A comprehensive gas chromatography procedure for measurement of drugs in biological materials.

Procedures have been developed for the determination in serum and urine of 21 drugs which include all of the barbiturates, methyprylon, glutethimide, meprobamate, propoxyphene, methaqualone, primidone, diphenylhydantoin, methadone, codeine, morphine, amphetamine and methamphetamine. The method involves pipeting 1.0 ml of serum or urine into an extraction tube, adding an internal standard together with either a pH 4.9 or pH 8.3 buffer and extracting with chloroform. The drugs in the extract are concentrated by exaporation of the organic layer and identified and quantitated by gas-liquid chromatography (GLC) using a column packed with 3 percent OV 17. GLC analysis times are kept below seven minutes by using temperature programming which allows good separations of drugs of widely varying volatilities. All calculations are based on peak areas determined by an electronic integrator. Losses of drugs during extraction, evaporation and chromatography steps are compensated for by the use of internal standards. Actual extraction efficiencies of drugs from serum or urine range from 75 to 100 percent although recoveries, based on standards carried through the entire procedures, are quantitative. Precision (s.d.) based on replicate determinations is approximately plus or minus 0.2 mg per dl.


A chemical approach to the fine structure of biomolecular complexes: the amino terminal region of the 50S ribosomal "A" protein from Bacillus stearothermophilus.

An experimental approach and methodology are described for determining the reactive properties and ionization constants of individual functional groups of proteins within biomolecular complexes. The ionization constants and reactivities of the methionyl-l amino terminus and the lysyl-3 residue of the alanine rich 50S ribosomal "A" protein from Bacillus stearothermophilus have been determined by an extension of the competitive labeling technique used by H. Kaplan, K. J. Stevenson, and B. S. Hartley ((1971), Biochem. J. 124, 289-299). This approach employs (1-14C)- and (3H)acetic anhydride in a double-labeling procedure. In 0.1 M KCl-0.02 M Mg2+-0.05 M Veronal at 10 degrees the methionyl-l amino terminus has a pKa of 7.5 and is exposed on the surface of the ribosome. The lysyl-3 has a pKa of 10 and is also exposed to solvent at the surface of the 50S subunit. Based on a linear free energy relationship (Bronsted plot) obtained with a series of standard amines the methionyl amino terminus has a substantially higher reactivity than expected from its ionization constant. The lysyl epsilon-amino group has the expected reactivity. The abnormally high reactivity of the methionyl amino terminus can only be accounted for by a specific interaction with other functional groups in the ribosome. These data support the proposal that the charged state of this residue is important in the structure and function of the "A" protein at the surface of the ribosome.


Synthesis of fluorescent organic phosphates and their equilibrium binding to bovine oxyhemoglobin.

Fluorescent organic phosphates, beta-naphthyl diphosphate, beta-naphthyl triphosphate, and beta-naphthyl tetraphosphate, were synthesized from beta-naphthyl monophosphate using Pi and N,N'-dicyclohexylcarbodiimide. These organic phosphates were interacted with bovine oxyhemoglobin, all in no buffer, 0.1 M NaCl, at 25 degrees and in the pH range 5.5-7.0. Equilibrium binding parameters were determined by measuring the fluorescence quenching upon their interaction. It is indicated that bovine oxyhemoglobin has more than one binding site, one of which is very strong. The strength of binding to the stronger site is in the order beta-naphthyl tetraphosphate greater than beta-naphthyl triphosphate greater than beta-naphthyl diphosphate. The logarithms of association constants of these phosphates depend linearly on the net charges of these phosphates at any pH. The results were accounted for by electrostatic effects using a simple charge model. In that model, the average positive net charges in oxyhemoglobin involved in the binding of beta-naphthyl phosphate are shown as a function of pH. It is shown that the binding of these fluorescent organic phosphates is prevented reversibly by the excess addition of nonfluorescent organic and iorganic phosphates, inositol hexaphosphate, tripolyphosphate, and pyrophosphate. Assuming competitive binding in a single strong site, the association constants of these nonfluorescent phosphates were also determined by measuring the recovery of the fluorescence intensity upon the release of fluorescent phosphates. At pH 6.18, the association constant of pyrophosphate is lower than that of tripolyphosphate by one order.


Conformational isomerism and effective redox geometry in the oxidation of heme proteins by alkyl halides, cytochrome c, and cytochrome oxidase.

In contrast to its lethargy at physiological pH, horse heart cytochrome c can be oxidized at room temperature by the axial inner sphere oxidant bromomalononitrile (BMN) at higher acidities. The following stoichiometry obtains: 2Fe11 c + BrCH(CN2) + H+ leads to 2FeIII c + CH2(CN)2 + Br-, and the rate law is given by: rate = k2(FeIIc)(BMN). At an ionic strength of 1.0 (KCl), second-order rate constants vary from 300 l. per mol per sec (pH 2-3) to 0(pH 9). Below pH 6 there is a noticeable increase in rate with ionic strength while there is no specific salt effect for the process. At pH 7.4 there is no influence of added salt (0.01-1.0 M) upon the slow rate of reaction. The vast changes in rate occur over a pH region (3-6) in which only very minor changes in the visible spectrum of the cytochrome are manifest. The results are interpreted in terms of a conformational isomerism of cytochrome c in which the effective redox geometry alters from a predominantly "short C" form (in which an axial position is available for substitution) at lower pH's to a predominantly "C" form (axial positions encumbered) in the physiological region. At 5 degrees, pH 7.4, both hemes of beef heart cytochrome oxidase are oxidized by the addition of BMN (k2 = 29 plus or minus 3 l. per mol per sec). However, the reaction is inhibited by potassium cyanide and the protein containing iron(II) cyt alpha along with the cyano adduct of iron(II) or iron(III) cyt alpha3 is inert. The results demonstrate cytochrome alpha3 as the site of reaction and that alpha reduces alpha3 in the process. Cytochrome oxidase does catalyze the oxidation of cytochrome c with BMN as substrate. Taken together the results provide additional support for a recent theory and they demonstrate BMN to be an efficient probe for the effective redox geometry of a hemoprotein in solution.


Interactions of aromatic residues of proteins with nucleic acids. Fluorescence studies of the binding of oligopeptides containing tryptophan and tyrosine residues to polynucleotides.

The binding of oligopeptides of general structure Lys-X-Lys (where X is an aromatic residue) to several polynucleotides has been studied by fluorescence spectroscopy. Two types of complexes are formed, both involving electrostatic interactions between lysyl residues and phosphate groups as shown by the ionic strength and pH dependence of binding. The fluorescence quantum yield of the first complex is identical with that of the free peptide. The other complex involves a stacking of the nucleic acid bases with the aromatic amino acid whose fluorescence is quenched. Fluorescence data have been quantitatively analyzed according to a model involving these two types of complexes. Association constants and the size of binding sites have been determined. Stacking interactions are favored in single-stranded polynucleotides as compared to double-stranded ones. A short oligopeptide such as Lys-X-Lys is thus able to distinguish between single-stranded and double-stranded nucleic acids. Fluorescence results are compared to those obtained by proton magnetic resonance and circular dichroism.


Interaction of aromatic residues of proteins with nucleic acids. Circular dichroism studies of the binding of oligopeptides to poly(adenylic acid).

The binding of peptides containing lysyl and aromatic residues to poly(A) in its single-stranded form at pH 7 leads to a change of its circular dichroism (CD) spectrum, which is mainly due to the stacking of the aromatic amino acid with the bases of poly(A). Comparison is made between the binding of peptides having different primary structures which gives indications on the way the peptides bind to poly(A). A method is described which allows the calculation of the binding parameters from CD data. The magnitude of the association constant depends on the size of the aromatic ring and decreases in the order tryptophan greater than tyrosine greater than phenylalanine. The CD amplitude decreases linearly with the concentration of bound molecules. These results are discussed with respect to the role played by aromatic amino acids in complex formation between nucleic acids and proteins.


Initial reactions in the oxidation of naphthalene by Pseudomonas putida.

A strain of Pseudomonas putida that can utilize naphthalene as its sole source of carbon and energy was isolated from soil. A mutant strain of this organism, P. putida 119, when grown on glucose in the presence of naphthalene, accumulates optically pure (+)-cis-1(R),2(S)-dihydroxy-1,2-dihydronaphthalene in the culture medium. The cis relative stereochemistry in this molecule was established by nuclear magnetic resonance spectrometry. Radiochemical trapping experiments established that this cis dihydrodiol is an intermediate in the metabolism of naphthalene by P. Fluorescens (formerly ATCC, 17483), P. putida (ATCC, 17484), and a Pseudomonas species (NCIB 9816), as well as the parent strain of P. putida described in this report. Formation of the cis dihydrodiol is catalyzed by a dioxygenase which requires either NADH or NADPH as an electron donor. A double label procedure is described for determining the origin of oxygen in the cis dihydrodiol under conditions where this metabolite would not normally accumulate. Several aromatic hydrocarbons are oxidized by cell extracts prepared from naphthalene-grown cells of P. putida. The cis dihydrodiol is converted to 1,2-dihydroxynaphthalene by an NAD+-dependent dehydrogenase. This enzyme is specific for the (+) isomer of the dihydrodiol and shows a primary isotope effect when the dihydrodiol is substituted at C-2 with deuterium.


The antibody-enzyme analogy. Comparison of enzymes and antibodies specific for phosphopyridoxyltyrosine.

Reduced Schiff base compounds of pyridoxal-P and tyrosine, which were used to induce specific antibodies described in the preceding article (V. Raso and B. D. Stolar, Biochemistry, 1975), caused active site-directed inhibition of tyrosine transaminase and tyrosine decarboxylase. The antibodies, studied as analogs of enzymes, were able to bind an unsaturated Schiff base catalytic intermediate, as shown by equilibrium dialysis and absorbance difference spectroscopy. Schiff base formation can proceed while the pyridoxal-P and tyrosine are within the antibody combining site, but the rate of this bimolecular condensation within the sites was not greater than the rate in free solution. Antibody did effect a small rate enhancement for the pyridoxal-P-catalyzed transamination of L-tyrosine. These results are discussed in light of current ideas in the mechanisms of enzyme catalysis.


Properties of the stable aerobic and anaerobic half-reduced states of NADPH-cytochrome c reductase.

