Endocarditis as a manifestation of Chlamydia B infection (psittacosis). A case of Chlamydia B (psittacosis) endocarditis is described in a patient with no known previous valve disease. After mitral valve replacement a fall in Chlamydia B antibody titre occurred. At repeat mitral valve replacement five months later for a paravalve leak no evidence of continuing endocarditis was present. Direct confirmation of infection in the patient's apparently healthy budgerigar was obtained. Aspects of the pathogenesis, diagnosis, and treatment are discussed.


Indirect blood pressure and heart rate measured quickly without observer bias using a semi-automatic machine (auto-manometer)--response to isometric exercise in normal healthy males and its modification by beta-adrenoceptor blockade. 1 In a double-blind crossover study, six volunteers performed sustained handgrip at 50% of maximal voluntary contraction before and 90 min following oral administration of 0.25 and 100 mg metoprolol tartrate, a beta1 selective adrenoceptor blocking agent. Blood pressure and heart rate were measured with the Auto-Manometer, an electronic semi-automatic device based on the principles of the London School of Hygiene and Tropical Medicine sphygmomanometer. It eliminates observer and digital bias completely, and also records heart rate at the same time as blood pressure is recorded. 2 Resting heart rate fell 15% after 25 mg, 21% after 100 mg and was unchanged after placebo. Systolic blood pressure fell 6% on both doses and was unchanged on placebo. Diastolic pressure did not change with any of the doses. 3 At 1 min of handgrip, heart rate was significantly lower after 25 and 100 mg than before drug or after placebo. There was no difference between the blood pressure levels attained before or after any of the dose levels. The rise of heart rate tended to be somewhat dampened after 100 mg only. The rise in blood pressure was unchanged after any dose compared with before.


Eye symptoms in patients taking propranolol and other hypotensive agents. 1 Four hundred and eighty-three hypertensive patients being treated with propranolol or other hypotensive drugs were questioned about symptoms of gritty feelings, sore or red eyes, photophobia and use of eye drops. 2 Between 11.6% and 29.8% gave positive answers but there was no difference between the different drug regimes. The complaint of 'red eyes' was more common in smokers.


Intravenous fluids in medical in-patients. 1 Data from the Boston Collaborative Drug Surveillance Program were reviewed to determine the proportion of patients receiving intravenous fluids in the participating hospitals. 2 Wide differences between hospitals in the United States and four other countries were observed: i.v. fluids being given to 54% of patients in one American hospital and only 7% of patients in an Israeli one. A two-fold difference in the frequency of i.v. fluid use in two otherwise comparable Scottish teaching hospitals was observed. This difference was not due to observed patient characteristics, did not arise from selection bias or observational differences between the two hospitals and was unlikely to have arisen by chance. 3 It is concluded that the findings were due to different policies on the part of the attending physicians. Although the study could not be used to evaluate the beneficial effects of the administered fluids, adverse effects were common (15% of recipients) and in some instances potentially serious. Adverse effects were reported more frequently after infusion of 5% dextrose (13% of recipients) than after isotonic saline (7%) perhaps because of the low pH of the former solution.


Determinants of anticoagulant control in patients receiving warfarin. 1 A hospital-based drug information system has been used to assess the time for which patients treated with warfarin were outside the range of Thrombotest values 5-10% and 5-15% and to examine possible contributory factors in situations where anticoagulation fell outside these ranges. 2 Anticoagulant control varied with the age of the patient and with concomitant drug therapy but not with patient sex or indication of anticoagulation. 3 Most patients were 'under-anticoagulated' at some stage but patients over 70 years spent significantly longer in the 5-10% range than those in the age range 30-59 years and Thrombotest values of less than 5% were found predominantly in the older group. 4 Patients given drugs known to interact with warfarin spent least time in the defined Thrombotest ranges. Those on drugs known to potentiate warfarin effect had significantly lower Thrombotest values than the other patients studied.


Age as a determinant of sensitivity to warfarin. 1 Parallel human and rat studies were carried out to confirm the previous suggestion of an increased sensitivity to warfarin in old age. 2 The anticoagulant response to warfarin was found to be greater in the elderly groups despite, in the case of the patient study, the elderly subjects being given a smaller weight-related dose. 3 At the same plasma warfarin concentrations there was greater inhibition of vitamin K-dependent clotting factor synthesis in the elderly. There was no difference in the rate of clotting factor degradation in the two age groups. 4 There was no appreciable difference in warfarin pharmacokinetics (plasma half-life, apparent volume of distribution, plasma clearance, plasma protein binding or plasma warfarin alcohol levels) in the two age groups. 5 There appeared to be no major age-related differences in warfarin pharmacokinetics and the increased effect of warfarin in the elderly seemed to result from an increased intrinsic sensitivity to warfarin.


Digoxin measurements in general practice. 1 As part of a prospective study on the use of digoxin in a general practice, plasma digoxin concentrations were measured in all patients receiving the drug. 2 A low mean plasma digoxin concentration was observed, suggesting a cautious approach to digitalization. 3 The correlation between plasma digoxin concentration and signs or symptoms commonly used in the assessment of digoxin effect was poor. 4 It was concluded that the measurement of plasma digoxin could be of assistance in a variety of clinical settings within a general practice.


Some effects of injected hyoscine butylbromide: a versatile class experiment in human pharmacology. 1 An experiment with hyoscine butylbromide has been used to demonstrate how drug effects differ in intensity and time course after intravenous (20 mg) and subcutaneous (40 mg) injection with and without massage of the injection site. It also demonstrates how objective and subjective observations may be related, and gives students practice in the interpretation of experimental data. 2 The experiment takes about 2 h, and the effects of the drug have worn off completely after about 3 hours. 3 The structure of the discussion of the experiment and its results with the students are described to draw attention to the questions that can be asked of the data. The results obtained with 41 subjects are summarized to illustrate this discussion. 4 Various ways of modifying the experiment to suit other teaching objectives are mentioned. The need to consult the local Ethics Committee before undertaking experiments on students is emphasized.


