnitude of contracture least in those septa perfused at pH 6.8 during hypoxia; calcium overload did not occur. By contrast, marked calcium overload (3.5 mumol/g wet wt) occurred in septa perfused at pH 7.4 during hypoxia. Reduction of pH to 6.6 during hypoxia did not result in a greater degree of recovery of developed tension or complete reversal of contracture in the posthypoxic period, and marked calcium overload was not prevented. These results indicate that: (1) partial recovery of mechanical function in the posthypoxic period can occur concurrent with a net gain of calcium; (2) the beneficial effects on recovery in the posthypoxic period in septa perfused at pH 6.8 during hypoxia may be in part released to prevention of calcium overload; (3) the beneficial effects of acidosis are lost when the perfusate pH is reduced to 6.6 during hypoxia.


Adrenergic- and cyclic nucleotide-induced glycerol release from brown adipocytes. Previous work has shown that although both alpha- and beta-agonists stimulate brown fat heat production, the beta agonist is quantitatively more effective. The finding in the present study that isoproterenol, a beta-agonist, significantly stimulates net glycerol release from isolated hamster brown adipocytes while the alpha-agonist, phenylephrine does not, may account (at least in part) for the differential thermogenic response to the two types of agents. The present study also compared lipolytic responses to AMP, cAMP, cGMP and theophylline, with the results indicating a greater rate of net glycerol release being induced by AMP and by cAMP than by theophylline or cGMP plus theophylline.


Effects of alternative transmitter amines on cyclic AMP formation in rat brain tissue. Aromatic amines and related compounds, some of which are taken up and released from nerve terminals, might act at brain receptors ordinarily stimulated by traditional amine neurotransmitters. Several of these compounds were evaluated for their ability to stimulate or impede synthesis of cyclic AMP in rat striatal homogenates and cortical slices. In contrast to catecholamines, most had no effect, consistent with their possible role as false transmitters.


Dopamine-sensitive adenylate cyclase in homogenates of rat nucleus accumbens: structure-activity studies and effects of agonists and antagonists. A study has been made of the structural requirements for activity on the dopamine-sensitive adenylate cyclase present in homogenates of rat nucleus accumbens. The only active phenylethylamine derivatives tested were those containing hydroxy groups at the 3 and 4 positions on the benzene ring, a two carbon side chain and a terminal nitrogen, either unsubstituted or containing a single methyl group. The alpha- and beta-adrenergic agonists, phenylephrine and isoprenaline respectively, were both inactive. Norsalsolinol was a weak agonist producing only a 50% stimulation of adenylate cyclase activity. The typical neuroleptic drugs, fluphenazine and cis-flupenthixol were both potent antagonists of the dopamine response as opposed to the atypical neuroleptics, metoclopramide and sulpiride, and the alpha- and beta-adrenergic blocking agents, phentolamine and propranolol respectively, which were all inactive. Our results indicate that the dopamine receptors associated with adenylate cyclase in the nucleus accumbens are similar to those in the corpus striatum.


The mode of action of phenobarbital on the excitable membrane of the node of Ranvier. Single myelinated nerve fibres of Rana esculenta were investigated under current and potential clamp conditions at 20 degrees C. Under 2.5 mM phenobarbital, the amplitude of the action potential was reversibly reduced to 85.5 +/- 5% (n = 6), and the threshold potential was raised by 32% of the control in Ringer solution. The resting potential remained constant. Solutions with 2.5 mM phenobarbital caused a decrease of the Na current to 38.3 +/- 5.6% (n = 10), when the fibre had its holding potential before the test step. The effect was reversible at wash out of the drug. The Na currents were only negligibly decreased (7 +/- 4.5%; n = 10) when the test pulse was preceded by a long lasting negative polarization to Em = -140 mV. The effect of the conditioning polarization could be described by two time constants, tau 1 = 7.1 +/- 2.0 msec and tau 2 = 44.5 +/- 9.5 msec (n = 5). Experiments with 500 msec conditioning pulses showed that the Na inactivation curve, h infinity(Em), was shifted in a negative direction along the potential axis. In the range between 0.5 and 5.0 mM phenobarbital there was a shift of 8 mV for an e-fold change in drug concentration. 15 mM Ca2+ caused a shift to the h infinity(Em) curve in a positive direction along the potential axis, while simultaneous application of 2.5 mM phenobarbital and 15 mM Ca2+ caused no shift of the h afinity(Em) curve. The undissociated drug seemed to be responsible for the effects (pK = 7.3).


Action of putative neurotransmitters in the guinea pig cochlea. Scala tympani of the guinea pig was perfused with artificial perilymph containing putative transmitter substances. Single auditory nerve fibre activity was recorded during these perfusions and the responses of these fibres were studied using computer-controlled routine. Acetylcholine significantly inhibited tone-induced responses of a large proportion of the fibres tested. Glutamate appeared to be a potent depolarising agent when applied in concentrations between 2 and 10 mM. Noradrenaline did not affect tone-induced activity, but inhibited the spontaneous activity of some fibres. The results support the view that acetylcholine is the transmitter at synapses of the olivocochlear bundle, and that glutamate may be involved in the afferent synapse. The data do not support the hypothesis that noradrenaline is the afferent transmitter.


Solubilization of unconjugated bilirubin by bile salts. Freshly precipitated unconjugated bilirubin (UCB) is solubilized rapidly and to a large extent by the sodium salts of di- and trihydroxy bile acids. The solubilization effect depending on bile salt concentration, pH and ionic strength is based on micellar mechanisms.


Protein methylase from calf liver nuclei: enzyme characterization and stimulation by serum albumins. A protein methylase from calf-lifer nuclei was partially purified by sonication of the nuclear pellet at high ionic strength, chromatin removal and ammonium sulphate fractionation of the solubilized activity.


Neurotransmitter synthesis in Limulus ventral nerve photoreceptors. Radiochemical precursor compounds for neurotransmitters were incubated with Limulus ventral nerve photoreceptor preparations. Octopamine was preferentially synthesized by a photoreceptor rich fraction of the nerve, acetylcholine was made by a photoreceptor poor fraction, and gamma-aminobutyric acid was made about equally well in both fractions. The possibility that the ventral nerve photoreceptor cells serve a neurosecretory function in the adult Limulus is discussed.


pH and calcium concentration changes in a molluscan egg during development. During development, the egg albumen of calcified land snail eggs becomes more and more acid, correlated directly with a constant rise in the calcium concentration of this albumen. It is suggested that the developing embryo releases some acid metabolite and the subsequent change in albumen pH aids in embryonic absorption of the CaCO3 (calcite) egg shell, used for making the embryonic body shell or skeleton (CaCO3 in the form of aragonite).