The microsomal flavoprotein, NADPH-cytochrome c reductase, has been reexamined to determine: (1) the nature of the flavine bound to the enzyme and (2) the oxidation-reduction state of the "half-reduced" form of the flavoprotein. Iyanagi and Mason (Iyanagi, T., and Mason, H.S. (1973), Biochemistry 12, 2297) have recently proposed that NADPH-cytochrome c reductase contains both FAD and FMN as prosthetic groups in lieu of FAD as the sole constituent, as suggested by all previous studies of this enzyme. The data presented herein, utilizing the recently published fluorometric procedure of Faeder and Siegel (Faeder, E. J., and Siegle, L. M. (1973), Anal. Biochem. 53, 332) for the determination of FAD and FMN in mixtures, confirm the conclusions of Iyanagi and Mason for both rat and pig liver reductase preparations. Data for other flavoproteins are also presented. Iyanagi and Mason have also concluded that the air-stable "semiquinone" is a form of NADPH-cytochrome c reductase reduced by one electron per two falvines (F-FH). The present studies, however, do not agree with this conclusion, but instead support our previous results which indicate that both the aerobic and anaerobic half-reduced states of this flavoprotein exist in the two-electron reduced form (FH-FH). Removal of NADP+ does not affect the spectrum of the air-stable half-reduced form of the flavoprotein, nor does it affect the back titration of this intermediate by potassium ferricyanide. The possible implications of these observations on the catalytic cycle of the flavines of NADPH-cytochrome c reductase are discussed.


Kinetic studies on the mechanism of pepsin action.

The linear noncompetitive inhibition of the pepsin-catalyzed hydrolysis of Ac-Phe-Phe-Gly at pH 2.1 by L-Ac-Phe, L-Ac-Phe-NH2, and L-Ac-Phe-OEt has been claimed to substantiate the ordered release of products specified by the amino-enzyme mechanism for pepsin action. According to this interpretation, the binding of inhibitor to free enzyme and the amino-enzyme intermediate (Scheme I) generates the observed inhibition pattern. The proposition is valid only if a simple alternative explanation for the kinetic data, Scheme II, can be disproved. Scheme II attributes the inhibition pattern to the binding of inhibitor to free enzyme and the enzyme-substrate (Michaelis) complex. The experiments reported here have enabled us to distinguish between the two mechanisms. The pepsin-catalyzed hydrolyses of Ac-Phe-Trp, Z-H'IS-Phe-Trp, Z-Gly-His-Phe-Trp, and Z-Ala-His-Phe-Trp at pH 1.8 occur exclusively at the Phe-Trp bond and must yield the same amino-enzyme, E-Trp, if it is implicated. Under these circumstances, Scheme I requires that a plot of 1/kc vs. (I)o for the four substrates and a given noncompetitive inhibitor provide a set of four parallel lines. Scheme II predicts that the four lines generally will not be parallel. L-Ac-Phe, L-Ac-Phe-NH2, L-Ac-Phe-OMe, and D-Ac-Phe act as linear noncompetitive inhibitors for the pepsin-catalyzed hydrolysis of the four Trp-containing substrates. The plot of 1/kc vs. (I)o for each inhibitor results in a set of four nonparallel lines. Therefore Scheme II must be correct and the detection of noncompetitive inhibition accompanying the pepsin-catalyzed hydrolysis of peptides offers no insight into the merits of the amino-enzyme hypothesis.


Resonance Raman investigation of an enzyme-inhibitor complex.

The resonance Raman spectrum has been recorded for two different binary complexes formed between 2-carboxy-2'-hydroxy-5'-sulfoformazylbenzene (zincon) and liver alcohol dehydrogenase. The shifts in the zincon spectrum upon complexation with enzyme in one complex are similar to those in model compounds containing azo or formazyl linkages upon complexation of these with zinc. The results are interpreted in terms of complexation of zincon to a zinc atom at the enzyme active site. Since zincon is a coenzyme competitive inhibitor, it is probably bound at or near the coenzyme binding site; the results of this study, therefore, are useful in understanding the chemistry of zinc at the enzyme active site.


Characterization of the mycoplasma membrane proteins. V. Release and localization of membrane-bound enzymes in Acholeplasma laidlawii.

The peripheral membrane protein fraction released by washing Acholeplasma laidlawii membranes with low-ionic strength buffers contained about 50% of the total membrane-bound ribonuclease and deoxyribonuclease activities. The ATPase, NADH oxidase and p-nitrophenylphosphatase activities remained bound to the membrane even when EDTA was added to the wash fluids, and thus appear to belong to the integral membrane protein group. Serving as a marker for peripheral membrane proteins, the membrane-bound ribonuclease activity was solubilized by bile salts much more effectively than the integral membrane-bound enzymes. On the other hand, the solubilized ribonuclease showed a much lower capacity to reaggregate with other solubilized membrane components to membranous structures. Yet, most of the ribonuclease molecules which were bound to the reaggregated membranes could not be released by low-ionic strength buffer. The reaggregated membranes differed from the native membranes in the absence of particles on their fracture faces obtained by freeze cleaving, and by their much higher labeling by the [125-I]lactoperoxidase iodination system. These results suggest that most of the proteins are exposed on the reaggregated membrane surfaces, with very little, if any, protein embedded in its lipid bilayer core. Enzyme disposition in the A. laidlawii membrane was studied by comparing the activity of isolated membranes with that of membranes of intact cells after treatment with pronase or with an antiserum to membranes. The data indicate the asymmetrical disposition of these activities, the ATPase and NADH oxidase being localized on the inner membrane surface, while the nucleases are exposed on the external membrane surface.


Metabolism and properties of 3-methoxy-4-hydroxyphenyl-pyruvate; a metabolite of dihydroxyphenylalanine.

The pathway of 3,4-dihydroxyphenylalanine undergoing metabolism via transamination and subsequent oxidative rearrangement to 2,4,5-trihydroxy-phenylacetate was investigated. 3-Methoxytyrosine does not pursue an identical course, since its corresponding keto acid is not subject to action of p-hydroxy-phenylpyruvate hydroxylase. The radiochemical synthesis of 3-methoxy-4-hydroxy[carboxy-14C] pyruvate was accomplished. This metabolite was used to demonstrate that this keto acid does not proceed through oxidative rearrangement both in vitro and in vivo. The keto acid was found to be a competitive inhibitor of the hydroxylase and can help account for some of the metabolites observed in the urine of patients treated with 3,4-dihydroxyphenylalanine.


[Isoenzymes of carbonic anhydrase of an euryhaline fish. Variations related to the osmoregulation].

A chemical study of carbonic anhydrase (EC 4.2.1.1) from the red blood cells and the gills of an euryhaline fish (Anguilla anguilla) is presented. Animals adapted to fresh water were compared to those adapted to sea water. The physiochemical constants of the various molecular formsisolated by chromatography and isoelectric focusing were determined; isoelectric pH, molecular weight, and the Km and V/E of the enzyme dehydration activity were compared. In both red cells and gills of fish adapted in either media various forms were isolated, characterized by different enzymatic kinetics (high- and low- activity forms) but having the same molecular weight (27 250). Some isoenzymes isolated from the gills differed significantly from those isolated from the red cells. Adaptation to fresh water or sea water is accompanied by modifications in the distribution of the isoenzymes in both red cells and gills: adaptation to sea water is characterized by a shift of the molecular forms towards an isoelectric pH higher than pH equals 6. The role of these enzymes is discussed under both a physiological and biochemical point of view in relation to the electrolyte exchange across fish gill. The origin of the different molecular forms of the carbonic anhydrase is discussed in relation to the prevailing theories on this subject.


[Solubilization and several properties of NAD-glucohydrolase isolated from rabbit heart].

Methods of isolation of NAD-glucohydrolase from the fraction of heart microsomes were searched for. NAD-glucohydrolase proved to pass into a soluble state under the effect of phospholipase A, triton X-100 and Na cholate. NAD-glucohydrolase was found to possess peculiar properties; it reversibly became denatured under the effect of 6M urea, but failed to become inactivated with own substrate (NAD) at pH 8.0. In case of  NAD-ases isolated from other sources the reversible denaturation by means of urea as a rule correlated with the enzyme inactivation in the presence of NAD at pH 8.0.


[Letter: Laser light scattering study of the effect of extracellular medium properties on the myofibrillar structure of muscle fibers].

Mean myofibrillar radii are calculated from the curvity of the correlation function computed from equatorial light scattering distribution by means of Hankel integral transformation. Light scattering is measured for the same muscle fibre in the media of various ionic strength or pH. The light scattering experimental results are compared with X-ray diffraction data and our theoretical predictions.


Cctionic selectivity of bimolecular membranes in the presence of nystatin and amphotericin B.

Ergosterin or 7-dehydrocholesterin-containing artificial membranes are selectively permeable for cations of alkaline metals in aqueous solutions of nistatin and amphotericine B. Membrane permeability changes spontaneously within several tens of minutes from the cation to the anion one. A decrease of temperature, an increase of medium pH or introduction of dimethylsulphoxide into aqueous solution inhibit, while the addition of calcium accelerate this transition.


[Effect of cation concentration and medium pH on the distance between 2 lipid bilayers in the contact zone].

Dependence of the distance between contacting bilayer lipid membranes on the concentration of mono-and bivalent cations and medium pH has been measured by an optimcal method. It has been show that the presence of cations in the medium decreases the equilibrium distance between contacting membranes, the effect being more pronounced for bivalent Ca.


Pathology of modified graft-versus-host disease in bone marrow allografted monkeys treated with antilymphocyte serum.

Lethally irradiated rhesus monkeys were used for bone marrow allografting and autografting. Monkeys receiving allogeneic bone marrow developed acute graft-versus-host reaction (GVHR) and had a mean survival time of 9.1 days as compared to autografted monkeys which survived above 500 days. Treatment with antilymphocyte sera (ALS) before allografting modified the GVHR and extended the survival time to an average of 43 days. Histologically, such animals showed evidence of "chronic" GVHR and septicemia secondary to a lack in lymphoreticular recovery. Subsequently, severe GI-tract infections followed which usually served as portal of entry for septicemia.


Experimental salicylate intoxication in young baboons. A preliminary report.

Salicylate intoxication has been investigated in young baboons. The results of these studies are similar to these previously obtained in man. Acidosis appears to be of considerable importance in the pathogenesis of infantile salicylism as it enhances the passage of salicylate into the CSF. The CSF concentration of salicylate seems to be of major physiologic importance in this condition. Moreover, the serum concentration of free salicylate correlates more closely with the CSF concentration of salicylate than does the total serum concentration of salicylate.


Fluorescent dye method for determination of the bilirubin-binding capacity of serum albumin.

A simple, rapid microfluorometric method utilizing a fluorescent dye, Direct Yellow 7, for quantitative measurement of serum albumin-binding capacity for bilirubin has been developed. The dye binds exclusively to serum albumin with enhancement of fluorescence. The dye shares bilirubin-binding sites on albumin with up to 2 mols of bilirubin per mol of albumin with differing affinities to the first and second sites. Lowered pH reduces albumin-binding capacity and exposure to light increases binding capacity of icteric sera. The binding capacity of adult human sera is greater than that of human cord sera or purified human serum albumin compared on albumin molar basis.


Aminopyrine--an effective modifier of liver and serum gamma glutamyl transpeptidase.