Amphetamines, growth hormone and narcolepsy. 1 Plasma amphetamine and growth hormone levels have been measured in eight normal and twenty-six narcoleptic subjects following a single dose of (+)-amphetamine (20 mg) or (-)-amphetamine (20 mg) by mouth. 2 Peak plasma levels and the shape of the plasma amphetamine-time curve were similar with both isomers in normal and narcoleptic subjects. 3 In most normal subjects both (+)-and (-)-amphetamine (20 mg) caused an increase in the plasma concentration of growth hormone. The two isomers were approximately equipotent in this respect. Neither (+)- nor (-)-amphetamine (20 mg) caused an increase in plasma growth hormone concentration in narcoleptics. 4 Following amphetamine (30 mg), two of six narcoleptic subjects had an increase in plasma growth hormone concentration. 5 Levodopa (250 mg) with (-)-alpha-methyldopa hydrazine 25 mg (Sinemet) by mouth, caused a rise in plasma growth hormone concentration in most normal subjects. The magnitude of the Sinemet-induced rise in plasma growth hormone concentration in narcoleptics was less than in normal subjects.


Plasma prednisolone levels in man following administration in plain and enteric-coated forms. 1 Prednisolone plasma levels were measured in seven patients by a competitive protein binding method after administration of prednisolone (20 mg) by mouth as plain or enteric-coated tablets. 2 The mean peak plasma prednisolone concentration after the enteric-coated preparation, 199 ng ml-1, was significantly lower than after the plain tablet, 268 ng ml-1, and occurred later, at 4.69 h compared with 1.72 h. The bioavailability of the two preparations was similar. 3 It is concluded that, although the plasma concentration versus time profiles are different, the absorption of prednisolone from plain and enteric-coated preparations is equal.


The absorption and elimination of ICI 74,917 in man. 1 The pharmacokinetics of ICI 74,917 were studied in both asthmatic patients and normal volunteers. 2 The tritiated compound was administered to the lungs by inhalation from an aerosol and a bronchoscope, and by intravenous, oral and buccal routes. Radioactivity was measured in plasma, urine, faeces, sputum and exhaled air. 3 After bronchoscopic administration 63% of the available dose was absorbed; after aerosol administration 8% was absorbed from the lung and more than 50% swallowed. 5 Intravenous studies indicated that the drug is excreted in the bile and urine in the ratio 2:1. 5 Minimal oral and no buccal absorption occurred. 6 There was no evidence of tritium exchange or drug metabolism. 7 The mean terminal half-life following administration by all route was 16.1 hours. However, the majority of the dose was rapidly excreted. 8 Aerosol administration is the method of choice for the clinical use of ICI 74,917.


Vascular reactions to histamine and compound 48/80 in human skin: suppression by a histamine H2-receptor blocking agent. 1 The ability of a specific competitive histamine H2-receptor antagonist, cimetidine, to inhibit vascular responses to histamine in human skin provides new evidence that skin blood vessels possess histamine H2 receptors. 2 Simultaneous systemic administration of cimetidine and chlorpheniramine (an H1-receptor antagonist) was more effective than either drug alone in inhibition of the erythematous reaction both to exogenous histamine, and endogenous histamine secreted by skin mast cells in response to compound 48/80. 3 These results suggest that combined therapy of histamine-mediated skin diseases included urticaria and dermatitis using a combination of H1- and H2-histamine receptor antagonists may be more effective than either class of drug alone.


Fluorimetric assay of cephradine, cephalexin and cephaloglycin. 1 A simple, rapid and reproducible fluorimetric assay for cephradine, cephalexin and cephaloglycin is described. 2 The method involves addition of formaldehyde which catalyses the formation of fluorescent derivatives. 3 The structural similarities between the side chains of these antibiotics and their identical excitation and emission spectra suggest that they may be forming similar fluorescent derivatives.


Plasma, bone, hip capsule, synovial and drain fluid concentrations of lincomycin during total hip replacement. 1 Lincomycin (600 mg) was given 6 h preoperatively by intramuscular injection, as an intravenous infusion over 30 min and for 72 h postoperatively in twelve patients having total hip replacement. 2 The plasma, bone, hip capsule, synovial and drain fluid concentrations of lincomycin were almost always above the M.I.C. of lincomycin against penicillinase producing Staphylococcus aureus. 3 There was a good correlation between the estimated concentrations of lincomycin in bone by the grinding and agitation methods of analysis. 4 Two patients developed pseudomembranous colitis after parenteral lincomycin.


Lignocaine kinetics in cardiac patients and aged subjects. 1 The pharmacokinetics following long term intravenous infusion of lignocaine to cardiac patients have been examined. 2 Plasma levels and half-lives of lignocaine and monoethylglycinexylidide (MEGX) showed wide inter-patient variability. 3 Toxicity reactions to therapy were associated with elevated lignocaine and/or MEGX plasma levels. 4 In a separate study the effect of age on the pharmacokinetics of lignocaine was examined using bolus doses (50 mg) of the drug to young and aged subjects. 5 Elderly subjects had significantly longer half-lives for lignocaine compared to younger individuals although no change in plasma clearance occurred. 6 The drug appeared to distribute differently in the aged as reflected by significantly increased apparent volumes of distribution. 7 The 24 h urinary recovery of the major metabolite (total 4-hydroxyxylidine) showed a significant reduction in the elderly when compared to the young. 8 The clinical significance of these studies with respect to lignocaine therapy has been discussed.


Comparison of quinidine plasma concentration curves following oral administration of some short- and long-acting formulations. 1 The time-course of quinidine plasma concentrations and selected kinetic parameters were studied in healthy male volunteers given single oral doses of four different quinidine formulations. Comparison was made with quinidine sulphate in a rapidly dissolving form. 2 Plasma half-lives did not significantly differ among treatments. 3 Quinidine polygalacturonate appears to be equivalent to quinidine sulphate in respect to both the absorption and elimination kinetics. When both quinidine bisulphate (Kinidin Durules) and quinidine arabogalactansulphate (Longacor) were administered plasma levels peaked significantly later than after quinidine sulphate. However, while the former seems to be absorbed to the same extent as quinidine sulphate, the latter exhibits lower bioavailability in respect to all other preparations.


Effect of clofibrate on glucose tolerance in maturity onset diabetes. 1 Fourteen maturity onset diabetics showed improvement of glucose tolerance while on treatment with clofibrate. Fasting blood glucose levels were reduced by 20% after treatment for 14 and 28 days. 2 The effect was found to be independent of concurrent treatment with oral hypoglycaemic drugs. 3 Plasma insulin levels were also lower during clofibrate treatment. 4 Clofibrate may prove to be a useful adjunct to the treatment of maturity onset diabetes.


Ciclazindol and mazindol on glucose uptake into human isolated skeletal muscle: no interaction of mazindol with methysergide. 1 Ciclazindol, like mazindol, produced a significant concentration-dependent increase in glucose-uptake into human skeletal muscle in both the presence and absence of insulin. 2 Methysergide, which inhibits the action of fenfluramine on glucose-uptake into skeletal muscle, did not influence the effect of mazindol.