Direct evidence for pH-dependent Fc receptors on proximal enterocytes of suckling rat gut. By means of an erythrocyte-antibody rosette technique, Fc receptors, functional at pH 6.0 but not at 7.2, were shown to be present on enterocytes isolated from duodenum and jejunum (but absent from ileum) of 12-20-day-old suckling rats.


Human cervical mucus. V. Oral contraceptives and mucus rheologic properties. Mucus viscoelasticity on individual samples obtained from patients using combination oral contraceptives was quantitated by microrheometry. These results, in conjunction with mucus chemical characterization, indicate that combination oral contraceptive use eliminates the cyclic variations in mucus chemical, physicochemical, and rheologic properties associated with the ovulatory menstrual cycle. A correlation was demonstrated between the mucus elastic modulus and mucus nondialyzable dry weight, and the mucins produced during oral contraceptive therapy were shown to be similar to those recovered from ovulatory donors. Differences in mucus properties were noted when donors using estrogenic contraceptives were contrasted with those using androgenic contraceptives. On the basis of established relationships between sperm penetrability and mucus solids content, it was concluded that the use of contraceptives, as examined in this study, provided a secondary degree of fertility control at the cervical level.


On stability of oral poliovirus vaccines. A stabilizer which prevents oral poliovirus from degradation is described. It contains mainly phosphate buffer 0.3 mol, at a pH of 6.7.


Report of IABS committee on avian product standardization. The International Association of Biological Standardization (IABS) organized an international symposium in August 1973 on the "Requirements for Poultry Virus Vaccines" at Lyon (France). The Proceedings of this symposium were subsequently published as "Developments in Biological Standardization", volume 25. At the conclusion of this symposium, it was resolved that an Avian Biological Products Standardization Committee should be established, this committee including four subcommittees, each under the direction of a chairman who would be responsible to make a report on his own subject. The four subjects are: specific pathogen freedom (SPF), infectious bronchitis, Marek's disease, Newcastle disease. The work established by the Committee is presented.


Studies on dehydrogenases of the glucuronate-xylulose cycle in the livers of diabetic mice and rats. In view of reports that accessory pathways of glucose oxidation are enhanced in the diabetic state, we have determined the levels of key enzymes of the glucuronate-xylulose cycle in the livers of diabetic mice and rats. Genetically diabetic mice (db/db) were found to have increased levels of two NADP-linked enzymes of this cycle [NADP-xylitol dehydrogenase and NADP-L-hexonate dehydrogenase (aldehyde reductase II)], whereas other NAD- and NADP-linked dehydrogenase activities of the pathway were not changed. On the other hand, the livers of streptozotocin-diabetic mice and rats showed normal levels of all these enzymes. In the course of this study, evidence was obtained for the presence in db/db mouse liver of low molecular weight material inhibitory for glucose 6-phosphate dehydrogenase. The use of these animal models in diabetes research is briefly discussed.


Treatment of severe diabetic ketoacidosis. A comparative study of two methods. Patients with severe diabetic ketoacidosis (pH less than 7.10) were treated according to two protocols. Protocol I consisted of high-dose insulin therapy by intravenous and intramuscular injections and bicarbonate infusion and was used in the first 12 patients; they received an average of 260 U insulin and 167 mmol bicarbonate in the first 6 h of treatment. Protocol II consisted of low-dose continuous intravenous insulin therapy, 8 U/hour, without bicarbonate in a further 12 patients. Rehydration and potassium-supplementation were the same in both methods. Basal data of both groups were not significantly different. The fall of plasma glucose concentration, rise in arterial pH and decrease in 3-hydroxybutyrate were similar in the two groups. The mean time to achieve a pH equal to or greater than 7.30 was 6.8 hours in the high-dose group and 7.6 hours in the low-dose group (p greater than 0.10). Potassium supplementation and potassium concentration during both treatments were the same. During the low-dose treatment the mean (+/- SD) plasma insulin concentration was 121 +/- 46 microU/ml. The presence of insulin binding antibodies did not result in lower free insulin concentrations. Thus, in the treatment of severe ketoacidosis continuous intravenous therapy with low-dose insulin is as effective as high-dose therapy and bicarbonate-administration is probably unnecessary.


Effect of Methylene Blue on pyridine nucleotides and insulin secretion of rat pancreatic islets. Methylene Blue, which is known to oxidise NADPH in red blood cells, was used to assess a possible role of NADPH in the glucose-stimulated secretion of insulin. When islets from rats were incubated with 3 mg/ml of glucose, Methylene Blue (0.5), 1.0, 2.0 or 5.0 micrograms/ml) significantly decreased the concentration of NADPH, increased that of NADP+ and decreased the NADPH/NADP+ ratio in a dose-dependent manner. This effect was associated with inhibition of the glucose-induced insulin release. No significant change of NADH, NAD+ and ATP could be observed. It is suggested that the secretory response of the pancreatic islet to glucose stimulation depends on the NADPH/NADP+ ratio.


Some determinants of latency of off-response to electrical field stimulation in circular layer of smooth muscle of opossum esophagus. Transverse strips from smooth muscle of opossum esophagus were subjected to electrical field stimulation of intramural nerves to study the latency of the off-response. Latency is the time from the end of the stimulus train to the beginning of the off-response. Latency increased from one end of the smooth muscle segment to the other; it was shortest proximally and longest distally. Variables altering this latency gradient were sought. Strip width and tension had no effect (P greater than 0.02). Longer stimulation trains shortened latency (P less than 0.02). Krebs solution was modified by changing potassium concentration and by the addition of: hexamethonium, 1 x 10(-4) M; D-tubocurarine, 1 x 10(-4) M; bethanechol, 5 x 10(-7) M; carbachol, 5 x 10(-7 M; physostigmine, 1 x 10(-7) M; propranolol, 1 x 10(-6) M; tolazoline, 5 x 10(6) M; norepinephrine, 1 x 10(-5) M; isoproterenol, 1 x 10(-6) M; and dopamine, 1 x 10(4) M. Increased potassium concentrations shortened latency, and decreased potassium concentrations lengthened it (P less than 0.02). Carbachol, bethanechol, physostigmine, and isoproterenol shortened latency (P less than 0.02). Hexamethonium, D-tubocurarine, tolazoline, propranolol, norepinephrine, and dopamine had no effect (P greater than 0.02).