Serum activities of gamma glutamyl transpeptidase, alanine and aspartate aminotransferases, and alkaline phosphatase were determined in children on long-term treatment with aminopyrine. Gamma glutamyl transpeptidase activity was increased up to 15 times above the upper normal limit in children, who received aminopyrine for two weeks or longer. Livers of rats treated with aminopyrine (600 mg/kg/day for 18 to 25 days) had an exceedingly increased activity of gamma glutamyl transpeptidase and a slightly elevated microsomal cytochrome P-450 content. Apparently isolated enhancement of serum gamma glutamyl transpeptidase during aminopyrine medication represents a drug-induced increase of microsomal liver enzymes without clinical relevance and without evidence of damage of liver cells.


Long-term beta blockade: possible protection from myocardial infarction.

The clinical behaviour of 90 patients on beta-blocking drugs for established coronary heart disease who were admitted to a coronary care unit with prolonged ischaemic myocardial pain was compared with that of 90 similar patients not on this therapy.  Transmural myocardial infarction was confirmed in 30 of the patients on beta-blockers and in 62 controls.  A diagnosis of myocardial necrosis without infarction was made in 20 patients on beta-blockers and in 14 controls.  Coronary insufficiency was diagnosed in 40 patients on beta-blockers and in 14 controls.  The incidence of simus bradycardia, hypotension, syncope, and radiological pulmonary oedema was similar in the two groups.  Established beta-blockade, therefore, has not been shown to prejudice the outcome of patients with coronary heart disease admitted to hospital with prolonged ischaemic myocardial pain.  On the contrary, it may protect some patients from the development of a myocardial infarction.


Failure of secretin release in patients with duodenal ulcer.

The release of secretin was studied in 12 normal subjects and 23 patients suffering from proved duodenal ulceration. After infusing acid directly into the duodenum mean plasma levels (plus or minus S.E.M.) of secretin rose in normal subjects to 52.6 plus or minus 4.8 ng/1 at six minutes but to only 37.5 plus or minus 3.6 ng/1 in duodenal-ulcer patients, a significant difference. The impairment of secretin release was as great in patients with a recent onset as in those who had had symptoms of a duodenal ulcer for a long time, raising the possibility of it being a primary defect. Patients who smoked 20 or more cigarettes a day had a particularly reduced secretin release, in accord with the greater incidence of ulcers in heavy smokers.


Results of adrenal surgery in patients with hypertension, aldosterone excess, and low plasma renin concentration.

Fifty patients with hypertension, aldosterone excess, and low plasma renin concentration underwent adrenal surgery.  There was a highly significant fall in mean systolic and diastolic pressures after the operation.  The mean postoperative diastolic pressure fell to strictly normal levels, however, in only 19 out of 38 patients from whom an adrenocortical adenoma was removed and in only two out of 10 non-tumour patients.  There was a significant correlation between the fall in blood pressure during spironolactone treatment and after adrenal surgery though levels were generally slightly lower during the former therapy. It is suggested that removal of an aldosterone-producing adenoma is the treatment of choice provided a good preoperative hypotensive response to spironolactone occurs, while the treatment of choice for non-tumour patients is often long-term spironolactone.


The pharmacological and anatomical substrates of the amphetamine response in the rat.

Bilateral 6-hydroxydopamine microinjections into the substantia nigra abolished both the locomotor and stereotyped responses to d-amphetamine in adult rats.  The lesions resulted in a depletion of over 99 per cent of striatal tyrosine hydroxylase activity (indicating a near total lesion of the nigro-striatal dopamine pathway) as well as severe noradrenaline depletions.  However, lesion of the dorsal or ventral noradrenergic pathways resulted in similar noradrenaline depletions but with no effect on striatal tyrosine hydroxylase levels and without the concomitant blockage of the amphetamine response.  The substantia nigra lesioned rats were behaviourally supersensitive to apomorphine and L-DOPA and did not show a locomotor response to cocaine.  The substantia nigra lesioned rats were not aphagic or adipsic.  It was concluded that both the locomotor and stereotyped responses induced by amphetamine are dependent on the functional integrity of the nigro-striatal dopamine pathway.


Compartmentation of amino acid metabolism in the rat dorsal root ganglion; a metabolic and autoradiographic study.

The incorporation of radioactivity into glutamate, glutamine, GABA and other amino acids was followed after incubation of desheathed rat dorsal root ganglia in media containing [14C]glucose or [14C]acetate. The results indicated that [14C]glucose was incorporated into a large pool of glutamate, but that this glutamate pool did not synthesize glutamine or GABA to any great extent. [14C]Acetate, on the other hand, was incorporated into a small glutamate pool which was readily converted to glutamine, and which synthesized GABA to a greater extent than the large pool. Light microscopic autoradiography of ganglia incubated with [14C]glucose or [14C]acetate confirmed that the small pool labelled by acetate was probably associated with satellite glial cells, while the large pool was located within the ganglion neurons. The results are discussed within the context of previous work on compartmentation of glutamate metabolism in the central nervous system.


Ionic excitation of a clone of mouse neuroblastoma.

The electrical properties and the possible regulation of these properties were studied by means of intracellular microelectrode recordings in cells of mouse neuroblastoma clone N1E-115. This clone has high levels of tyrosine hydroxylase and regulates this enzyme. Cells treated for 24 h with 4 muM aminopterin followed by at least 5 days in culture developed rhythmic discharge of action potentials when superfused with phosphate-buffered saline containing less than 0.2 mM calcium or less than 0.2 mM calcium and zero potassium. This ionic excitation occurred in no cells at less than 5 days after treatment with aminopterin but at 5 days or more after treatment, 20% of cells responded to low calcium while 52% responded to low calcium and zero potassium. Concomitant with the development of a susceptibility to ionic excitation was an increase in the average resting membrane potential and morphologic maturation. This ionic excitation of cultured mouse neuroblastoma cells may be useful for studying biochemical events associated with repetitive discharge of action potentials.


Comparison of the effects of biogenic amines on cyclic GMP and cycle AMP levels in mouse cerebellum in vitro.

Norepinephrine (NE) elevates levels of both 3',5'-guanosine monophosphate (cyclic GMP) and 3',5'-adenosine monophosphate (cyclic AMP) in incubated slices of mouse cerebellum. As little as 1 muM NE is capable of increasing the level of either cyclic nucleotide. Maximal elevations of cyclic AMP and cyclic GMP levels produced by NE are 15- to 40-fold and 2- to 4-fold, respectively. Dopamine, serotonin and histamine, other biogenic amines considered to be neurotransmitters in CNS, have no effect on mouse cerebellum cyclic nucleotide levels except at relatively high (1 mM) concentrations. NE-induced accumulation of cyclic GMP, but not cyclic AMP, is blocked by omission of Ca2+ from the incubation media. Theophylline does not alter the effect of this catecholamine on either cyclic nucleotide. In tissue slices incubated in buffered sucrose or in choline-Krebs buffer, NE is still capable of increasing both cyclic GMP and cyclic AMP levels; however, these elevations are less than those observed in brain slices incubated in Krebs-Ringer buffer. NE, in combination with glutamate, produces supra-additive elevations of both cyclic GMP and cyclic AMP levels. However, NE in combination with adenosine or high levels of K+ only has a synergistic effect on cyclic AMP and not on cyclic AMP and not on cyclic GMP combination. The elevation of cyclic AMP levels produced by NE appears to be mediated via both alpha- and beta-adrenergic receptor sites. In contrast, the receptor site(s) mediating cyclic AMP accumulation do not appear to be either typical alpha- or beta-adrenergic receptors.


The effects of psychomotor stimulants on stereotypy and locomotor activity in socially-deprived and control rats.

Using measures of locomotor activity and stereotypy, dose-response curves to several psychomotor stimulant drugs were obtained on rats reared in deprived or normal environments. At both 0.5 and 1.5 mg/kg d-amphetamine, the deprived rats exhibited more intense stereotyped behavior than the control rats. At 5.0 mg/kg, both groups showed maximum response. However, there was no significant difference between the two groups in locomotor response. A similar pattern of results was found for pipradrol, cocaine, and apomorphine. The findings show that different social and environmental experience can modify the response to dopaminergic stimulating agents. The results also suggest that stereotyped behavior should not be considered on the same continuum as locomotor activity. These two behaviors may be mediated by different mechanisms.


Effects of dopaminergic receptor agonists and antagonists on the activity of the neo-striatal cholinergic system.

The effects of various neuroleptics and of apomorphine on the metabolism of ACh were examined in the neostriatum of the rat. For this purpose, a specific radio-enzymatic assay for brain ACh was used. This method is based on the preliminary purification of the choline esters by liquid cation exchange, separation of choline and ACh on thin layer chromatography plates, hydrolysis of ACh then reactylation of the choline moiety with a purified and stabilized rat brain choline acetyltransferase. The rat neostriatal ACh levels were decreased by neuroleptics of the phenothiazine and butyrophenone type and increased by apomorphine. An "in vivo" estimation of the rate of utilization of ACh was obtained by measuring the decline in neostriatal ACh content following the local microinjection of hemicholinium-3. This compound blocked almost totally the synthesis of ACh in these conditions. Chlorpromazine (15 mg/kg) enhanced neo-striatal ACh utilization and apomorphine (10 mg/kg) antagonized this effect. Neuroleptics did not effect ACh levels in the parietal cerebral cortex and the hippocampal formation. The modifications of the activity of neostriatal cholinergic neurons by chlorpromazine and apomorphine were still observed following the degeneration of the nigro-neostriatal dopaminergic fibers induced by the injection of 6-hydroxydopamine into the substantia nigra. The results strongly suggest that dopaminergic receptors as defined by their pharmacological interaction with neuroleptics and apomorphine are localized on neostriatal ACh neurons.


Ontogeny of the induction of tyrosine hydroxylase by reserpine in the superior cervical ganglion, nucleus locus coeruleus and adrenal gland.

The ontogeny of the induction of tyrosine hydroxylase by reserpine has been studied in the superior cervical ganglion, adrenal gland and nucleus locus coeruleus of the rat. The inductive response developed gradually over a period of days in all 3 areas. However, the onset of induction occurred at markedly different times in these regions, being present from day 2 of life, the earilest time tested, in the adrenal, day 6 in the locus coeruleus and day 24 in the ganglion. In the ganglion even extremely high, toxic doses of reserpine failed to induce the enzyme during the first 3 weeks of life. Decentralization studies indicated that the ganglion was functionally innervated at this time. Moreover, the onset of induction was not time-locked to a specific phase of the postnatal development of tyrosine hydroxylase activity in the areas examined. It is probable that the development of inducibility reflects maturation of mechanisms intrinsic to the adrenergic cell, and this timetable is different for cells in different areas.


On GABA metabolism in the gliocyte cells of the rat pineal gland.