Phase 1 tolerability and searching dose studies with 4-isothiocyanato-4'-nitrodiphenylamine (C.9333-Go/CGP 4540), a new anthelmintic. 1 Phase 1 and 2 studies were carried out in thirteen volunteers and thirty patients with hookworm anaemia with a new anthelmintic, 4-isothiocyanato-4'-nitrodiphenylamine (C.9333-Go/CGP 4540). Single and multiple doses were administered by mouth under careful supervision by the staff of the clinical pharmacology unit. 2 4-Isothiocyanato-4'-nitrodiphenylamine (C.9333-Go/CGP 4540) was well tolerated in volunteers up to a single dose of 1750 mg and in patients up to 3000 mg in divided doses. Side effects were rare, transient and mild. One patient had moderate giddiness for 24 h after the compound. The laboratory investigations did not show any adverse effects on organ functions. Transient non-specific changes in the T-wave were occasionally observed in the e.c.g. 3 Multiple doses (125 mg, 250 mg or 1000 mg X 3) of the compound were highly effective in eradicating hookworm infection, as judged by almost 100% reduction in faecal egg counts and significant recovery of parasites from 24 h stool samples. The compound also showed activity against A. lumbricoides, T. trichiura and E. vermicularis. The compound appears to be promising and more studies, in a field situation, are desirable.


The effect of quadruple chemotherapy on the peripheral blood lymphocyte populations in Hodgkin's disease. 1 Quadruple chemotherapy with cytotoxic drugs in advanced (Stage IIIB) Hodgkin's disease causes a fall in the blood lymphocyte count with recovery 1 month after stopping treatment. 2 No selective effect on the T or B lymphocyte populations was shown.


Serum concentrations of lithium after three proprietary preparations of lithium carbonate (Priadel, Phasal and Camcolit). 1 The serum lithium concentration was measured before dosing, and at 2, 4, 6, 8, 10, 20, 22, and 24 h after three proprietary preparations of lithium carbonate (Camcolit, Phasal or Priadel). 2 A total of twenty-eight studies were performed on eleven patients. In seven studies the patients received Camcolit, in ten studies patients received Priadel and in eleven the patients took Phasal. 3 The dose of each preparation was adjusted during a 10 week run-in period to maintain the serum lithium within the normal therapeutic range (0.6-1.2 mmol/1). Five patients received one lithium preparation, one patient received three lithium preparations and five patients received four lithium preparations consecutively. 4 After a single daily dose of Phasal and Priadel, the serum lithium remained within the therapeutic range for 24 h. 5 After a single daily dose of Camcolit, the serum lithium remained within the therapeutic range for only 12 h. 6 After three doses of Camcolit (given at 0.800 h, 12.00 h, and 18.00 h) each day, the serum lithium was maintained in the therapeutic range for 24 h.


Characterization of chromatin modified with ethyl acetimidate. Thymus chromatin was extensively modified with ethyl acetimidate, substituting up to 90% of the lysyl residues of the histones while retaining the positive charge of the basic amino acid. Physiochemical and immunochemical characterization of this derivative chromatin indicates a high degree of retention of the native structure of the nucleoprotein even after extensive modification. The alterations which are detected are most simply interpreted as resulting from a weakening of the interactions of histone H1 with DNA in the modified chromatin. The near-native character of amidinated chromatin contrasts with the more extensive structural alterations observed in acetylated chromatin. Our data demonstrate the suitability of this reagent for mapping available lysyl residues in this and other nucleoproteins and suggest that the related bisimido esters may be reagents of choice for cross-linking of chromatin histones.


Binding studies on anti-fructofuranan mouse myeloma immunoglobulins A47N, A4, U61, and E109. Four murine myeloma immunoglobulins, A4, A47N, U61, and E109, have been studied for their binding affinities with inulin and a series of oligosaccharides derived from inulin. The results indicate that the combining site of these immunoglobulins shows highest complementarity for a trifructofuranosyl sequence (A4 and A47N) and a tetrafructofuranosyl sequence (U61 and E109). The size of the combining area of the immunoglobulin E109 derived from the antigenic determinant (approximately 15 X 14 X 10 A) agrees well with the size observed on a hypothetical space model of the Fv portion of E109 (Potter, M., Rudikoff, S., Padlan, E. A., and Vrana, M. (1976), Antibodies in Human Diagnosis and Therapy, Haber, E., and Krause, R.M., Ed., New York, N.Y., Raven Press).


Malate dehydrogenase isolated from extremely halophilic bacteria of the Dead Sea. 1. Purification and molecular characterization. The complete purification of malate dehydrogenase (EC 1.1.1.37) from extremely halophilic bacteria of the Dead Sea is described. The purification procedure includes (a) precipitation by ammonium sulfate, (b) fractionation on Sepharose 4B using a decreasing concentration gradient of ammonium sulfate, (c) gel permeation chromatography on Sephadex G-100, (d) chromatography on hydroxylapatite, and (e) affinity chromatography on 8-(6-aminohexyl)amino-NAD+-Sepharose at 4.26 M NaCl. The absorption and fluorescence spectra of the native and denatured enzyme were measured, and the extinction coefficient at 280 nm in 4.26 M NaCl was found to be 0.803 cm2mg-1. The amino acid composition analysis showed an excess of 10.4 mol % of acidic amino acids. The apparent specific "volume" phi' of the active enzyme at 4.26 M NaCl was found to be 0.680 +/- 0.015 mL/g. The molecular weight of the native enzyme was found to be 84 000 +/- 4000 determined in 4.26 M NaCl from equilibrium sedimentation data. The molecular weight of the subunits is 39 000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, the native enzyme is composed of two subunits.


Oxidation of corticosteroids to steroidal carboxylic acids by an enzyme preparation from hamster liver. Enzyme activity which catalyzes the oxidation of 11-deoxycorticosterone to 21-oic acids accompanies the "detritiating" enzyme (isomerase) of hamster liver recently isolated by Martin, K. O., et al. ((1977) Biochemistry 16 (preceding paper in this issue)). The metabolites isolated were 20alpha- and 20beta-hydroxy-3-oxo-pregn-4-en-21-oic acid and 3,20-dioxo-pregn-4-en-21-oic acid. When 21-hydroxy[4-14C, 21-3H]pregn-4-en-3,20-dione was the substrate, about half of the tritium was retained in position 20 of the hydroxy acids. The system which catalyzes the conversion of the ketol side chain of corticosteroids to acid metabolites appears to be a cluster of closely related enzymes. As a result of these studies, we believe that the hamster liver enzyme preparation provides a useful model system for studies on the biosynthesis of acid metabolites of the corticosteroids in man.