National Cooperative Crohn's Disease Study: study design and conduct of the study. The design and execution of the National Cooperative Crohn's Disease Study are described in this paper. The Study incorporated several noteworthy features developed to meet specific demands of the disease and its therapy. A standard clinical grading system, the Crohn's Disease Activity Index (CDAI) was developed to allow uniform decentralized clinical evaluation and decision-making throughout the 5 yr of the study. All three drugs in widespread clinical use in Crohn's disease were studied both for suppressive and prophylactic efficacy and for toxicity. The study employed a scheme for double-blind evaluation of patient progress which allowed adjustment of prednisone dose according to the degree of illness and ensured continuous monitoring for serious toxicity of any study drug. Results were analyzed primarily by ranking the clinical outcome of every patient according to a uniform and detailed scheme and applying Wilcoxon nonparametric statistics. Outcome was also analyzed by life-table methods. Eleven hundred nineteen patients were entered and 604 patients were randomized at 14 study centers during the 5-yr duration of the study. Twenty patients were eliminated from analysis as not meeting diagnostic criteria for Crohn's disease, and another 15 patients were eliminated as not meeting other preestablished criteria for analysis. Nine percent of randomized patients, equally distributed in the four treatment groups, withdrew as noncompliant. Ninety percent of patients completed all or all but one protocol-specified visits, and 95% completed the final radiologic and sigmoidoscopic evaluation.


National Cooperative Crohn's Disease Study: results of drug treatment. The response of active and quiescent Crohn's disease to prednisone, sulfasalazine, or azathioprine has been studied in 569 patients in a placebo-controlled, randomized, multicenter cooperative trial. The response of active symptomatic disease to prednisone or sulfasalazine was significantly better than to placebo. Response to azathioprine was better than to placebo, but the difference did not reach conventional levels of statistical significance. Patients with colonic involvement were especially responsive to sulfasalazine, and those with small bowel involvement were especially responsive to prednisone. Patients' drug therapy immediately before entry to the study significantly affected subsequent response. For patients with quiescent disease, none of the drugs was superior to placebo in prophylaxis against flare-up or recurrence. There is less than a 5% risk that a clinically significant prophylactic effect of any of the drug regimens was missed.


National Cooperative Crohn's Disease Study: adverse reactions to study drugs. Adverse reactions to the drugs employed in the National Cooperative Crohn's Disease Study were sought prospectively at each patient visit and by retrospective review of all patient charts. Prednisone caused evident side effects in over 50% of patients on high-dose suppressive therapy and in approximately one-third of patients on prophylactic dose. Thirty-two percent of patients on high-dose, and 26% on prophylactic-dose prednisone required dose reduction or withdrawal because of side effects. Comparable figures for sulfasalazine were 14% and 12%, and for azathioprine 32% and 20%. The incidence of nausea, vomiting, or anorexia among patients taking sulfasalazine was 46% and 34%, on high and low dose respectively; however, this incidence was no different than that observed among patients taking placebo. These symptoms occasioned withdrawal from the study of only 4% and 3% of patients on high and low doses of sulfasalazine, respectively. Azathioprine produced leukopenia at a dose of 2.5 mg/kg body weight in 15% of patients and the mean white cell count, lymphocyte count, granulocyte count, and hematocrit all fell significantly in patients on this dose. Pancreatitis occurred in 5% of patients taking azathioprine but in no other patients. Sulfasalazine proved to be the safest effective suppressive drug for Crohn's disease. Prednisone toxicity, though substantial, is acceptable in view of its demonstrated suppressive efficacy. Azathioprine was approximately as toxic as prednisone but no more effective than placebo in suppressing active disease. None of the drugs was effective prophylactically, and all showed appreciable long-term toxicity.


Azathioprine-related pancreatitis in patients with Crohn's disease. Pancreatitis developed in 6 patients in the National Cooperative Crohn's Disease Study. In five of these the diagnosis was confirmed by elevated levels of seum amylase or lipase. All cases were in the 113 patients who received azathioprine and occurred within the first 21 days of treatment. This incidence of pancreatitis was significantly greater than in the patients treated with sulfasalazine, prednisone, or placebo (P less than 0.01).


A trial of sulfasalazine as adjunctive therapy in Crohn's disease. The effect of the combination of sulfasalazine and prednisone has been compared with that of prednisone and placebo in 89 actively symptomatic patients with Crohn's disease in a double-blind, randomized, multicenter controlled trial. The combination was less effective than prednisone alone in treatment of active symptomatic disease. The probability of obtaining this result, if sulfasalazine truly has a clinically useful effect equal to or greater than that specified in the calculation, is less than 1%. Patients who were in remission at the end of 8 wk were rerandomized to receive either the two drugs together or prednisone plus placebo while repeated systematic attempts to withdraw prednisone were made over the next 6 mo. Sulfasalazine showed no prednisone-sparing effect as judged either by outcome ranking or total dose of prednisone consmed by the two treatment groups. However, in this comparison the probability is greater than 5% that, given the results observed, a clinically useful effect of sulfasalazine of specified minimum degree truly exists. It was possible to withdraw prednisone from 25% of patients at the first attempt and ultimately in 37%.


Correlations between clinical, blood leukocyte, and skin test data in the National Cooperative Crohn's Disease Study. Peripheral blood leukocyte counts, percentages of lymphocytes and granulocytes, serum albumin levels, and results of skin tests with five antigens were examined in patients randomized in the National Cooperative Crohn's Disease Study. Inspection for differences from normal and correlations with a variety of variables showed no unusual characteristics except for a high incidence of anergy (31%) at randomization, and ranging up to 47.5% after treatment.