The uptake of the inhibitory transmitter substance gamma-aminobutyric acid (GABA) into the adult rat pineal gland was studied autoradiographically using both light and electron microscopy. The sites of GABA uptake were shown to be exclusively present in the gliocyte cells of the gland following both in vitro incubation with tritiated GABA and after in vivo administration of the amino acid by intra-arterial injection. Both the pinealocyte cells and the numerous sympathetic axons in the gland were devoid of silver grains. Preliminary biochemical studies indicated that the gliocyte uptake process for GABA resembles that in the satellite glia of the sensory ganglia but differed from that in slices of the cerebral cortex. Evidence is also presented which shows the pineal gland to contain endogenous GABA and the enzymes directly associated with its in vivo metabolism, L-glutamate-1-carboxylase (EC 4.1.1.15) (GAD) and GABA-2-oxoglutarate aminotransferase (EC 2.6.1.19) (GABA-T). Furthermore, a 3-fold rise in endogenous GABA occurred in the pineal after inhibition of GABA-catabolism as would be expected if the GABA-shunt pathway was functionally active in the oxidative metabolism of the pineal gland.


The detection of transmissible gastroenteritis viral antigens by immunodiffusion.

Parathyroid (PT) glands from 20-day-old embryonic chicks cultured in a chemically defined medium secreted a stimulator of in vitro bone resorption. This stimulator was presumed to be parathyroid hormone (PTH) because: 1) the in vitro dose response curve was parallel to that obtained with bovine PTH; 2) the activity was eluted on Sephadex G-1--chromatography at a postition similar to that for PTH; and 3) the material produced hypercalcemia in vivo in chicks. The amount of PTH-activity secreted was inversely proportional to the calcium concentration of the medium over the range of 0.75-2.25 mM. The chick PT glands also secreted an inhibitor of PTH-stimulated bone resorption in vitro. This inhibitor was presumed to be calcitonin (CT) because: 1) the in vitro dose-response curve was parallel to that obtained with synthetic salmon CT; 2) the activity was eluted on Sephadex G-50 chromatography at a position similar to that for salmon CT; and 3) the material produced hypocalcemia in vivo in rats. In contrast to what would be expected for CT secretion, the CT-activity was secreted by the PT glands in response to a low, not high calcium concentration. The data suggest that the secretion of avian PTH is similar to that of the mammalian hormone, and that the ultimobranchialectomized chick with an intact parathyroid gland may not be deficient in CT.


Pharmacokinetics of phenylbutazone and oxyphenabutazone in the pig.

The kinetics of phenylbutazone (pbz) and its main metabolite oxyphenbutazone (oxpbz) were investigated in seven pigs in order to determine if this animal might serve as a model for human investigations. The study was performed in two stages, one as single dose experiments using intravenous injection, the second as infusion experiments. The rate of elimination for both compounds was demonstrated to be much faster than in man. Similar results have been observed in several other animal species. The degree of protein binding (pbz 98%, ox-pbz 97%), the apparent specific volume of distribution (pbz 0.18 1/kg, ox-pbz 0.28 1/kg) and the renal clearance were found to be similar to results obtained in man and rat. Ox-pbz had a higher renal clearance (0.13 ml/min/kg) than pbz (0.003 ml/min/kg) but still the renal excretion constituted only a small fraction of the total elimination (pbz 0.5%, ox-pbz 5%). Provided real steady state conditions were obtained during the infusion experiments calculations based on the results from the single dose experiments in the same animal revealed that between 20 and 60% of the injected pbz was metabolized to the ring-hydroxylation product (ox-pbz). It is concluded that the pig model is not superior to other animal models for studies of pbz/ox-pbz. The rapid elimination pattern is the main problem in relation to human investigations.


Cultivation and maintenance of Sphaerophorus necrophorus. Part II: Influence of pH on maximum growth and acid production in Medium 156.

Sphaerophorus necrophorus was grown in Medium 156 at 40 pH values ranging from 4.93 to 9.46. Growth occurred between pH 5.30 and 9.28. The optimum pH for maximum growth was 6.84 while the optimum pH for acid production was 7.70. At pH 7.70 acid production was more than double that at pH 6.84 during a 12 hour incubation period. Thus, maximum growth occurs at one pH and maximum acid producation at another. Since the two optima are different, acid production should be evaluated only when: 1. an optimum medium is employed, 2. the optimum pH for acid production is employed. 3. the incubation period necessary for maximum acid production has elapsed before the test is conducted.


In vitro activity of clindamycin and other antimicrobials against gram-positive bacteria and Hemophilus influenzae.

Seven antimicrobials--clindamycin, penicillin, ampicillin, cloxacillin, erythromycin, lincomycin and cephalexin--were found to have a high degree of activity in vitro against 256 isolates of gram-positive bacteria and Hemophilus influenzae. Clindamycin was clearly superior against staphylococci and 3.12 mug/ml or less of clindamycin inhibited all 35 isolates of H. influenzae. Synergism was not demonstrated when clindamycin was tested in combination with sulfisoxazole or sulfamethoxazole by either the agar dilution or 24-hour growth curve method. This was true for penicillin as well, and the effect was independent of sulfonamide sensitivity. The erythromycin-sulfonamide combination was synergistic against 6 of 10 strains studied by the growth curve method; this effect was not demonstrable by the agar dilution method.


Some aspects of medical management of gastrointestinal disease. I.

Among drugs recently introduced into Canada for treatment of gastrointestinal disease are carbenoxolone sodium (used in treating gastric ulcer) and metoclopramide hydrochloride (which modifies upper gastrointestinal tract motility). A third drug, lactulose (useful in treating hepatic encephalopathy), will soon be available. Clinical experience with these drugs has been most extensive in Europe. In a new class of agents are histamine H2-receptor antagonists, which are currently under clinical trial. These drugs are the most potent inhibitors of gastric acid secretion yet investigated, and are potentially the most exciting agents to appear in many years. Part I of this paper is a review of the actions, clinical use, side effects, and dosage and administration of these new drugs.


Renal tubular acidosis during therapy for diabetic ketoacidosis.

A young woman presented with typical diabetic ketoacidosis. Five hours after insulin had been given hyperchloremic metabolic acidosis developed. This could not be attributed to gastrointestinal loss of bacarbonate, ingestion of HCI or carbonic anhydrase inhibitor, or the administered fluids and electrolytes. The combination of hyperchloremic metabolic acidosis and a urine pH of 5.6 during acidemia prompted specific studies that established the presence of disorders of renal acidification. A transient defect of hydrogen ion secretion in the distal nephron was suggested by the decrease in urine-blood Pco-2 gradient after administration of sodium bicarbonate. Proximal renal tubular acidosis was indicated by the reduced bicarbonate threshold that persisted for approximately 7 weeks.


Immunotherapy of melanoma with BCG: two fatalities following intralesional injection.

Two fatalities are reported following intralesional injection of BCG for immunotherapy of malignant melanoma. In both cases the terminal events were characterized by high fevers, major clotting defects, hypotension, and anuria, suggesting that the mechanism was hypersensitivity to BCG.


Adrenal cortical carcinoma. A study of 32 patients.

Fifteen males and 17 females with adrenal cortical carcinoma were reviewed. Their average age at diagnosis was 50.33 years plus or minus 12.26 SD for males and 39.76 years plus or minus 12.02 SD for females. The 5-year survival rate in this series was 31.25%. Abdominal pain, weakness, and weight loss were the primary presenting symptoms, whereas abdominal mass, distant lymphadenopathy, hepatomegaly, and edema of the lower extremities were the main presenting signs. The major sites of distant metastatic involvement were the lungs, liver, lymph nodes, and bones. The best available mode of therapy was surgery. The use of ortho para 'DDD (o,p'DDD), either in addition to surgery or as the only mode of therapy, was not associated with any significant advantage to the patient. Female patients had a longer survival than males, probably because of their younger age at diagnosis.


Management of recurrent melanoma of the extremity.

Two forms of therapy employed for treatment of patients with recurrent melanoma limited to the extremity, and carried out during different intervals of time, are presented. Perfusion of the involved extremity with phenylalanine mustard has resulted in a 5-year survival rate of 28% of 43 patients. A second group of 25 patients has been treated by a four-stage immunotherapy program consisting of sensitization with intradermal BCG, followed in 6 weeks by intra tumor injection of BCG. A third stage involved the activation of the patients's lymphocytes, after removal by a blood cell separator, incubated in vitro with irradiated neuraminidase-treated melanoma cells and reintroduced into the patient either by subcutaneous or intratumor injection. The fourth stage of immunotherapy involves injection of an inoculum of irradiated neuraminidase-treated autochothonous tumor cells plus BCG injected intratumorally or subcutaneously. Sixteen of 24 patients receiving immunotherapy treatment program have experienced arrest of their disease lasting from 5 to 42 months.


Sequential evaluation of general immune competence in cancer patients: correlation with clinical course.

An evaluation of general immunologic reactivity was performed in 116 patients with malignant melanoma and in 40 patients with skeletal and soft tissue sarcoma who received adjunctive immunotherapy. An excellent correlation was observed between delayed cutaneous hypersensitivity to DNCB and the clinical extent of malignancy. Eighty percent of patients with Stage I disease were DNCB positive, whereas only 37% of Stage III patients were reactive on initial testing. A method for sequential evaluation of DNCB response was established, and revealed that variations in immune reactivity occurred that also correlated with the patient's clinical course. Persistence of nonreactivity to DNCB or conversion from a reactive to an anergic status was associated with postoperative recurrence in more than 80% of the patients. Conversely, conversion from an anergic to a reactive status was observed if tumor control was achieved. These results indicate that the defect in systemic immunity is closely associated with tumor cell burden, and that sequential evaluation of DNCB reactivity is a clinically useful monitor of disease progression.


The immunologic and histopathologic changes of BCG-mediated tumor regression in patients with malignant melanoma.

Six patients with intradermal metastases of malignant melanoma were treated with intralesional bacile Calmette-Guérin (BCG). Four patients showed a good response with regression of injected, and in some cases, uninjected lesions, whereas two developed metastatic viscereal disease and died. Three of the six patients had complete regression of all lesions, and one exhibited complete regression of untreated lesions. All remain free of disease. The fourth patient had complete regression of injected and of some untreated lesions, but developed widespread dissemination and died. Preliminary experiments suggest the presence of a blocking factor in his sera which abrogates the lymphocyte stimulation in response to melanoma antigens. Three of four responders (i.e. those patients in whom treated lesions decreased in size by more than 50% for more than 1 month) showed a dramatic increase in lymphocyte stimulation to melanoma antigens. All responders (four out of four) had a marked increase to phytohemagglutinin (PHA), whereas non responders had no increase in lymphocyte stimulation either to melanoma antigens or PHA. Two of four responders showed inhibition of leukocyte migration to melanoma antigens before BCG, and two of four responders were positive after BCG. Of the nonresponders, one was positive and one negative before BCG; this remained the same after. There was a marked increase in active rosette forming cells in all responders and in one of the two nonresponders. Histopathologic studies at 3 hours, 6 hours, 24 hours, 14 days, and 4 weeks after BCG showed a definite sequence of events occurred, progressing from 1) inflammatory cell response at the periphery of the lesion, disruption of melanogenesis, extensive dumping of pigment from melanoma cells, proceeding to actual cell death at 24 hours, to 2) macrophages containing melanin and granulomas replacing tumor by 2 weeks. These studies suggest that BCG activates both specific and nonspecific immune responses. Thus, in vitro parameters of cellular immunity, including migration inhibitory factor production and inhibition of leukocyte migration, are affected by intralesional BCG, and some, particularly the lymphocyte stimulation and rosette test, seem to correlate with the clinical response of the patients.