Trypsin-like neutral protease associated with soluble elastin. Isolation of tropoelastin is complicated by the presence of a neutral protease closely associated with tropoelastin that is capable of sequentially degrading tropoelastin to small peptides. Substrate and inhibitor specificities of this neutral protease associated with purified tropoelastin were examined. The enzyme displayed proteolytic activity against casein, and esterase activity was detected when assayed against N-tosyl-L-arginine methyl ester but not against tert-butyl-oxycarbonyl-L-alanine p-nitrophenyl ester. No appreciable elastinolytic activity was detectable against either insoluble sodium dodecyl sulfate treated elastin or maleylated tropoelastin. The enzyme was not inhibited by the chymotrypsin inhibitor toluenesulfonylphenylalanine chloromethyl ketone. The enzyme was inhibited by phenylmethanesulfonyl fluoride and, to various degrees, by metal chelators. Tosyllysyl chloromethyl ketone, epsilon-aminocaproic acid, and Aprotinin (pancreatic trypsin inhibitor--Kunitz type), all inhibitors of trypsin-like enzymes, were very effective inhibitors, as were soybean trypsin inhibitor and human alpha-1-antitrypsin. The data suggest that the tropoelastin-associated enzyme is a neutral serine protease with trypsin-like specificity.


Lateral diffusion in phospholipid multibilayers measured by fluorescence recovery after photobleaching. The method of fluorescence recovery after photobleaching has been used to measure the temperature dependence of the lateral diffusion coefficients (D) of two fluorescent lipid analogues in phospholipid multibilayers of various compositions. The probes employed were 3,3-dioctadecyloxocarbocyanine (diO-C18(3) and N-4-nitrobenz-2-oxa-1,3-diazole phosphatidylethanolamine (NBD-PE). In fluid egg phosphatidylcholine multibilayers at 25 degrees C, D was about 4 X 10(-8) cm2/s for NBD-PE and 1.5 X 10(-7) cm2/s for diO-C18(3) and was moderately temperature dependent (2-fold change over 10 degrees C). Equimolar cholesterol reduced D for NBD-PE in these multibilayers by a factor of 2. A greater than 100-fold decrease in D was detected in dimyristoylphosphatidylcholine multibilayers at approximately 23 degree C, which coincides with the gel-to-liquid-crystalline transition temperature, Tm (D  5 X 10(-8) cm2/s at T greater than Tm to D less than 5 X 10(-10) cm2/s at T less than Tm). Equimolar cholesterol abolished this transition behavior, raising D below Tm and decreasing D above Tm. These results confirm and extend previous studies of lateral diffusion employing magnetic resonance and other optical techniques and give additional confidence in the fluorescence methods.


Kinetics of association of amphotericin B with vesicles. Amphotericin B associates with vesicles prepared from phosphatidylcholines. The influence of lipid composition on the initial rate of amphotericin B association with vesicles was examined using stopped-flow kinetic measurements. A relationship was found between the tightness of packing of phosphatidylcholine molecules in the vesicles and the initial rate of amphotericin B association. Shortening of the fatty acyl chain length of saturated phosphatidylcholines and increasing the number of double bonds in the fatty acyl chains of unsaturated phosphatidylcholines enhance the initial rate, whereas addition of cholesterol of the bilayers reduces the rate. The initial rate of association with phosphatidylcholine-sterol vesicles follows the order, thiocholesterol greater than androst-5-en-3beta-ol greater than epicholesterol greater than ergosterol greater than cholesterol and is thus inversely related to the order of phospholipid-sterol affinity, as revealed by permeability, surface area, and magnetic resonance measurements. These data suggest that the initial rate of amphotericin B uptake into vesicles depends on competition between lipid-lipid and amphotericin-lipid interactions.


Equilibria involved in prothrombin- and blood-clotting factor X-membrane binding. The study of prothrombin- and factor X-membrane interaction by light-scattering intensity measurements at 90 degrees is reported. This technique, which uses a fluorometer as a light-scattering photometer, can be applied to measurement of free and membrane-bound protein concentrations, from which equilibrium constants can be obtained. The following equilibria adequately describe the observed properties of prothrombin-membrane interaction (formula: see text) where P and PL are protein and phospholipid, PiCa and PLjCa are the calcium complexes, P'iCa is the protein after undergoing a calcium dependent transition, and P'-PLi+j+mCa is the protein-membrane complex. Several lines of evidence indicate that i, j, and m are interrelated and m decreases to 0 when i and j are saturated. In agreement with this, direct calcium binding measurements indicate m values of 3.2 +/- 1.5 and 1.1 +/- 1.5 at 0.5 and 1.2 mM calcium, respectively. The total number of functional calcium ions in the complex (i + j + m) is 6 to 9 based on Hill coefficients for the reactions and direct calcium binding measurements. In reaction 3, the maximum stoichiometry of calcium per acidic phospholipid is 1:2. While the details of factor X-membrane binding were not determined in quite as great detail, the equilibria (identified) appear the same but a major difference is the calcium concentration needed to initiate protein-membrane binding.


Interaction of prothrombin and blood-clotting factor X with membranes of varying composition. The interaction of prothrombin and factor X with membranes of widely varying composition is reported. These protein-membrane interactions require the presence of acidic phospholipid; phosphatidylserine is the most effective from the standpoint of protein-membrane affinity. The maximum protein binding capacity is proportional to the phosphatidylserine content of the membrane up to about 15% and the stoichiometry is 9.2 +/- 1.3 phosphatidylserine residues per prothrombin molecule and 5.2 +/- 1.5 per factor X molecular. The protein apparently causes clustering of the phosphatidylserine residues in these membranes of low phosphatidylserine content. Above about 20% phosphatidylserine, the factor limiting protein-membrane interaction appears to be protein packing density on the membrane surface. The maximum protein binding capacity at 50% phosphatidylserine is 1.2 g of protein per g of phospholipid. Phosphatidic acid is similar to phosphatidylserine in its ability to bind these proteins except for somewhat larger dissociation constants. Phosphatidylethanolamine and phosphatidylglycerol are less effective in all respects for promoting these protein-membrane interactions. Small amounts of phosphatidylserine mixed with these latter phospholipids, however, have a major effect on protein-membrane binding so that the dissociaction constants are more characteristic of membranes of high phosphatidylserine content.