Radiographic findings of the National Cooperative Crohn's Disease Study. On-study barium radiographs of 535 patients in the National Cooperative Crohn's Disease Study have been analyzed for the pattern of distribution of bowel disease and the individual features that characterized bowel involvement. On-study and off-study radiographs of 403 of these patients were compared under code to judge radiographic response to drug treatment and discover correlations of radiographic findings with clinical response. Patients with more clinically active disease had more colonic disease on x-ray. Duodenal abnormalities were recognized in 22% of the patients and radiographically typicaly Crohn's disease of the duodenum in 8%. Recurrent Crohn's disease and that characterized by small bowel obstruction each displayed a characteristic appearance. Overall there was little evidence of radiographic improvement during the study, and little correlation between clinical response and evidence of radiologic improvement. Only patients treated with prednisone for more than 6 mo showed statistically significant radiologic improvement. Patients with definite radiographic progression or regression were found in each treatment group. Both fistula and stricture with obstruction were associated with a poor clinical response to all therapies. In view of the evidence from this study that radiographic findings do not correlate with clinical symptoms or response, the ritual use of x-ray to follow patients with Crohn's disease is unnecessary.


Dementia: differential diagnosis and treatment. With careful differential diagnosis and judicious treatment, you may be able to reverse certain aspects of dementia in the elderly. Drug toxicity, depression, metabolic and endocrine disorders, and other systemic problems are all potentially treatable causes of mental deterioration. Even when the cause cannot be identified, you can still treat the patient's symptoms. Antidepressants, antipsychotics, and antianxiety agents, plus supportive measures, often effect marked improvement.


Involuntary movement disorders--each has a meaning all its own. Degenerative processes in the corpus striatum, neuroleptics drugs, thyroid disease, and Parkinson's disease are all causes of abnormal spontaneous movements in the elderly. Differential diagnosis is essential for selecting the proper therapy, and the patient's history often provides the key.


Gastric acid secretion rate and buffer content of the stomach after a rice- and a wheat-based meal in normal subjects and patients with duodenal ulcer. The study describes gastric acid secretory response to a rice-based and a wheat-based meal over a prolonged period of five hours and buffer content of the stomach in five normal and seven duodenal ulcer subjects from the rice-eating eastern Indian population. The results suggest that (1) the meal-mediated gastric acid secretory response in duodenal ulcer subjects is much higher than in the controls, even though the histamine stimulated response is similar, (2) the type of meal, whether rice and fish based or wheat and meat based, does not influence the acid secretory response, and (3) the duodenal ulcer subjects in this area, two hours after a meal, have a buffer capacity similar to the controls.


Antacids and peptic ulcer--a reappraisal. Antacids can reduce gastroduodenal acidity for long periods if taken in substantial quantities after food. Their healing effect on gastric ulcer is minimal, if present at all, and easily overwhelmed by the benefit obtained from admission to hospital. Intensive antacid therapy appears effective in healing duodenal ulcer and preventing haemorrhage from stress ulcer, and is comparable in these respects with cimetidine but with a higher incidence of side-effects. Clinical impression strongly suggests that antacids relieve pain in peptic ulcer but objective confirmation is lacking.


Partial purification and properties of glucosyltransferase from Streptomyces aureofaciens. Differential centrifugation, precipitation with ammonium sulphate and chromatography on DEAE-cellulose led to a twenty-fold purification of glucosyltransferase from Streptomyces aureofaciens B 96. The Michaelis constants for glucosyluridyl diphosphate (UDP-glucose) was 10.8 microM for 1,2-dihydroxy-9,10-anthraquinone (alizarin) 110 microM; the maximum rate of glucosylation reaction was 5.32 mumol per s per mg protein. The pH optimum was at 7.1; the flat temperature optimum was at 30 degrees C. Using some hydroxy derivatives of 9,10-anthraquinone it was found that the production of glucosides from aglycones with alpha-hydroxyl groups was about 1/8 of the values obtained with beta-hydroxyl substrates. In both types of aglycones the presence of another hydroxyl group led to a higher glucoside production.


Properties of cellulase from Trichoderma viride. Cellulase produced by Trichoderma viride acted on carboxymethyl cellulose with a Km of 4.9 g substrate per litre, showing a pH optimum at 4.5 and a temperature optimum at 55 degrees C. Ag+, Hg2+, Zn2+, Cu2+ and N3- were inhibitory..


[Differential diagnosis with adrenergic beta blockers]. Several beta-adrenergic blocking agents are on the market in Western Germany. They differ not only in their beta-blocking potency and selectivity but also in their unspecific effects, as intrinsic activity and membrane stabilizing properties. Also the pharmacokinetic behaviour varies widely. From the clinical point of view the selectivity is important for avoiding an aggravation of an underlying obstructive lung disease of effects in the peripheral vascular bed. The intrinsic activity on the one hand might be responsible for some side effects like nightmare or headache; the slowing of resting heart rate on the other hand might be less pronounced. The discrepancies of bio-availability might be overcome by increasing the oral dose.


Study of testicular feedback in male rats using artificial cryptorchidism as a model. Plasma LH, FSH and testosterone were measured in testosterone-treated and untreated cryptorchid and castrated male rats. Exogenous testosterone prevented the increase in basal LH but not FSH levels seen in the untreated cryptorchids. Increases in plasma LH and FSH in response to LH-RH were greater in the cryptorchid as compared to the control group and this could not be reversed by exogenous testosterone, suggesting that spermatogenesis-related feedback factors regulate LH as well as FSH at the pituitary level in the intact rat. The results were consistent with a reduced but nevertheless significant secretion of inhibin by the cryptorchid testis. Basal plasma testosterone levels and ventral prostate weights were not significantly different from intact animals.


Meyenburg complexes of the liver and bile cysts as a consequence of hepatic ischemia. Meyenburg complexes and simple bile cysts are described in a patient having polyarteritis nodosa with involvement of the intrahepatic arterial tree. Similar lesions are found in the liver of monkeys subjected to experimental occlusion of the peripheral hepatic arterial tree. It is proposed that a pathogenetic mechanism conducive to the formation of Meyenburg complexes may be hepatic ischemia.


Confirmation of the assignment of the gene for arylsulfatase A to chromosome 22 using somatic cell hybrids. Human/hamster hybrid cell cultures were examined for the presence of ARSA and other marker enzymes. Many of these hybrids were also analyzed for human chromosomes. Our results confirm the assignment of ARSA to chromosome 22.