New looks in leukemia.

Immunologic markers are now available for acute leukemia. These show that most patients now diagnosed as having acute lymphatic leukemia have blast cells which react with antihuman T antisera. In addition, about 20% of patients have blast cells which form E rosettes. These patients tend to be those with high white counts and aggressive disease. Tumor-associated antigens have been identified in acute leukemia. Antisera raised in other species by immunization appear to detect some antigens common to all acute leukemias and other antigens which can distinguish the myeloid from the lymphoid leukemias. The reactivity to leukemia antigens can be related to prognosis. The immune manipulations required to produce cure, however, are not understood. These antigenic markers, by allowing us to detect smaller amounts of tumor than can be detected by morphology alone, may help us to predict relapse and define cure.


Purification, properties, and mode of action of hemicellulase I produced by Ceratocystis paradoxa.

A culture isolate (CP2) of the fungal plant pathogen Ceratocystis paradoxa produces at least five extra-cellular hemicellulases when grown on a medium containing a commercial hemicellulose as inducer. One of the five enzymes, hemicellulase I (HC-I), was purified by ammonium sulphate preceipitation, ion-exchange chromatography (DEAE-Sephadex and then Cellex-CM), and iso-electric focusing at pH 3-10 and 8-10. HC-I behaves as a single protein on a electrophoresis at pH 6.0 and 8.4. The enzyme degrades hemicellulose B (an arabino-4-O-methylglucurono-xylan) and arabinoxylanto arabinose, xylose, xylobiose (Xyl2; beta-D-Xylp-(1 leads to 4)-D-Xyl), and a mixture of arabinose-xylose and xylose oligosaccharides (AraXyln and Xyln, where n=3, 4, or 5). The enzyme is deduced to be an endo-enzyme. Xylotetraose (Xyl4) was the lowest homologue of the xylose oligosaccharides attacked, yielding xylobiose and xylotriose (Xyl3) only. A mechanism is postulated for this reaction. AraXyl5 were slowly hydrolysed to arabinose and the respective xylose saccharide (Xyl2-Xyl5), and thence to Xyl2 and Xyl3. Hydrolysis of the arabinofuranosyl linkage probably does not occur at the same active site as for the xylose oligosaccharides. Hemicellulose B fractions from different sources appeared to be degraded by HC-I. The enzyme showed optimum activity at pH 5.5 and 40 degrees, and Km was 4.24 mg of hemicellulose/ml.


Pulmonary edema related to changes in colloid osmotic and pulmonary artery wedge pressure in patients after acute myocardial infarction.

Pulmonary artery wedge and plasma colloid osmotic pressures and their relationship to pulmonary edema were investigated in 26 patients with acute myocardial infarction of whom 14 developed pulmonary edema. In the absence of pulmonary edema, both the pulmonary artery wedge pressure and plasma colloid osmotic pressure were in normal range; after onset pulmonary edema, a moderate increase in pulmonary wedge pressure and reduction in plasma colloid osmotic pressure were observed. When the gradient between the plasma colloid osmotic pressure and the pulmonary artery wedge pressure was calculated, highly significant differences were demonstrated (P less than 0.002). In the absence of pulmonary edema, this gradient averaged 9.7 (plus or minus 1.7 SEM) torr; following appearance of pulmonary edema, it was reduced to 1.2 (plus or minus 1.3) torr. During therapy with digoxin and furosemide, reversal of pulmonary edema was closely related to a concomitant change in the colloid osmotic-hydrostatic pressure gradient. These observations indicate that both increases in pulmonary capillary pressure and decreases in colloid osmotic pressure may follow the onset of pulmonary edema. Such decline in colloid osmotic pressure and especially the reduction in colloid osmotic-hydrostatic capillary pressure gradient may favor transudation of fluid into the lungs.


Vidicon spectrometer applied to simultaneous enzyme determinations.

Lactate dehydrogenase and alkaline phosphatase activities in the same medium can be determined simulataneously, at 350 and 550 nm, with a vidicon spectrometer. Substrate concentrations and ph have been made optimum for the combined analysis. These conditions result in activities for lactate dehydrogenase that are equivalent to those found by methods in common use, and activites for alkaline phosphatase that are about 31% below the maximum values that could be obtained with its substrate used at the same ph and temperature in the absence of NAD+ and lactate. However, activites measured by the simultaneous analysis were proportional to those obtained by other methods used in clinical laboratories, and the coefficients of variation were 2.3% for lactate dehydrogenase and 3% for alkaline phosphatase.


Determination of serum thyroxine after dissociation from thyroxine-binding globulin in alkaline solution and absorption on dextran-coated charcoal.

A competitive protein-binding procedure for determining serum thyroxine is described in which thyroxine is described in which thyroxine is first dissociated from serum thyroxine-binding globulins in an alkaline solution. After binding equilibration, the unbound thyroxine is separated by dextran-coated charcoal. The procedure takes only 100 mul of serum, requires no evaporation, and is sensitive and reproducible. Results by this procedure correlate well with those for a generally accepted method.


Evidence supporting a proposed mechanism explaining the inverse relationship between guanidinoacetate and guanidinosuccinate in human urine.

A proposed mechanism [Clin. Chem. 19, 668 (1973)] for the inverse relationship between guanidinoacetate (I) and guanidinosuccinate (II) in human urine is explored. The mechanism proposes that canavaninosuccinate (III) may be reduced to form homoserine and II or, alternatively, that the III may be acted upon by a lyase to form canavanine and fumarate. The canavanine would then proceed to transamidinate to glycine to form I. This study demonstrates for the first time that lyase activity for converting III to canavanine and fumarate exists in human liver and kidney extracts. Transamidination from canavanine to glycine to form I is also readily accomplished with human tissue. Reductive cleavage of III to II and homoserine has been demonstrated before [Clin. Chem. 15, 397 (1969)]. The optimum pH for the lyase reaction is 6.5, for the reductive cleavage it is 8.7. In following the course of the lyase reaction, we developed a technique whereby the fumarate formed was hydrated with fumarase (EC 4.2.1.2) and then dehydrogenated with malate dehydrogenase (EC 1.1.1.37). The changes in absorbance of NADH formed in the reaction were then measured and used to determine the amount of fumarate formed, as a measure of lyase activity. Canavanino-succinate lyase activity follows pseudo-first-order reaction kinetics. The Michaelis constant of this lyase was 6.16 X 10-4 mol/liter, for argininosuccinate lyase 9.74 X 10-4 mol/liter. These data suggest that the binding affinity for III to the enzyme is greater than that for argininosuccinate. Glycine added to the reaction acts as an activator, probably because it removes the canavanine from the reaction mixture. On the other hand, arginine acts as an inhibitor of III-lyase. Other substances tested, such as canavanine, fumarate, and argininosuccinate had no effect on the reaction kinetics.


Evidence supporting a proposed mechanism explaning the inverse relationship between guanidinoacetate and guanidinosuccinate in human urine.

A proposed mechanism [Clin. Chem. 19, 668 (1973)] for the inverse relationship beteen guanidinoacetate (I) and guanidinosuccinate (II) in human urine is explored. The mechanism proposes that canavaninosuccinate (III) may be reduced to form homoserine and II or, alternatively, that the III may be acted upon by a lyase to form canavine and fumarate. The canavanine would the proceed to transamidinate to glycine to form I. This study demonstrates for the first time that layse activity for converting III to canavanine and fumarate exists in human liver and kidney extracts. Transamidination from canavine to glycine to form I is also readily accomplised with human tissue. Reductive cleavage of III to II and homoserine has been demonstrated before [Clin. Chem. 15, 397 (1969)]. The optimum pH for the lyase reaction is 6.5, for the reductive cleavage it is 8.7. In follwing the course of the lyase reaction, we developed a technique whereby the fumarate formed was hydrated with fumarase (EC 4.2.1.2) and then dehydrogenated with malate dehydrogenase (EC. 1.1.1.37). The changes in absorbance of NADH formed in the reaction were then measured and used to determine the amount of fumarate formed, as a measure of lyase activity. Canavanino-succinate lyase activity follows pseudo-first-order reaction kinetics. The Michaelis constant of this lyase was 6.16 X 10-4 mol/liter, for argininosuccinate lyase 9.74 X 10.4 mol/liter. These data suggest that the binding afficinity for III to the enzyme is greater than that for argininosuccinate. Glycine added to the reaction acts as an activator, probably because is removes the canavanine from the reaction mixture. On the otherhand, arginine acts as an inhibitor of III-lyase. Other substances tested, such as canavaine, fumarate, and arginissuccinate had no effect on the reaction kinetics.


PH measurement problems affecting assay of carcinoembryonic antigen.

Measurments with combination pH electrodes of the pH of the dilute buffers used in a commercial kit (CEA-Roche) for assay of carcinoembryonic antigen resulted in pH values 0.1 to 0.3 unit lower than pH values measured on an electrode system with a capillary junction. If the pH values of these buffers were adjusted, based on such measurements, an error in the assay of 0.2 to 0.6 ng/ml in the 1.5-3.0 ng/ml range would result. We recommend that the pH of dialyzed samples and of the working ethylenediaminetetraacetate and ammonium acetate-acetic acid buffers be monitored with pH electrodes that have a capillary junction between sample and saturated KCI, as is true of most blood-pH instruments. We also recommend use of a 1 mol/liter rather than 2.5 mol/liter stock ammonium acetate-acetic acid buffer, because of the closer similarity of the pH of buffers at this molarity to those at 0.01 mol/liter.


Assay of L-tyrosine in serum by amperometric measurement of tyrosinase-catalyzed oxygen consumption.

A new enzymatic method for rapid direct measurement of serum tyrosine is described, based on the amperometric measurement of the rate of oxygen consumption when tyrosine is oxidized in the presence of the enzyme tyrosinase in a phosphate buffer (0.1 mol/liter, pH 7.4). A Beckman Glucose Analyzer with a Clark electrode is used for the measurement. The procedure does not require removal of serum protein, incubation nor extraction, and results are obtained in less than 1 min. Results agree closely with those obtained by a fluorometric procedure and with an amino acid analyzer.