Structure of the prothrombin- and blood clotting factor X-membrane complexes. The configuration of the prothrombin- and factor X-membrane complexes was investigated by the technique of quasielastic light scattering. It is concluded that the fragment 1 region of prothrombin is located at one end of the prothrombin molecule and that the membrane binding site is at the tip of the fragment 1 region. Prothrombin binds to the surface of the membrane with no detected penetration into the lipophilic region of the membrane. The remainder of the prothrombin molecule extends radially from the membrane surface with maximum protrusion into solution. Factor X also binds to the membrane at one end of the molecule and extends into solution. Based on the evidence presented here and in other communications [Nelsestuen, G. L., and Lim, T. K. (1977), Biochemistry 16, and Nelsestuen, G. L., and Broderius, M. (1977), Biochemistry 16 (respectively the first and second in a series of three papers in this issue)] a model for prothrombin-membrane interaction is given. Quasielastic light scattering appears to be a valuable new method for studying protein-membrane interactions.


Characterization of bovine factor XIIa (activated Hageman factor). Factor XIIa (activated Hageman factor) was isolated from bovine plasma by ammonium fractionation followed by heparin-agarose, carboxymethylcellulose, and arginine-agarose column chromatography. It was separated from factor XII in the final step by chromatography on benzamidine-agarose. Factor XIIa has a molecular weight of approximately 74 000 and is composed of a heavy and light chain held together by a disulfide bond(s). The amino-terminal sequence of the heavy chain is Thr-Pro-Pro-Trp--Lys-Gly-Pro-Lys-Lys-His-Lys-Leu- which is the same as the precursor protein. The carobyl-terminal residue in this polypeptide chain is arginine. The amino-terminal sequence of the light chain is Val-Val-Gly-Gly-Leu-Val-Ala-Leu-Pro-Gly-Ala-?-Pro-Tyr-Ile-. This latter sequence is homologous with the amino-terminal sequence of a number of plasma serine proteases when compared with the chain containing the active-site serine residue. These data suggest that factor XII is converted to factor XIIa by the cleavage of a specific internal arginyl-valine peptide bond. Factor XIIa, in contrast to factor XII, has hydrolase activity toward arginine-containing substrates and is readily inhibited by antithrombinIII and diisopropyl phosphorofluoridate. The inhibitors, in each case, are bound to the light chain of factor XIIa which contains the active-site serine residue.


Activation of bovine factor VII (proconvertin) by factor XIIa (activated Hageman factor). Bovine factor VII (proconvertin) is a plasma glycoprotein that participates in the extrinsic pathway of blood coagulation. It has a molecular weight of 45 500 and is composed of a single polypeptide chain with an amino-terminal alanine residue. Factor VII is readily converted to factor VIIa by factor XIIa (activated Hageman factor) employing an enzyme to substrate weight ratio of 1:50. Factor VIIa is composed of a light and a heavy chain held together by a disulfide bond(s). The heavy chain, which is formed from the carboxyl-terminal region of the precursor, contains an amino-terminal sequence of Ile-Val-Gly-Gly-. The heavy chain also contains the active-site sequence of -Phe-Cys-Ala-Gly-Tyr-Thr-Asp-Gly-Thr-Lys-Asp-Ala-Cys-Lys-Gly-Asp-Ser-Gly-Gly-Pro-His-. This sequence is homologous with the active-site region of a number of plasma serine proteases. These data indicate that factor VII is a typical precursor of a serine protease which is converted to an enzyme by factor XIIa by the cleavage of a single, internal peptide bond.


Synthesis and biological activity of a lambda pseudo operator. The chemical and enzymatic syntheses of bacteriophage lambda pseudo operator DNA are described. The 17 base-paired duplex contains the DNA which has been proposed as the binding site for cI repressor protein. The synthetic duplex is twofold symmetric and represents the best possible nucleotide summation of the six proposed operator sites in the leftward and rightward operators. However, it does not correspond exactly to any single proposed operator sequence. The chemical synthesis includes the deoxyoligonucleotides d(T-A-T-C-A-C), d(C-G-C-C-G-G-T-G-A-T-A), d(T-A-T-C-A-C-C), and d(G-G-C-G-G-T-G-A-T-A). These deoxyoligonucleotides were joined with T4 DNA ligase to form d(T-A-T-C-A-C-C-G-C-C-G-G-T-G-A-T-A) and d(T-A-T-C-A-C-C-G-G-C-G-G-T-G-A-T-A). The cI repressor protein was found to bind to the duplex formed from these two segments.


Properties of an altered RNA polymerase II activity from an alpha-amanitin-resistant mouse cell line. alpha-Amanitin-resistant clones were selected in the mouse lymphoblastoid cell line L5178Y. One resistant clone, named A169b, was recloned and the properties of its DNA-dependent RNA polymerases were examined. The RNA polymerase II activity from A169b differs from the parental cell line in that approximately half the activity is resistant to 0.5 microgram/mL alpha-amanitin, while the parental enzyme is 50% inhibited at 0.005 microgram/mL. The enzymes from A169b and the parental line were purified free of polymerase III and their properties compared. The two preparations were identical in their apparent affinities for the four nucleoside triphosphates, in their salt and divalent cation preferences, and in their preference for denatured over native DNA. They differed in their response to alpha-amanitin. The apparent K1 for the parental enzyme was 3.5 X 10(-9) M; plots of 1/V vs. alpha-amanitin concentration gave a biphasic curve with A169b enzyme. The two apparent K1 values were 4.1 X 10(-9) and 2.1 X 10(-6) M. In addition, the enzyme from A169b showed a twofold higher activity on poly [d(AT)] as template, compared to native DNA, than that of the parental enzyme. Other template preferences may be affected, but differences were marginal. These results indicate that mutation to alpha-amanitin resistance may alter other enzymatic parameters; such mutations may be helpful in elucidating structure-function relationships in these complex enzymes.


Synthesis and properties of nucleoside 5'-phosphoazidates derived from guanosine and adenosine nucleotides: effective on elongation factors G and Tu dependent reactions. A new type of nucleoside poly (5'-phosphate) analogue, nucleoside 5"-phosphoazidate, with an azido group on the terminal phosphate of GTP, ATP, GDP, GMP, AND AMP, has been synthesized by nucleophilic displacement of the corresponding activated nucleosides by azide ion in yields of 25-45%. Guanosine 5-phosphoazidates is readily photolyzed by ultraviolet light; the corresponding adenosine derivative photolyzes more slowly. Guanosine 5'-O-(2-azidodiphosphate) and guanosine 5"-O-(3-azidotrophosphate) are competitive inhibitors of the formation of the ribosome-EF-G-GDP-fusidic acid complex and of the ribosome-EF-G GTPase. The dissociation constants of the former reaction are calculated to be 27 and 7 micrometer, respectively, or 270 and 73 times that of GDP. In the latter reaction, which is conducted in the absence of fusidic acid, the Ki values are 330 and 28 micrometer, respectively, or 12 and 1 times that of GDP. Guanosine 5"-O-(2-azidodiphosphate) and guanosine 5'-O-(3-azidotriphosphate) also complete with GTP in the formation of the binary complex. EF-Tu-GTP, with respective Kd values of 750 and 75 relative to GTP.