Differential depletion of T lymphocytes in the spleen of dengue virus-infected mice. Following the i.c. inoculation of dengue type 2 virus (DV) the spleen weight of infected mice was reduced, as was the proportion of cells killed by ATS and complement (T lymphocytes) in spleen-cell suspensions. In DV-infected mice the mean haemolysin titre, 16 days after i.p. inoculation of 4 x 10(8) SRBC, was 47 compared with 406 in normal mice and spleen cells from DV-infected mice produced significantly reduced direct GVH reactivity in Parker strain (PS) infant mice. Adoptive transfer of spleen cells obtained from mice given three weeks i.p. doses of DV or a single i.c. dose, suppressed antigen-specific antibody secretion as detected by Jerne plaque technique. This suppression was abrogated by pretreating the transferred cells with ATS and complement. Thus DV selectively depletes T-lymphocyte subpopulations responsible for helper and effector functions and spares suppressor T cells in the spleen of infected mice.


Adherence of Escherichia coli to human urinary tract epithelial cells. The adherence of Escherichia coli to human uroepithelial cells obtained from midstream urine specimens of healthy women was studied. Bacteria labeled with [(3)H]uridine were used, and unattached organisms were separated from the epithelial cells by vacuum filtration with 5-mum-pore-size Nucleopore membrane filters. These techniques allowed adherence to be measured in large numbers of epithelial cells and overcame the problem of distinguishing experimental bacteria from the indigenous organisms present on uroepithelial cells. Adherence was not appreciably affected by temperature. Adherence was maximal at pH 4 to 5 and at bacterial-to-epithelial-cell ratios of 5,000 or more. The latter observation suggested that there are a limited number of receptors on the epithelial cell surface, an idea which was supported by competition experiments. Adherence occurred within 1 min and then decreased gradually or quickly, depending on the type of bacterial growth medium, to a stationary level of adherence, approximately 50% of that observed initially. The ability of epithelial cells from a single individual to bind E. coli varied in a cyclical and repetitive pattern. Adherence tended to be higher during the early phase of the menstrual cycle and diminished shortly after the time of expected ovulation; adherence frequently correlated with the value obtained on the same day of the menstrual cycle during the preceding months. Adherence was markedly enhanced by bacterial incubation in broth for 72 h and inhibited by alpha-d-mannose. These results suggest that adherence is a complex phenomenon perhaps mediated in part by bacterial pili and mannose residues on uroepithelial cells.


Properties of extracellular neuraminidase produced by group A streptococcus. Extracellular neuraminidase production by group A streptococci was examined in 92 strains. Fourteen of these strains produced appreciable amounts of enzyme; 12 of the neuraminidase-producing strains belonged to T types 1, 4, and 12. Production of the enzyme paralleled bacterial growth in culture and was maximal in medium containing 0.2% glucose. The enzyme produced by one of these strains was partially purified by ammonium sulfate fractionation and filtration on G-200 Sephadex. Its molecular weight was estimated at 90,000. Activity was optimal at pH 5.7 and in the presence of 0.01 to 0.03 M calcium and magnesium cations. The enzyme was stable at temperatures of 4 and 37 degrees C for at least 24 h but was inactivated within 10 min at temperatures of 50 and 65 degrees C. The enzyme hydrolyzed 40% of the sialic acid in bovine submaxillary mucin, but was inactive on sialyl-lactose, porcine submaxillary mucin, oligosaccharides derived from porcine mucin, or human orosomucoid. The Km value for this enzyme with bovine submaxillary mucin as substrate was in the order of 3.6 x 10(-4) M.


The removal of hypnotic drugs from human serum. A comparative investigation of the adsorptive properties of native and agarose-encapsulated resins and charcoal. The adsorptive capacities of some ion-exchange resins and activated charcoal towards the hypnotic drugs, phenobarbital, glutethimide, carbromal, and methaqualone, have been investigated. Furthermore, the properties of some of these resins and charcoal, encapsulated in an agarose matrix in the form of beads, have been compared with those of the adsorbent phases in their native states. In general, the adsorptive capacities of charcoal were at least as good as, and frequently better than those of the resins. Amberlite XAD-type resins had a higher affinity than Dowex-type resins for all the drugs tested, except for phenobarbital, which was not adsorbed to a satisfactory extent onto Amberlite XAD-4. The encapsulation of adsorbent phases into agarose beads of diameter 5-10 mm results in a reduction of the adsorbtion of the drugs. Nonetheless, the remaining adsorptive capacity is sufficient for application in an extracorporeal detoxification system. Moreover, the overwhelming advantage of good haemocompatibility is provided by the agarose-encapsulated adsorbents.


Intracranial heterotransplantation of human hematopoietic cells in nude mice. A panel of established cell lines and many primary cell specimens from lymphomas and leukemias as well as from normal lymphatic tissues were tested for tumorigenicity by intracranial heterotransplantation in nude mice. Not only lymphoma and leukemia cell lines, but also lymphoblastoid cell lines, lacking markers of malignancy, were tumorigenic in the brains of nude mice. These findings indicate that tumorigenicity following intracranial heterotransplantation in nude mice cannot be used as proof for the malignant nature of established cell lines. Heterotraplantation of primary cell specimens yielded only a few tumor takes. When primary cells were infected with exogenous Epstein-Barr virus prior to the transplantation procedure, tumorigenicity could be significantly increased. Cytogenetic evaluation of tumors growing after intracranial transplantation of human hematopoietic cells showed, in some cases, a selection of cytogenetically aberrant cell clones.


Neighborhood health center patients who use minor tranquilizers. Thirty-four ambulatory medical patients who had used minor tranquilizers for varying lengths of time were interviewed to determine their medical and psychiatric statuses, and to learn their own perspective of their medical care. The population was elderly of lower socioeconomic status, and chronically ill (a significant minority with serious and disabling illness). Depressive equivalents, depression, and anxiety were prominent, but clinical states requiring psychiatric care were not. Slightly less than one-half were alcoholic. Forty-one percent took the medications for target symptoms other than anxiety, and 76% believed these agents were efficacious. Chronic users had significantly more chronic medical illness, and significantly more somatization, anxiety, and fatigue.


Pharmacopsychological investigations concerning the combined effects of dipotassium clorazepate and ethanol. The effect of a single dosage of 20 mg dipotassium clorazepate (DPC) on reaction times, with and without administration of ethanol (1 g/kg body weight), was studied in 14 male subjects. When administered in the evening, the sort-term effect of DPC consisted of slightly increased optic reaction times and decreased endurance and concentration. With simultaneous administration of ethanol, all types of performance studied were significantly impaired; impairment was more pronounced than with the ethanol tests. On the following morning at the second retest, performance levels almost completely returned to those at the onset of testing. This restitution, however, was not as pronounced with subjects who had only received DPC.