Effect of lactate dehydrogenase isoenzymes on the coupled enzymatic assay for alanine aminotransferase activity.

We show an example of the importance of specifying the form of isoenzyme and source of indicator enzymes to be used in coupled enzymatic assays. When we compared H-4 (pig heart) and M-4 (rabbit muscle) isoenzymes of lactate dehydrogenase for their suitability as indicator enzymes in the assay for alanine aminotransferase activity, we found that about fourfold as much M-4 as H-4 was required in terms of lactate dehydrogenase activity to reflect accurately equivalent amounts of alanine aminotransferase activity. Moreover, the substrate specificities of the two isoenzymes differed quantitatively.


Use of leuco-dyes in the quantitative colorimetric microdetermination of hemoglobin and other heme compounds.

Sensitivity, stability, and specificity of the color-producing reaction of hydrogen peroxide with bezidine, leuco-malachite green, or o-dianisidine were tested in numerous systems containing hemoglobin and other hemoproteins. Use of urea or low temperature (to minus 12 degrees C), or both, was highly beneficial, especially with leuco-malachite green, for which the color reaction was stable, after about 15 min, for longer than 24 h, with a colorless bank. Absorbance was 0.3 at a final hemoglobin concentration of 0.27 mg/liter. Nonspecific color produced by substances such as FeCl-3 and ascorbic acid was completely eliminated. Of the three leuco-dyes studied, only benzidine yielded a completely linear calibration curve, but its relative instability and reported carcinogenicity are serious disadvantages. No system tested eliminated completely the known inhibition by plasma of the peroxidase activity of these leuco dyes.


pH dependence of the binding of folates to milk binder in radioassay of folates.

We compare the binding of folic acid and N-5-methyltetrahydrofolic acid to milk binder in the pH range 7.4 to 10.1. At pH 7.4 the relative affinities are quite disparate, with folic acid showing the greater affinity for milk binder. As the pH is increased from 7.4 to 9.3, the difference in affinities becomes smaller, and at pH 9.3 the affinities are nearly the same. As the pH is increased from 9.3 to 10.1, the relative affinities again begin to differ, with N-5-methyltetrahydrofolic acid displaying the greater affinity. These results indicate that at a pH of 9.3 the more stable folic acid may be used as the standard in radioassay of endogenous folates.


The distribution of ammonia between extracellular and intracellular compartments of the rat brain.

1. The distribution of ammonia between extra-and intra-cellular compartments of the brain was evaluated in anaesthetized rats exposed to sustained hyperammonaemia and superimposed acute hypo- or hyper-capnia. 2. It is concluded that the concentration gradient for ammonia between arterial plasma and cerebro-spinal fluid (CSF) cannot be explained by pH-dependent non-ionic diffusion of ammonia. A continuous uptake by the brain tissue of ammonia from the CSF is postulated and possible mechanisms are discussed.


Partial pressures of oxygen and carbon dioxide, and pH of blood sampled from wedged pulmonary artery.

1. Po2, Pco2 and pH were examined in twenty-seven pulmonary "wedge" samples of blood obtained from seventeen patients with various heart diseases. 2. High Po2 and pH, and low Pco2, values were found in comparison with those in simultaneous samples of peripheral arterial blood. In the pulmonary "wedge" samples of blood, the following mean values were found: Po2, 17.65 kPa (132.2 mmHg); Pco2, 3.10 kPa (23.2 mmHg); pH 7.47. An explanation of these findings is considered.


Crosslinking and carbohydrate studies on the hydroxylated glycopeptides which accumulate in alveoli and lamellar bodies.

These data suggest that the alveolar lining layer and lamellar body membranes contain the same saline-insoluble proteins, two of which are unusual in that they contain hydroxyproline and large amounts of glycine. Neither the function nor the origin of these glycopeptides has been established, and we are currently evaluating methods of stimulating their production in animals for further study.


Comparison of the cardiopulmonary effects of subcutaneously administered epinephrine and terbutaline in patients with reversible airway obstruction.

The cardiopulmonary effects of epinephrine and terbutaline were compared in a doubleblind crossover study in 23 subjects with chronic obstructive airway disease. On each of three days each subject received a single subcutaneous dose of saline, 0.25 mg of epinephrine or 0.5 mg of terbutaline. Treatment with epinephrine produced significant increases in forced vital capacity (FVC), forced expiratory volume in one second (FEV-1), maximal expiratory flow rate (MEFR) and maximal mid-expiratory flow (MMEF). Terbutaline caused even more pronounced increases in all four parameters and exhibited a longer duration of action. Neither drug altered arterial pH, arterial oxygen pressure (PaO-2), or arterial carbon dioxide pressure (PaCO-2). With regard to cardiovascular effects, no alterations in either systolic or diastolic pressure were observed. Administration of epinephrine and terbutaline caused statistically significant increases in heart rate. The effect of terbutaline was more pronounced that that of epinephrine. In addition, terbutaline caused a heart rate-related depression of the T-wave of the lead 2 ECG. Neither drug altered any of the hematologic, hemochemical or urinary parameters monitored before and after treatment. Side effects were seen in eight subjects after administration of saline solution, in 13 subjects after epinephrine and in 19 subjects after terbutaline. None of these side effects was considered clinically serious and none required treatment. It is concluded from this study that subcutaneously administered terbutaline is a more effective bronchodilator than epinephrine.


Neurolept analgesia for bronchoscopic examinations.

In 38 patients requiring diagnostic bronchoscopy, three different procedures of anesthesia have been investigated. Of the three, neurolept analgesia combined with topical anesthesia of the airway proved by far the best form. It is followed by psychic indifference without lessening the patient's voluntary control, thus facilitating intricate technical manipulations on the bronchial system. No severe side effects arise when suitable equipment for diagnosing and treating respiratory and cardiovascular depression is available.


Evidence for pulmonary microembolization during hemodialysis.

The generation of microaggregates during the extracorporeal circulation of heparinized blood through two types of hemodialyzers was demonstrated by increased screen filtration resistance. Arterial blood deoxygenation and widening of the alveolar-arterial oxygen difference occurred following either unfiltered or inadequately filtered dialysis. These changes were consistent with pulmonary microembolization. The blood gas abnormalities and the increased screen filtration resistance were reversed or improved when venous line microemboli filters were used.


[Landry-type motor polyneuropathy and spinal transection syndrome with flaccid paraplegia. Rare neurological syndromes in panarteritis nodosa].

In a 59-year-old man in complete health a pure motor polyneuropathic syndrome with quadruplegia, swallowing and respiratory paralysis developed over a few hours. A few days later he died with the clinical picture of septicaemia. In a 59-year-old woman who had been investigated and treated for a suspected carcinoma for several months a predominantly distal sensomotoric polyneuropathy syndrome developed. The further course of disease was completely misleading as regards the diagnosis due to an acutely occurring transection syndrome with flaccid paraplegia, loss of reflexes, and bladder and rectal paralysis. Due to the very unusual neurological symptoms panarteritis nodosa was only diagnosed at autopsy and by histology in both cases. Panarteritis nodosa must be considered as a differential diagnosis in Landry-type polyneuropathy as well as in an acute spinal transection syndrome. The first patient demonstrates toxic damages, the second vascular damages of the nervous system which in general determine the neurological symptomatology of this vascular disease.


[Antral morphology and serum-gastrin levels in achlorhydria].

38 patients were found to have achlorhydria after maximal stimulation with pentagastrin and on multiple biopsies (atrophy of gastric mucosa). It was demonstrated that the sequence pH-antroreceptors-G cells-parietal cells-pH antroreceptors was interrupted already in mild or moderately severe superficial gastritis of the antral mucosa involving more than half of the antral surface. Reduction of specific functional epithelium is unlikely in this form of inflammation so that it is probably an effect of the pH receptors.


[Prenatal diagnosis of Sandhoff's disease (GM2-gangliosidosis, type 2)].

The diagnosis of GM2-gangliosidosis type 2 (Sandhoff's disease) was made prenatally (23rd week of pregnancy) by amniocentsis. A sibling with "Tay-Sachs disease" had died shortly before. Severe deficiency of total beta-hexosaminidase was found in amniotic fluid and amnion-cell culture. After interruption of the pregnancy the enzyme defect was also found in the fetal brain tissue and the concentration of ganglioside GM2 was three times normal, confirming the diagnosis.


[Alpha-antitrypsin deficiency in infancy].

Clinical, histological (including electron-microscopic), immunohistochemical and genetic studies were performed on two infants with alpha1-antitrypsin deficiency. The clinical picture was one of neonatal biliary stasis. Liver biopsies revealed multiple cytoplasmic acidophilic bodies within many cells of the liver parenchyma which were strongly periodic acid-Schiff-positive, diastase-resistant and stained selectively with fluorescein-labelled rabbit antihuman alpha1-antitrypsin. Ultrastructurally, the bodies were situated within enlarged cisterns of the endoplasmatic reticulum. Both infants were of the protease inhibitor (Pi) phenotype ZZ, having inherited on PiZ gene from each parent. Results of Pi typing of both families were consistent with an autosomal co-dominant inheritance. Both infants are clinically well except for slight hepatomegaly at one year of age. But transaminase and gamma-glutamyl transpeptidase activities have remained elevated.


[Contamination of human breast milk with chlorinated hydrocarbon residues].

136 out of 137 human breast milk samples which were investigated between June 1973 and January 1974 contained remains of the insecticide DDT and its metabolite DDE, Some of them in considerable amounts. On average the quantities of DDE were double those of DDT. In 73 samples DDD could be found at the same time. DDD can be formed during metabolization of DDT. Less frequently other chlorinated hydrocarbons were found, mainly hexachlorobenzene, lindane (gamma-HCH), TECHNOLOGICAL HCH and heptachloroepoxide. On the other hand quite frequently found in animals were rarely observed. Only one of the investigated samples of human breast milk was in accordance with the recently introduced legal maximum allowed for cow's milk and milk products, It seems particularly serious that, assuming normal conditions, the ADI value for DDT in adults suggested by the WHO was exceeded on average fourfold and in the highest value 16-fold. Also noteworthy is the not infrequent contamination with polychlorinated biphenyles. These are remains of industrial products which are very similar to DDT in their toxic and physico-chemical behaviour.


[The purity of commercial insulin preparations].

Insulin preparations available in Germany were tested by polyacrylamide-disc electrophoresis. A large proportion of preparations contained proteins additional to insulin. Insulin purified chromatographically was cleaner than that by crystallization. Insulin purified by ion-exchange chromatography had the highest degree of purity: in addition to the insulin band only deamidation products of insulin were demonstrated. Deamidation occurs both in neutral and acid solvents. These observations indicate that commercial full purification of insulin is possible but not always practised.