Differential scanning calorimetry and enzymic activity of rat liver microsomes in the presence and absence of delta1-tetrahydrocannabinol. The thermal transitions of rat liver microsomes and isolated lipids were investigated by using differential scanning calorimetry. Endothermic transitions at approximately-5 degrees C and between approximately18 degrees and 40 degrees C were detected in the membranes and at approximately-10 degrees C and between approximately 10 degress and 20 degrees C in the extracted lipids. Interaction with delta1-tetrahydrocannabinol of microsomal membranes and of extracted lipids influences the thermotrophic behaviour as revealed by differential scanning calorimetry and eliminates the break in the Arrhenius plot of the enzymic activity of O-demethylase.


The interaction of hepatocyte plasma membranes with an azide derivative of procaine. A photoreactive derivative of procaine, p-azidobenzoyldiethylaminoethanol hydrochloride, has been synthesized and used as a site-directed probe to label hepatocyte plasma membranes. The procaine derivative was shown to have membrane binding and Ca2+ displacement characteristics quite similar to that of procaine. Photolysis of the derivative in the presence of hepatocyte plasma membranes resulted in the covalent incorporation of the probe into both the protein and lipid fractions. Analysis of the labeled membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that one membrane protein was significantly labeled with a molecular weight of 21 400 in addition to membrane lipids. Both binding and labeling could be inhibited in the presence of an excess of procaine. The labeled membrane components may be involved in the binding of Ca2+ to the membrane system.


Refinement of the fluid-mosaic model of membrane structure. Certain molecular packing criteria previously employed in a quantitative analysis of lipid micelles and bilayers are here extended to biological membranes. The inclusion of both thermodynamic and packing considerations point to a highly complex self-assembly mechanism in which the organization of lipids and proteins is highly coupled, with far reaching consequences as regards the structure and function of biological membranes.


Intra- and intermolecular cross-linking of membrane proteins in intact erythrocytes and ghosts by SH-oxidizing agents. In intact human erythrocytes, SH-oxidizing agents exclusively cross-link spectrin via disulfide bonds. In ghosts, additional dimerization of the major intrinsic protein, band 3, is observed. After blockade of intracellular GSH the agents dimerize band 3 in the intact cell too, indicating that GSH may prevent band 3 dimerization under physiological conditions. The oxidizing agents reversibly oxidize 80% of the membrane SH-groups, suggesting that these groups are arranged close enough to each other to form disulfide bonds. This arrangement may protect other cell cell structures against free radicals or oxidative stress.


Membranes undergoing phase transitions are preferentially hydrolyzed by beta-bungarotoxin. beta-Bungarotoxin preferentially hydrolyzes choline phospholipids (dilauroyl, dimyristoyl, dipalmitoyl) at their respective gel to liquid crystalline phase transition temperatures. Cholesterol markedly reduces the rate of phospholipid hydrolysis; at 0.33 mol percent cholesterol:phospholipid, the toxin's phospholipase activity is completely inhibited.


Cation diffusion selectivity in a pore model. The phosphatidylcholine/water lamellar phase. The diffusion coefficients D (cm2/s), of four monovalent cations K+, Na+, Rb+ and Cs+ and of Ca2+ have been measured in phosphatidylcholine/water lamellar phase as a function of phase hydration and temperature and in the presence of divalent cations. Diffusion rates vary strongly with phase hydration, between 10(-7) and 10(-6) cm2/s for monovalent and 10(-8) and 10(-7) for Ca2+. The activation energies obtained are relatively small (5--10 kcal/mol). As the phase water content increases, a series of diffusion sequences is obtained, corresponding to the sequences predicted by Eisenman's theory of alkali ion equilibrium selectivity. This diffusional selectivity, which depends exclusively upon non-equilibrium parameters (mobility) within the hydrophilic path is discussed in respect to current theories of pore selectivity.


Monte Carlo studies of the hydrocarbon region of lipid bilayers. We present the results of a Monte Carlo study of systems of hydrocarbon chains attached to a plane interface and interacting through hard core repulsive forces only. The chain-order parameters which we find in our studies are compared to experimental results (NMR and ESR). The role of "kink" states and the relevance of our studies to theoretical models are also discussed.


Mechanism of the inhibition by RNA of the RNA polymerases of Histoplasma capsulatum. 4 S RNA isolated from the dimorphic fungus Histoplasma capsulatum inhibited the DNA-dependent RNA polymerase activity of the yeast phase of this fungus. Inhibition was specific for initiation, and resulted from binding of the RNA to the enzyme. Among a variety of synthetic polynucleotides tested, only poly(G) and oligo(dG) were effective inhibitors, suggesting a role for guanines or guanine-rich sequences of RNA in the inhibition reaction.


A molecular orbital investigation of the conformation of transfer RNA. The PCILO method has been used for a theoretical exploration of the conformational properties of tRNAPhe with respect to the phosphodiester torsion angles. The computations were based on the utilisation of the dinucleoside triphosphate model and took into account the different combinations of sugar puckers and different conformations about the C4'-C5' bond. The dependence of the (omega'-omega) conformational energy maps upon these factors was specified. A detailed comparison is carried out between the theoretical results and experimental data on the crystal structure of tRNAPhe produced by four different groups of investigators.


Fetal utilization of maternally derived ketone bodies for lipogenesis in the rat. When D-beta-[3-14C]hydroxybutyrate was injected via the femoral vein into pregnant Sprague-Dawley rats at 21 days of gestation, D-beta-[3-14C]hydroxybutyrate was enzymatically detected in fetal plasma within 5 min. The time course of the incorporation of DL-beta-[3-14C]hydroxybutyrate into fetal lipids was studied. Lipid extracts of brown adipose tissue exhibited the greatest relative incorporation followed by pancreas, liver and lung. Less radioactivity was incorporated into brain and placenta. The incorporation into fetal lipids was several-fold greater than into maternal lipids. The labelling of the individual phospholipids was similar in the different tissues with phosphatidylcholine accounting for more than 50%. 75% of the radioactivity in brown adipose tissue was in the triacylglycerol fraction. In brain, liver and placenta, approximately half of the neutral lipid radioactivity was in cholesterol. Experiments in which D-beta-[3-14C]hydroxybutyrate was directly injected into fetuses in utero confirmed that this substrate was directly used by the fetuses without maternal intervention. These studies demonstrate that the rat placenta is permeable to beta-hydroxybutyrate and suggest that this ketone body is rapidly used by the fetus for the synthesis of fatty acids and cholesterol.