Alpha- and beta-adrenoceptor blocking properties of labetalol in renin release. The effect of labetalol, an alpha- and beta-adrenergic receptor blocking antihypertensive, on plasma renin activity (PRA) and the hemodynamics of healthy volunteers at rest and during an ergometric exercise test was studied. Oral doses of 200 and 400 mg labetalol were tested against a placebo in a crossover manner. The labetalol plasma concentrations were determined. Systolic and diastolic blood pressures in the supine position decreased after 400 mg labetalol as did the response of the heart rate to exercise. The lower dose decreased the resting heart rate, but had no effect on the heart rate during exercise. The ergometric exercise induced an increase in PRA which was partly inhibited after 200 mg labetalol in a manner similar to that induced by beta-blockers in our earlier studies. After 400 mg labetalol PRA was already increased at one hour at sitting rest and this higher basal level was maintained for four hours. After this higher dose of labetalol the reaction of PRA to exercise was not significantly inhibited. In renin release the vasodilating alpha-blockade thus dominated the beta-blocking property of labetalol at the dose which decreased the blood pressure.


Effects of beta-blocking agent Metipranolol on metabolic variables in patients with ischemic heart disease, hyperkinetic syndrome, hyperthyreosis and in healthy subjects. Metabolic effects of Metipranolol, a new beta adrenergic blocking agent, have been studied in patients with ischemic heart disease, hyperkinetic syndrome, hyperthyreosis and in healthy subjects. Administration of the drug (30 mg per day for one week) resulted in the decrease of noradrenaline excretion, blood free fatty acid level, and in lowering of blood pressure and heart rate, particularly in patients with ischemic heart disease and hyperkinetic syndrome. These alterations were accompanied by alleviation of clinical symptoms. It is suggested that Metipranolol by suppressing the activity of sympathetic nervous system and thereby diminishing lypolysis, exerts favourable clinical effects, most probably related to diminution of myocardial oxygen consumption.


Cutaneous rheumatoid vasculitis.

Cutaneous rheumatoid vasculitis. Rheumatoid arthritis is a multisystem disease, with many clinical forms bearing close resemblance to systemic lupus erythematosus, dermatomyositis or polyarteritis nodosa. Although the involvement of the integument is not as disabiling as the joint disease, the extent of disability may be of sufficient magnitude to necessitate therapeutic intervention. Two patients are presented whose skin involvement was suggestive of cutaneous angiitis and who responded dramatically to treatment using sulfasalazine.


Effects of D and L amino acid residues in linear peptides on carbon-13 nuclear magnetic resonance parameters. The 13C spectra of the linear tripeptidyl diastereoisomers, Gly-Gly-Leu, Gly-Gly-D-Leu, Leu-Gly-Gly, D-Leu-Gly-Gly, Ala3, Ala-Ala-D-Ala, Ala-D-Ala-Ala, Val3, and Val-Val-D-Val are very similar or even identical at pH meter readings of 1.0, 7.0 and 12.0 in D2O. The spectra of Pro-Leu-Gly-NH2 and Pro-D-Leu-Gly-NH2 likewise show only minor differences in 13C chemical shifts (less than 0.4 p.p.m.) under similar conditions. This contrasts significantly with previous findings comparing 13C chemical shifts of cyclo(Pro-Leu) and cyclo(Pro-D-Leu) where major differences in chemical shifts were observed for both residues due to differences in conformational constraints present in these cyclic proline-containing peptides. The least-squares fit of spin-lattice relaxation times (T1) for Pro-Leu-Gly-NH2 and Pro-D-Leu-Gly-NH2 show that it is not possible to fit all the T1 values to a unique and rigid structure whether folded or extended. The glycyl residue undergoes enhanced motion when compared with the prolyl and leucyl residues. Internal motion must be postulated within the proline ring and for the CH3 groups of leucine.


Separation of opioid peptides utilizing high performance liquid chromatography. Chromatographic procedures have been developed for resolving all of the known enkephalins and endorphins on a single column. The effect of eluant pH on the retention times and separation of the enkephalins and beta-endorphin was determined. By combining these separations with a sensitive radioreceptor assay it is possible to assay all of the opioid peptides in the pituitary gland or in various regions of the brain from individual small laboratory animals.


Interaction of radical anion probes with glucoamylase I from Aspergillus niger. The radical anions (SCN)2.- and Br2.- produced during a pulse radiolysis of the respective potassium salts have been used to study the tryptophan residues of the glucoenzyme, glucoamylase I (EC 3.2.1.3.). At neutral pH, Br2.- reacted with the tryptophan residues of glucoamylase I as expected from previous studies of proteins and free amino acids. However, (SCN)2.- at neutral and high pH was surprisingly unreactive towards the native enzyme. Reaction did occur, however, between (SCN)2.- and glucoamylase from which one-third of the covalently bound carbohydrate had been removed, producing a tryptophyl radical. Reaction also occured between (SCN)2.- and glucoamylase I inactivated by treatment with sodium dodecyl sulphate, but the tryptophan residues were not involved. It is concluded from the results that two 'types' of tryptophan residues are found in glucoamylase I; both are attacked by Br2.- but only one type is attacked by (SCN)2.-.


The reaction of solvated electrons with cytosine, 5-methyl cytosine and 2'-deoxycytidine in squeous solution. The reaction of the electron adduct intermediates with water, p-nitroacetophenone and oxygen. A pulse spectroscopic and pulse conductometric study. Using conductivity detection, pulse radiolysis experiments showed that solvent protonation of the electron adducts of cytosine, 5-methyl cytosine and 2'-deoxycytidine occurs with rate constants k greater than or equal to 2 x 10(4) M-1S-1. The protonated electron adducts transfer an electron to p-nitroactetophenone (PNAP) with rate constants ranging from 3.5 x 10(9) to 5.3 x 10(9) M-1S-1. The transfer is quantitative (G = 2.7), as shown by conductometric and spectroscopic measurements. In the presence of O2 no electron transfer to O2 takes place, implying that O2 adds to the protonated electron adduct radicals. No electron transfer from the H- and OH-adducts of the cytosine derivatives, either to PNAP or to O2, takes place near neutral pH. It is suggested that the differences in the reaction behaviour of the H-adduct radicals and the protonated electron adduct radicals towards PNAP can be accounted for if different radicals are formed by H-addition and protonation of the electron adduct. The H atoms most probably add to the C-5-C-6 double bonds, whereas the electron adducts are protonated at N-3 and/or 0-2.