Patterns of LH and FSH release from perifused rat pituitaries in response to infusions of hypothalamic extract.

A continuous flow incubation (perufusion) system was developed in which the secretory responses of pools of hemipituitaries from adult male rats to hypothalamic extract (HE) were characterized by serial radioimmunoassay of LH and FSH in the effluent medium. There was in initial massive release of LH and FSH which, in the absence of HE, declined to low basal levels at a rate which depended on the flow rate. Thereafter, the baseline for LH continued to decline gradually while that for FSH was stable. The rate of LH and FSH release rose abruptly after addition of the HE to the medium and returned promptly to baseline after withdrawal of the HE. During continuous infusion of HE for five hours, LH secretion was maintained at a relatively constant, elevated level. The responses to repeated identical pulses of HE were highly reproducible. The variability between responses by any one pool of tissue was significantly less than between responses of separate pituitary pools including pools comprising right and left halves of the same glands. For any given pool of pituitaries of the relationships were linear between: 1) duration of HE pulses (concentration constant) and increases in LH output, and 2) log of concentration of HE (pulse duration constant) and increases inLH and in FSH output. Consistent responses were obtained for up to 12 hr, the maximum period tested.


The influence of adrenergic, dopaminergic, cholinergic and serotoninergic drugs on plasma prolactin levels in ovariectomized, estrogen-treated rats.

Levels of plasma prolactin were estimated in ovariectomized, estrogen-treated rats following the systemic administration of several neural blocking and stimulating drugs. Phenoxybenzamine, an alpha-adrenergic blocker, at high doses, increased plasma prolactin. Phenotlamine, another alpha-adrenergic blocker, and propranolol, a beta-adrenergic blocker, also increased prolactin but the responses were small and transient. Clonidine, an alpha-adrenergic stimulating drug, elevated prolactin levels whereas the beta-adrenergic stimulator isoproterenol had no effect. Dopaminergic blockade by pimozide increased levels of prolactin while stimulation of dopamine receptors by apomorphine decreased prolactin release. Atropine (a muscarinic chilinergic blocker), arecoline (a muscarinic stimulator) and nicotine (a nicotinic cholinergic stimulating drug) did not affect basal prolactin levels. Mecamylamine (a nicotinic blocker) produced a small transient elevation in plasma prolactin. Methiothepin, an alleged serotoninergic blocker, markedly increased prolactin secretion, as did serotonin. The data suggested the involvement of several neurotransmitters in the control of basal secretion of prolactin.


Characterization and comparison of receptors for 17 beta-estradiol and progesterone in human proliferative endometrium and endometrial carcinoma.

Sedimentation coefficients of cytoplasmic estradiol and progesterone receptors of human proliferative endometrium and endometrial carcinoma were determined by sucrose gradient centrifugation. In the absence of KCl, receptors from proliferative endometrium sedimented as single bands in the 8 S region and in the presence of 0.3M KCl in the 4 S region of the gradients. Receptors from endometrial carcinoma sedimented in several bands (between 3 and 9 S). When chromatographed on agarose gel comumns, the receptors (from both normal and neoplastic tissue) showed different molecular weights in the presence and absence of KCl (approximately 40,000 and 120,000, respectively). Elution profiles from agarose gel and ion exchange columns, as well as electrophoretic patterns from isoelectric focusing, revealed a similarity between biochemical properties of the receptors from endometrial carcinoma and proliferative endometrium. While the concentration of binding sites for estradiol and progesterone in normal endometrium depended on the day of the cycle, in endometrial carcinoma it depended on the degree of differentiation of the tumor. The binding of estradiol was highest at the beginning of the proliferative phase and declined continuously towards the 14th day of the cycle. In contrast, the concentration of progesterone binding sites was relatively low throughout the proliferative phase. In endometrial carcinoma low binding of estradiol was obtained in well differentiated tumors and high binding (as high as in proliferative endometrium) in undifferentiated tumors. For progesterone the contrary was the case. There was no difference in pH sensitivity between cytoplasmic receptors from normal and neoplastic tissue, optimal binding occurring at pH 7. Dissociation constants (Kd) for estradiol and progesterone depended on the degree of tumor differentiation. Kd values increased for E2 and decreased for P with increasing differentiation of the tumor. Competition studies with various unlabeled steroids revealed no significant difference between the specificity of the receptors from proliverative and neoplastic endometrium.


Cyclic nucleotide phosphodiesterase from bone: characterization of the enzyme and studies of inhibition by thyroid hormones.

Studies were carried out to characterize the cyclic nucleotide phosphodiesterase from rat calvaria. 25-40% of the total enzyme activity was membrane-bound. pH, magnesium, and temperature requirements conformed closely to those established for phosphodiesterase from other tissues. Kinetic evidence was found for dual enzyme activities with different substrate affinities for both the particulate and soluble enzyme. Apparent Kms for the soluble enzyme (3.5 times 10-6 and 2.5 times 10-5M) approximated those for the particulate enzyme (5.7 times 10-6 and 2.5 times 10-5M). L-Thyroxine, 10-5M, inhibited competitively the low- and high-Km enzymes from both the particulate and soluble fractions (Ki equals 1.7 times 10-5M). T4 was more potent an inhibitor than T3 with all enzyme fractions, but this relationship could be altered by adding protein to the incubation mixtures. Tests of diverse thyroid hormone analogues showed that 1) T4 and its derivatives were more potent than T3 and its analogues; 2) acetic and propionic acid derivatives were more potent than the thyronines; 3) "reverse T3," an antagonist of some T3 actions, also inhibited phosphodiesterase. These effects were not attributable to chelation, and were not duplicated by iodide or by other physiologically inactive thyroid hormone analogues.


Pathway from progesterone to 5alpha-reduced c19 steroids not involving androstenedione and testosterone in immature mouse testes in vitro.

Testicular homogenates from mice of 23 and 70 days of age were incubated for 3-120 min with either 3-H-progesterone, 14-C-progesterone, 3-H-5alpha-pregnane-3,20-dione or 14-C-progesterone plus 3H-5alpha-pregnane-3,20-dione in the presence of NADPH. After incubation, radioactive products were purified and identified by column and paper chromatography, with derivative formation and recrystallization to constant specific activity. In immature mouse testes, the results indicate two biosynthetic pathways leading to C19 steroids from progesterone, one from progesterone via 17-hydroxyprogesterone and androstenedione to testosterone and a second via 5alpha-reduced C21 steroids to 5alpha-reduced C19 steroids such as androsterone and 5alpha-androstane-3alpha,17beta-diol. In adult mouse testes, very few 5alpha-reduced metabolites of all the delta-4-3-ketosteroids are shown to be produced from progesterone, while evident 17alpha-hydroxylation of 5alpha-pregnane-3,20-dione followed by C17-20 lyase reaction is demonstrated.


Temperature-determined enzymatic functions in octopine dehydrogenase.

We investigated the temperature dependence of several functions of octopine dehydrogenase, a monomeric enzyme extracted from the shell fish Pecten maximus L. We found that six enzymatic functions are temperature independent or change only negligibly with temperatue. These are the dissociation constants of three coenzyme complexes and the Michaelis Km values for NAD, NADH and one of the substrates (D-octopine). This is taken as an indication of a temperature-regulatory mechanism which enables the enzyme to maintain a constant level of NAD, NADH and D-octopine in binary and ternary complexes independent of fluctuations of the external temperature. This is discussed with reference to enzymes from other poikilotherms, which reportedly display similar biologically meaningful response to temperature. We also discuss the meaning of our data from a thermodynamic viewpoint. Considering that in a temperature-independent binding process only entropy changes contribute to the standard free-energy change, we speculate on possible molecular models which might account for our results. We also investigate the activation-energy parameters for the reaction catalyzed by octopine dehydrogenase, as obtained from the temperature dependence of V. It is found that octopine dehydrogenase, relative to other dehydrogenases, is provided with a rather low delta H not equal to, which enables the enzyme to change its turnover number by only a small factor in the temperature range 5--35 degrees C.


Lipid peroxidation in isolated hepatocytes.

Intracellular lipid peroxidation was initiated by the addition of ADP-complexed ferric iron to isolated rat hepatocytes and the reaction monitored by the thiobarbituric acid method or by measurement of the formation of conjugated dienes. Both the production of malondialdehyde (thiobarbituric-acid-reacting substances) and of conjugated dienes was dependent, on the ADP-Fe-3+ concentration in a dose-related fashion. Malondialdehyde formation stopped spontaneously within 20 min after the initiation of the reaction and the plateau reached was also related to the ADP-Fe-3+ concentration. Control experiments revealed that more than 90% of the malondialdehyde accumulating during the incubation period could be ascribed to intracellular production. The cellular NADPH/NADP+ ratio was always high and only slightly decreased upon ADP-Fe-3+-induced lipid peroxidation which, however, was associated with a marked decrease in the cellular glutathione concentration. The rate of accumulation of malondialdehyde as well as the final level reached during ADP-Fe-3+-initiated lipid peroxidation was increased by the addition of chloral hydrate. This apparent stimulatory effect could, however, be ascribed to the inhibition of the mitochondrial oxidation of the malondialdehyde formed during cellular lipid peroxidation, thus allowing more malondialdehyde to accumulate during the process. ADP-Fe-3+-induced cellular lipid peroxidation was associated with a decrease in the concentration of glutathione. Also, lowering of the intracellular glutathione level by the addition of diethyl maleate or by simply preincubating the hepatocytes (up to 50 min) promoted the ADP-Fe-3+ malondialdehyde production and formation of conjugated dienes. Furthermore, when cellular glutathione concentration had been lowered by preincubation of the hepatocytes, significant malondialdehyde production could be observed even at ADP-Fe-3+ concentrations which were too low to induce measurable lipid peroxidation in fresh hepatocytes. It is thus concluded that glutathione has an important role in the cell defence against lipid peroxidation and suggested that the isolated hepatocytes provide a suitable experimental model system for the characterization of this and other possible cellular defence mechanisms and how they are affected by the nutritional status of the donor animal.


Hyporeactivity to interferon induction: characterization of a hyporeactive factor in the serum of encephalomyocarditis virus-infected mice.

Mice infected with encephalomyocarditis virus develop a severe state of hyporeactivity to interferon induction. One mechanisms possibly responsibile for development of hyporesponsiveness in these animals is a circulating factor which can be detected in their serum 96 h after encephalomyocarditis virus infection (at the time of peak hyporeactivity in vivo). This report describes some of the physiocochemical characteristics of this serum hyporeactive factor (SHF). SHF is a protein with a molecular weight between 20,000 and 40,000 that was extremely labile at 56 C, losing greater than 90% of its biological activity in 8 min, but stable at 37 c for at least 4 h. Hyporeactive factor was also stable over a pH range of 2 to 11 for 48 h at 4 C. These results suggest that SHF is physicochemically similar to interferon. However, no interferon could be detected in the SHF preparation, and no loss in biological activity was observed when the serum factor was incubated with anti-interferon antibody, suggesting that they are separate substances.