A novel packing of the hydrocarbon chains in lipids. The low temperature phases of dipalmitoyl phosphatidyl-glycerol. The system dipalmitoyl phosphatidylglycerol-water displays several phases in the temperature-concentration range explored in this work. All the phases are lamellar; they differ by the organization of the hydrocarbon chains. In the high temperature phase the conformation of the chains is liquid-like. In the low temperature phases the chains are stiff and parallel and they interdigitate (in other words the CH3 ends of the chains of one layer are near to the polar groups of the opposite layer). Moreover, several types of packings of the stiff chains are observed which differ by the symmetry of the two-dimensional lattices. The observed lattices are p6, cmm and pgg.


Carotenoid biosynthesis in Rhodomicrobium vannielii. Experiments with nicotine and 2-(4-chlorophenylthio)triethylammonium chloride (CPTA). Nicotine and 2-(4-chlorophenylthio)triethylammonium chloride (CPTA) each inhibit production of the normal carotenoids of Rhodomicrobium vannielii (Rhodospirillaceae), especially rhodopin, beta-carotene and spirillixanthin, and cause the accumulation of lycopene. The inhibition of hydration of the C-1,2 double bond as well as cyclization is in agreement with proposals that these two reactions involve similar mechanisms. After removal of nicotine, cells reincubated in buffer solution or in the presence of diphenylamine convert accumulated lycopene into rhodopin. Under other conditions rhodopis is synthesized, on removal of nicotine, not from accumulated lycopene but from early precursors. The pathway of rhodopin and spirilloxanthin biosynthesis in Rm. vannielii is discussed briefly, and the possible involvement of enzyme aggregates in carotenoid biosynthesis is considered.


Lipid metabolism in inositol-deficient yeast, Saccharomyces carlsbergensis. The influence of temperature and anaerobiosis on the cellular lipid composition. Saccharomyces carlsbergensis ATCC 9080 was grown at temperatures of 9, 25, 30 and 35 degrees C, respectively. At all temperatures inositol-deficient cells contain less phosphatidylinositol than supplemented cells. Total acylglycerols increase in supplemented cells as the temperature decreases whereas in deficient cells total acylglycerols are higher at 35 degrees C than at 9 degrees C. Temperature has little influence on the fatty acid composition of the supplemented as well as on that of the deficient cells. Under anaerobic growth conditions two major effects of inositol deficiency observed under aerobic conditions, i.e. accumulation of cellular triacylglycerols and increased production of acetoin, do not occur. Other effects of inositol deficiency, such as increased glucose utilization, increase of ethanol production, decrease of phosphatidylinositol and aggregation of cells, are observed under anaerobic conditions as well as under aerobic conditions.


Reversible changes in circular dichroism spectra of cattle rhodopsin and isorhodopsin. When the disk membrane of rod outer segment is treated with detergents, the alpha-band CD of rhodopsin decreases and the gamma-band CD increases. This tendency of CD change is most prominent in the purified rhodopsin in cholic acid obtained by the ammonium sulfate fractionation of disk membranes, and the gamma-band CD is three times larger than the alpha-band CD. The beta-band CD of rhodopsin is only slightly influenced by detergents. The gamma-band of isorhodopsin shows two CD bands, one negative and one positive. Both in rhodopsin and isorhodopsin the gamma-band CD is lost by light irradiation. It is supposed that both chromophore retinal and aromatic amino acid residues of opsin are responsible for the gamma-band CD. When ammonium sulfate is added to the sonicated disk membranes suspended in cholic acid solution, the alpha-band CD of rhodopsin decreases to about a third and the gamma-band CD increases remarkably. The CD spectrum goes back to the original one on eliminating ammonium sulfate from the solution with dialysis. However, the purified rhodopsin recovers native CD spectrum on addition of lipids extracted from disk membranes. The retinal-opsin interaction that induces optical activity depends upon the property of a local environment formed by lipid and detergent.


Reinvestigation of extremely acidic proteins in bovine brain. Analysis of bovine brain extract by disc electrophoresis on a 20% polyacrylamide gel indicated the existence of three extremely acidic proteins. These proteins were isolated by column chromatography on DEAE-Sephadex A-50 and Sephadex G-75. The isolated proteins (PAP I-a, PAP I-b and PAP II) were homogeneous in various methods including 7.5% and 20% gel electrophoresis or gel chromatography, and share, in the extract, 85% of the total of the acidic proteins that migrate with the bromophenol blue marker in 7.5% gels. Their physicochemical properties, including molecular weight, ultraviolet absorption spectra or amino acid composition were similar, especially those between PAP I-a and PAP I-b where a part of primary structure appeared to be common in their tryptic peptide maps. These two proteins were identified to be the nervous system specific protein S 100 by immunochemical and electrophoresis methods as well as by amino acid analysis, and the other protein PAP II was revealed to be a calcium-binding protein. The existence and properties of the isolated proteins are discussed with relation to the heterogeneity problem of S 100 protein.


Lactoperoxidase-coupled iodination of cardiac sarcoplasmic reticulum proteins. The peptide compositions of rabbit skeletal- and canine cardiac-muscle sarcoplasmic reticulum preparations have been compared by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The cardiac preparations contain many proteins in addition to the 105 000 dalton peptide which has been previously identified as the Ca2+ stimulated ATPase. Four peptide components iodinated in the presence of either free or Sepharose 4B-bound lactoperoxidase have molecular weights of 130 000 (component I), 105 000 (component II), 52 000 (component III) and 47 000 (component IV). Comparison of the labelling patterns in the presence of the detergent Triton X-100 suggests that components I, III and IV have part of their peptide internally located. Although part of component II is externally accessible to free lactoperoxidase, its iodination is decreased by Triton X-100. Iodination of phospholamban, the 22 000 dalton substrate for cyclic AMP-dependent protein kinase, was not observed under the conditions investigated.