Radioisotopic evaluation of the treatment of Sudek's syndrome with calcitonin. The authors report their investigations in ten patients suffering from Sudek's syndrome of the foot, studied by the use of the radioosteotrope 99mTc-Pirophosphate. Through this radioindicator it has been possible to evaluate qualitatively (scintigraphy) as well as quantitatively (scintimetry) the modifications of the metabolism induced by the administration of calcitonin. The evaluation, whose radioisotopic method is described in detail, has been carried out at various intervals during treatment with calcitonin (from two to six months). The normalization of the readjustment of the bone generally corresponded with marked clinical recovery of the patient.


[Prevention and therapy with immunoglobulin SRK]. In a clinical study the tolerance and efficacy of a gamma-globulin, treated at pH 4, has been studied. This preparation manufactured by the "Zentrallaboratorium des Blutspendedienstes SRK" can be given intravenously without any risk of untoward reactions. It has been applied in high dosages up to 99 g per week. In 15 cases with primary humoral immunodeficiency, the frequency and the severity of acute bacterial infections were markedly reduced or completely absent. In 16 patients without antibody deficiency but suffering from severe septic-toxic infections, results with Immunglobulin SRK were encouraging and warrant further controlled studies.


Cytochemical studies on the localization of 5'-nucleotidase in the acinar cells of the rat salivary glands. The cytochemical localization of 5'-nucleotidase (5'-AMPase), and its validity, were investigated in parotid and submandibular acinar cells of a rat. Biochemical determinations showed that adequate treatment with glutaraldehyde could minimize the loss of enzymatic activity, and that 5'-AMPase and non-specific alkaline phosphatase (beta-GPase) possessed different pH optima. The cytochemical distribution of the reaction products from the 5'-AMPase activity was distinct from those of beta-GPase. 5'-AMPase activity was localized on the surface membranes of acinar, ductal and myoepithelial cells of both salivary glands. beta-GPase activity was evenly distributed on the entire plasma membranes of myoepithelial cells and on the basal plasmalemma of acinar cells. The reaction products, which appeared on the luminal and lateral plasma membranes of the acinar cells, were presumed to reflect the presence of 5'-AMPase, while those on the myoepithelial surface and basal plasma membranes of the acinar cells demonstrated both 5'-AMPase and beta-GPase. The results indicate that 5'-AMPase activity can be utilized as a reliable marker enzyme of plasma membranes in the salivary acinar cells.


PS-5, a new beta-lactam antibiotic. II. Antimicrobial activity. PS-5, a new beta-lactam antibiotic, has relatively potent antimicrobial activity against Gram-positive and Gram-negative bacteria, especially the Enterobacter groups, Serratia marcescens, the Proteus groups and Klebsiella pneumoniae. The activity of PS-5 against many beta-lactamase-producing organisms is greater than that of cefoxitin or cefazolin. PS-5 has good therapeutic activity in mice infected with Staphylococcus aureus Smith or Enterobacter cloacae 45.


Active groups of bicyclomycin and the reaction with thiols. The binding of [14C]bicyclomycin to whole cells of E. coli and to the inner membrane proteins was inhibited by dithiothreitol and 2-mercaptoethanol. The reactivity of the drug with the sulfhydryl group was further studied, using methanethiol as a model compound. The kinetics revealed that the reaction was of pseudo-first-order in excess of thiolate anion. Analysis with gas chromatography-mass spectrometry showed that the main product was an adduct of thiol with bicyclomycin in an equal molar ratio. The structure of the adduct was determined by 1H-NMR spectrometry, showing that thiolate attacked the olefinic double bond of the antibiotic. 3'-Acyl derivatives of bicyclomycin did not significantly affect the binding of [14C] bicyclomycin to inner membrane proteins of E. coli. The results suggested that 4,5-double bond hydrocarbons and 3'-hydroxy group of bicyclomycin participate in the binding to E. coli inner membrane proteins, which are presumably the receptors of the antibiotic. The olefinic double bond seems to be the active center of bicyclomycin, reacting with the sulfhydryl group of the receptor protein, although the whole molecular is needed for the activity.


Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). Buthionine sulfoximine (S-n-butyl homocysteine sulfoximine), the most potent of a series of analogs of methionine sulfoximine thus far studied (Griffith, O.W., Anderson, M.E., and Meister, A. (1979) J. Biol. Chem. 254, 1205-1210), inhibited gamma-glutamylcysteine synthetase about 20 times more effectively than did prothionine sulfoximine and at least 100 times more effectively than methionine sulfoximine. The findings support the conclusion that the S-alkyl moiety of the sulfoximine binds at the enzyme site that normally binds the acceptor amino acid. Thus, the affinity of the enzyme for the S-ethyl, S-n-propyl, and S-n-butyl sulfoximines increases in a manner which is parallel to those of the corresponding isosteric acceptor amino acid substrates, i.e. glycine, alanine, and alpha-aminobutyrate. Buthionine sulfoximine did not inhibit glutamine synthetase detectably, nor did it produce convulsions when injected into mice. Injection of buthionine sulfoximine into mice decreased the level of glutathione in the kidney to a greater extent (less than 20% of the control level) than found previously after giving prothionine sulfoximine. alpha-Methyl buthionine sulfoximine was also prepared and found to be almost as effective as buthionine sulfoximine; this compound would not be expected to undergo substantial degradative metabolism. Buthionine sulfoximine and alpha-methyl buthionine sulfoximine may be useful agents for inhibition of glutathione synthesis in various experimental systems.