Effect of gold on the immune response of mice.

Separate concurrent injection of organic gold compounds and antigen into mice resulted in immunoenhancement that could be measured by direct and indirect plaque-forming cells, rosette-forming cells, and serum antibody assays. Kinetics of the immune response showed variable effects through day 9 of the experiment. Studies with British anti-lewisite, a gold antagonist, showed that the gold must stay in the system 1 day to obtain immunoenhancement.


Studies on the differentiation of retinal pigmented epithelium cells in culture.

Sephadex G-25 fractions of chick embryo extract affect the differentiation of cloned embryonic pigmented epithelial cells (PE) in culture. Both tyrosinase activity and amounts of melanin are markedly affected by the extract fractions. 5-Hydroxyindole decreased melanin accumulation in PE cells while alpha-methyl-p-tyrosine had no apparent effect on the process. Melanin disposal as well as biosynthesis may be important in controlling the accumulation of pigment in PE cells under culture conditions. Triiodothyronine greatly increased visible pigmentation and affected the morphology of cultured PE cells.


Effects of monosodium glutamate on chick embryo retina in culture.

Monosodium glutamate added to 12-day chick embryo retinas in culture causes severe morphologic damage to the retina as judged by light microscopic examination. Damage is evident after a few hours with concentrations as low as 0.3 mM. Glutamyltransferase induction is also appreciably inhibited by the amino acid. General protein synthesis and RNA synthesis appear to be less affected.


Differential effects of inhibitors of monoamine oxidase Types A and B on the adrenergic system of the rabbit iris.

Topical instillation of inhibitors of monoamine oxidase Types A and B into the rabbit eye, followed by the administration of a releaser of monoamines (Ro 4-1284), induces strong and sustained dilation of the iris. The mydriatic pattern differs markedly with A- and B-inhibitors and with the duration of the inhibitor treatment. The Type B inhibitor, deprenyl, prevents the degradation of exogenous 2-phenylethylamine and possibly of the endogenous amine X capable of releasing monoamines from a compartment less accessible to Ro-1284.


The effect of intraocular ablation of the pecten oculi of the chicken.

The pecten oculi of 20 chickens was unilaterally ablated by intraocular electrocautery. Although the pecten was totally destroyed in 13 of the 20 birds, the retina remained histologically normal. All of the birds sustaining total lesion of the pecten retained the pupillary reflex and could peck up food and avoid objects when the control eye was covered. The intraocular pH of 5 operated birds was measured in vivo and averaged 7.35 (range 7.40 to 7.33) in operated eyes and 7.56 (range 7.65 to 7.50) in control eyes. It is suggested that the function of the pecten is not primarily nutritive and may instead be related to intraocular pH regulation.


Stability studies with amphotericin B and amphotericin B methyl ester.

In solid form, amphotericin B and amphotericin B methyl ester free base exhibit similar stability. Acid salts of the methyl ester derivative stored under identical conditions are less stable. In solution, amphotericin B is generally more stable than its methy ester salts. However, when pH is adjusted to 6.0 and storage temperature held at 5 degrees C the methyl ester salts reflect the stability exhibited by the parent compound, amphotericin B.


In vitro activity of sisomicin, an aminoglycoside antibiotic, against clinical isolates.

Sisomicin, a new aminoglycoside antibiotic which is produced by Micromonospora myoensis, was studied against 565 clinical isolates of gram-negative bacilli and gram-positive cocci. With the exception of Serratia marcescens, over 90% of isolates of gram-negative bacilli were inhibited by 1.56 mug/ml or less of sisomicin. Sisomicin was slightly more active than gentamicin and tobramycin aganist isolates of Escherichia coli, Proteus mirabilis and Klebsiella spp. It was substantially more active than butirosin and kanamycin against all gram-negative bacilli. Isolates of gram-negative bacilli which were resistant to gentamicin and tobramycin were also resistant to sisomicin. Most of these isolates were sensitive to amikacin.


Assay of deoxyribonucleic acid homology using a single-strand-specific nuclease at 75 C.

We investigated the conditions under which a crude preparation of endonuclease S1 gives maximal hydrolysis of denatured deoxyribonucleic acid (DNA) while giving minimal hydrolysis of native DNA. The hydrolysis was measured by filtering and determining the acid-insoluble reaction product using 3H-labeled substrates. We also investigated various parameters in making this measurement. Under appropriate conditions (in 1 mM ZnSO-4, 0.168 M NaCl at pH 4.8) denatured DNA is hydrolyzed within 3% of completion whereas native DNA is essentially unaffected. The reaction was applied to assay plasmid DNA homoand heteroduplexes for which the method proves to be simple, fast, and reproducible.


Isolation of super-repressor mutants in the histidine utilization system of Salmonella typhimurium.

Two super-repressor mutations in the histidine utilization (hut) operons of Salmonella typhimurium are described. Cells bearing either of these mutations have levels of hut enzymes that do not increase above the uninduced levels when growth is in the presence of either histidine or the gratuitous inducer imidazole propionate. Both mutations lie in the region of the gene for the hut repressor, hutC, and reverse mutations of both are to the constitutive (repressor-negative) rather than to the inducible (wild type) phenotype. In hybrid merodiploid strains the super-repressor mutations are dominant over either wild-type (hutC+) or repressor-negative (hutC-) alleles. Whereas both super-repressor mutations cause the uninducible synthesis of hut enzymes, the degree of repression is different. One mutation causes repression of enzyme synthesis in one of the two hut operons to a level below the basal, uninduced level of wild-type cells. The other mutation causes repression to a lesser degree than in wild-type cells, so that the hut enzymes are present at a level above the normal basal level; this partially constitutive synthesis is greater for the enzymes of one of the hut operons than for the enzymes of the other. Thus, both mutations apparently result in repressors with altered operator-binding properties, in addition to altered inducer-binding properties.


Production of racemic lactic acid in Pediococcus cerevisiae cultures by two lactate dehydrogenases.

Nicotinamide adenine dinucleotide (NAD)-dependent d(minus)-and l(plus)-lactate dehydrogenases have been partially purified 89- and 70-fold simultaneously from cell-free extracts of Pediococcus cerevisiae. Native molecular weights, as estimated from molecular sieve chromatography and electrophoresis in nondenaturing polyacrylamide gels, are 71,000 to 73,000 for d(minus)-lactate dehydrogenase and 136,000 to 139,000 for l(plus)-lactate dehydrogenase. Electrophoresis in sodium dodecyl sulfate-containing gels reveals subunits with approximate molecular weights of 37,000 to 39,000 for both enzymes. By lowering the pyruvate concentration from 5.0 to 0.5 mM, the pH optimum for pyruvate reduction by d(minus)-lactate dehydrogenase decreases from pH 8.0 to 3.6. However, l(plus)-lactate dehydrogenase displays an optimum for pyruvate reduction between pH 4.5 and 6.0 regardless of the pyruvate concentration. The enzymes obey Michaelis-Menten kinetics for both pyruvate and reduced NAD at pH 5.4 and 7.4, with increased affinity for both substrates at the acid pH. alpha-Ketobutyrate can be used as a reducible substrate, whereas oxamate has no inhibitory effect on lactate oxidation by either enzyme. Adenosine triphosphate causes inhibition of both enzymes by competition with reduced NAD. Adenosine diphosphate is also inhibitory under the same conditions, whereas NAD acts as a product inhibitor. These results are discussed with relation to the lactate isomer production during the growth cycle of P. cerevisiae.


Marker discrimination in deoxyribonucleic acid-mediated transformation of various Pneumococcus strains.

The function responsible for discrimination among markers (point mutations) in Pneumococcus (hex) was traced back to the early strains used to demonstrate the chemical basis of transformation in the early 1940s. Those currently used laboratory strains failing to manifest this function arose from a single subline of the original strain. The function was also evident in other independently isolated strains including a number of different serological types. The hex function was not evident in transformation between heterologous pneumococcal strains probably as a result of the sensitivity of the function to saturation in the presence of deoxyribonucleic acid from closely related but nonisogenic strains.


Changes in composition, biosynthesis, and physical state of membrane lipids occurring upon aging of Mycoplasma hominis cultures.

During the progression of Mycoplasma hominis cultures from the early logarithmic phase to the stationary phase of growth, the total phospholipid content of the cell membranes decreased. Measurement of the amount of the various phospholipids during the growth cycle showed that a decrease in the phosphatidylglycerol (PG) content, accompanied by an increase in the phosphatidic acid content, occurred upon aging of the culture. Pulse labeling experiments revealed that the PG, once formed, is relatively stable, undergoing no detectable turnover in growing cultures of M. hominis. No changes in the fatty acid composition of the membrane phospholipids were observed on aging of the culture, with palmitic acid predominating throughout the growth cycle. The preferential incorporation of palmitate into the phospholipid fraction is apparently caused by the higher activity of the membrane-bound acyl-coenzyme A (CoA):alpha-glycerophosphate transacylase with palmityl-CoA rather than with oleyl-CoA as substrate. The activity of the soluble acyl-CoA synthetase was the same whether palmitate or oleate served as substate. M. hominis membrane preparations contained a PG-synthetase system catalyzing the incorporation of L-alpha-glycerol-3-phosphate into PG. The activity of the PG synthetase system was markedly dependent on the age of the culture, being highest in cells from the early exponential phase of growth while declining sharply thereafter, and thus probably responsible, in part, for the decrease in PG content upon aging of the cells. Electron paramagnetic resonance spectra of a spin-labeled fatty acid incorporated in M. hominis membranes revealed a marked decrease in the freedom of motion of the spin label on aging of the culture. It is proposed that this decrease is due primarily to the decrease in the lipid-to-protein ratio of the membranes and has a marked effect on the activity of membrane-associated enzymes and transport systems.


l-amino acid oxidases of Proteus rettgeri.

Proteus rettgeri has been found to contain two separable 1-amino acid oxidases. Both enzymes are particulate in nature, neither being ribosomal bound. One of these enzymes appears to have broad specificity, being active toward monoaminomonocarboxylic, imino, aromatic, sulfur-containing, and beta-hydroxyamino acids. The other enzyme has more limited specificity, catalyzing the oxidative deamination of the basic amino acids and citrulline. The affinity of this oxidase for the various substrates at pH 7.6 in decreasing order is arginine, histidine, ornithine, citrulline, and lysine. This enzyme has a particularly high affinity for arginine (Km equal to 0.27 mM), and anomalous kinetics are observed with increasing substrate concentrations. When concentrations of arginine greater than 1.0mM were added to the reaction containing histidine, imidazole pyruvate formation was completely inhibited.