The enthalpy of protolysis of liver alcohol dehydrogenase upon binding nicotinamide adenine dinucleotide. The binding of NAD+, NADH, and ADP-ribose to horse liver alcohol dehydrogenase has been studied calorimetrically as a function of pH at 25 degrees C. The enthalpy of NADH binding is 0 +/- 0.5 kcal mol-1 in the pH range 6 to 8.6. The enthalpy of NAD+ binding, however, varies with pH in a sigmoidal fashion and is -4.0 kcal mol(NAD)-1 at pH 6.0 and +4.5 kcal mol(NAD)-1 at pH 8.6 with an apparent pKa of 7.6 +/- 0.2. The enthalpy of proton ionization of the group on the enzyme is calculated to be in the range 8.8 to 9.8 kcal mol(H+)-1. In conjunction with the available thermodynamic data on the ionization of zinc-bound water in model compounds, it is concluded that the group with a pKa of 9.8 in the free enzyme and 7.6 in the enzyme . NAD+ binary complex is, most likely, the zinc-bound water molecule. Our studies with zinc-free enzyme provide further evidence for this conclusion. Therefore, the processes involving a conformational change of the enzyme upon NAD+ binding and the suggested mechanism of subsequent quenching of the fluorescence of Trp-314 implicating the participation of an ionized tyrosine group must be re-evaluated in the light of this thermodynamic study.


Proton magnetic resonance studies on Escherichia coli dihydrofolate reductase. Assignment of histidine C-2 protons in binary complexes with folates on the basis of the crystal structure with methotrexate and on chemical modifications. The effects of pH upon the C-2 resonances of the 5 histidine residues of Escherichia coli MB 1428 dihydrofolate reductase in binary complexes with methotrexate, aminopterin, folate, methopterin, and trimethoprim were studied by 300-MHz 1H nmr spectroscopy. Three of the five histidine residues, labeled 1, 2, and 3, exhibited similar pK' values and chemical shifts for their C-2 protons in the five binary complexes. One histidine, 4, was quite different in the folate complex and the last histidine, 5 was quite different in the trimethoprim complex. For all five binary complexes, each histidine had a pK' which was significantly different from the other 4 histidines of that complex. Titration of the binary methotrexate complex of a 5,5'-dithiobis(2-nitrobenzoate)-modified enzyme showed that 2 histidines were not perturbed by this modification of Cys 152, and that the alkaline form of histidine 2, the acid form of histidine 4, and, to a lesser extent, the acid form of histidine 3 were slightly perturbed. Titration of the binary methotrexate complex of a N-bromosuccinimide-modified enzyme demonstrated that this modification slightly affected all of the histidines and drastically affected histidine 5. Histidines 3 and 5 of the binary methotrexate complex reacted rapidly with the histidine-specific reagent, ethoxyformic anhydride, while histidines 2 and 4 reacted at a moderate rate and histidine 1 reacted slowly if at all. The local electrostatic environments of the 5 histidine residues as deduced from the crystal structure of the binary complex of the enzyme with methotrexate (Matthews, D.A., Alden, R.A., Bolin, J.T., Freer, S.T., Hamlin, R., Xuong, N., Kraut, J., Poe, M., Williams, M.N., and Hoogsteen, K. (1977) Science 197, 594-597) were used as the basis for proposed assignments of the five histidine C-2 nmr resonances. The assignments were: 1, pK' 7.9 to 8.2, His 124; 2, pK' 7.2 to 7.4, His 141; 3, pK' 6.5 to 6.7, His 149; 4, pK' 5.7 to 6.3, His 114; and 5, pK' 5.2 to 5.9, His 45. The effect of the chemical modifications upon the enzyme's histidine residues were consistent with the assignments, but no direct chemical evidence in support of the assignments was obtained. It was proposed that, since the crystallographic data provided consistent assignments of the histidine nmr data for both native and chemically modified enzyme, the local environment of each of the 5 histidine residues was similar in the crystal and in solution.


Electron spin resonance study of the role of NO . catalase in the activation of guanylate cyclase by NaN3 and NH2OH. Modulation of enzyme responses by heme proteins and their nitrosyl derivatives. The role of NO . catalase in the activation of partially purified soluble guanylate cyclase of rat liver by NaN3 and NH2OH was examined by electron spin resonance (ESR) spectroscopy. Equilibration of bovine liver catalase with NO resulted in formation of a paramagnetic species exhibiting a three-line ESR spectrum similar to that of NO . catalase. This paramagnetic complex produced concentration-dependent stimulation of preparations of partially purified guanylate cyclase that were devoid of detectable endogenous heme content. The stimulation of partially purified guanylate cyclase by NO . catalase was similar to that obtained with NO . hemoglobin and with NO . cytochrome P-420 prepared by reaction of hepatic microsomes of phenobarbital-treated rats with NO. By contrast, these same enzyme preparations did not respond to NO or catalase alone. Addition of hematin or hemoglobin plus a reducing agent to purified guanylate cyclase restored enzyme responsiveness to NO and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but not to NaN3 or NH2OH. Responses to the latter agents were restored by catalase and potentiated by a H2O2-generating system. Formation of the NO . catalase complex was evident by ESR spectroscopy in test solutions containing NaN3 or nh2oh, catalase, and a glucose-glucose oxidase, H2O2-generating system. The presence of NO . catalase correlated well with the ability of test solutions to activate purified guanylate cyclase. These results provide evidence for catalase-dependent NO generation from NaN3 and NH2OH under conditions leading to guanylate cyclase activation. Preformed NO . hemoglobin or NO . cytochrome P-420 also activated heme-deficient partially purified guanylate cyclase. The ability of several preformed NO . heme protein complexes, but not NO, to stimulate heme-deficient guanylate cyclase supports the concept that formation of the paramagnetic nitrosyl . heme complex, mediated by either enzymatic or nonenzymatic reactions, is a common and essential step in the process by which NO or NO-forming compounds activate guanylate cyclase. In the absence of the NO ligand, both hemoglobin and catalase suppress the stimulatory effects of the corresponding NO . heme proteins on guanylate cyclase. Release of each heme protein from the NO . heme protein complex occurs more rapidly under aerobic compared to anaerobic conditions. However, hemoglobin is approximately 2000 times more effective as an inhibitor of NO . hemoglobin stimulation of guanylate cyclase than is catalase as an inhibitor of NO . catalase action. This finding may explain the more pronounced decline in the rate of cGMP generation in air in the presence of NO . hemoglobin compared to NO . catalase. The results imply that guanylate cyclase responses to activators that can form NO are determined by both the stimulatory activity of the endogenous heme acceptors of NO and the relative inhibitory effects of the unliganded heme proteins present.