Second derivative spectrophotometry as an effective tool for examining phenylalanine residues in proteins. The second derivative absorption spectra of N-acetyl ethyl esters of phenylalanine, tyrosine and tryptophan, as models of the aromatic amino acid residues in proteins, were measured. The second derivative spectra of tyrosine and tryptophan were found to have no influence on the spectrum of phenylalanine over the range of 245 to 270 nm, where characteristic absorbance bands of phenylalanine were observed. Thus the second derivative spectrum is a good tool for examining the optical properties of phenylalanine residues in proteins.


A spectrum of partially carboxylated prothrombins in the plasmas of coumarin-treated patients. Prothrombin has been purified from the plasmas of normal human donors and from patients ingesting coumarins. Equimolar amounts of normal prothrombin or coumadin-induced prothrombin were incubated with phospholipid micelles in the presence of Ca2+. Subsequent gel filtration of the incubation mixtures demonstrated that all of the normal prothrombin was bound to the phospholipid; only half of the coumadin-induced prothrombin was bound. Normal prothrombin contains approximately 10 mol of gamma-carboxyglutamic acid per mol of prothrombin. The coumadin-induced prothrombin which was bound to phospholipid contains approximately 6.5 mol of gamma-carboxyglutamic acid per mol of prothrombin while that which failed to bind to phospholipid has only 2.8 mol of gamma-carboxyglutamic acid per mol of zymogen. Thus, partially carboxylated prothrombins exist in the plasmas of coumarin-treated patients. Furthermore, there is a striking electrophoretic heterogeneity in the prothrombin preparations from the plasmas of the coumadin-treated patients. This most likely results from a wide spectrum of abnormal prothrombins with varying quantities of gamma-carboxyglutamic acid per mol of zymogen.


Purification and partial amino acid sequence of the cyanogen bromide fragments of muconolactone isomerase from Pseudomonas putida. Muconolactone isomerase is shown to be resistant to proteolytic cleavage by trypsin. Cyanogen bromide cleavage at the methionine residues of the polypeptide is at least 95% complete. Six cyanogen bromide fragments are separated on DEAE-cellulose. One fragment is shown by amino acid analysis and carboxyl-terminal analysis to be an incomplete cleavage product. The five remaining fragments represent the entire polypeptide and have been ordered with respect to the entire muconolactone isomerase sequence. Approximately 50% of the polypeptide sequence could be determined from these fragments by the dansyl-Edman technique. The possible evolutionarily homologous origins of muconolactone isomerase and two analogous isomerases, carboxymuconolactone decarboxylase and sigma5-3-ketosteroid isomerase, are discussed.


Chemical characterization of a new haemoglobin variant haemoglobin J Cubujuqui (alpha2141(HC3)Arg replaced by Ser beta2). Haemoglobin J Cubujuqui (alpha2141(HC3)Arg replaced by Ser beta2) was found during a screening for abnormal haemoglobins carried out in Costa Rica. This variant is clinically silent, although its substitution is in one of the residues involved in the stabilization of the deoxy form of the haemoglobin molecule.


A phytohemagglutinin from Sunn hemp seeds (Crotalaria juncea). II. Purification by a high capacity biospecific affinity adsorbent and its physicochemical properties. A galactose-specific lectin from seeds of Sunn Hemp (Crotalaria juncea) has been purified by fractional precipitation with ammonium sulfate followed by biospecific affinity chromatography and preparative isoelectric focusing. The adsorbent was prepared by coupling galactose to Sepharose 6B activated with divinyl sulfone. The lectin was homogeneous as judged by ultracentrifugation and by electrophoresis in cellulose acetate strips and in polyacrylamide gradient gel. Its isoelectric point is pH 8.8 and the molecular weight is about 120 000. It is a glycoprotein containing 9.8% also carbohydrate (mannose, N-acetyl-D-glucosamine, fucose, and xylose). The lectin contains 3.2 mol Ca2+, 2.2 mol Mg2+ and 0.2 mol Mn2+ per 120 000 g. No sulphur-containing amino acids were detected.


Isolation and identification of a trypsin-resistant fragment of human serum albumin with bilirubin- and drug-binding properties. Extensive digestion of human serum albumin with trypsin at pH 8.8 yields essentially one main fragment which is resistant to further tryptic degradation. The fragment has been characterized by amino acid analysis, N- and C-terminal analyses, cyanogen bromide digestion, electrophoresis, ultracentrifugation and gelfiltration, and circular dichroism measurements. The results indicate that the main fragment consists of the amino acids 182-585. Repeated digestion did not degrade the isolated fragment further. The fragment mainly retains the secondary and tertiary structure of intact human serum albumin as well as its capacity to bind bilirubin and diazepam. The localization of the binding sites for these substances is discussed.


[Photochemoluminescence of tryptophan-containing peptides and proteins during photooxidation. VI. Kinetic patterns of eosin sensitized photochemoluminescence of solutions of human serum albumin]. Regulariteis of sensitized photochemoluminescence of serum albumine solutions have been studied. The dependence of luminescence intensity on the dye cancentration is of an extremal character; at low concentrations it is determined by the Lambert--Beer law; at high concentrations quenching processes proceed. The shape of kinetic curves of after-luminescence and rise of luminescence is studied. Photochemoluminescence intensity is in a direct proportion to the irradiation intensity. Luminescence is found to be determined by the photodissociation of triptophanile groups of protein. On the basis of experimental and calculated data it is concluded that during sensitization the secondary processes are similar those which proceed under UV-irradiation; the differences lie in the primary processes of the free radical formation.


[Gel-permeating chromatography of interacting multi-component systems. I. Study of protein isomerization by the method of gel-permeation chromatography. II. Method of determining the kinetic constant of isomerization]. A technique is suggested of determining the isomerization constants by means of gel-permeation chromatography. Chromatograms of proteins and expressions for statistical moments of their distribution along the chromatographic column obtained in the authors previous work [1], are used. A necessary condition for optimal setup of the GPCh experiments is found. Fulfilment of this condition provides a maximal accuracy of determination of the isomerization constants.


[Changes in the concentration of free radicals in the organs of rats during experimental chemotherapy of Guérin tumor]. Kinetic description of the changes of free radical concentration in Geren tumour tissue and in the tissues of liver, lungs, brain at chemotherapeutic application of licopine, selenium and cyclophosphane was obtained by ESR method. Licopine application decreases free radical concentration in the tumour (after the stage of a short-time rise). Similar effects during some periods of the action of carotinoids and of a valuable inhibitor of oxidation--selenium, as well as the citostatics of cyclophosphane allow to suggest that the application of the drug of vitamine A group is expedient in tumour therapy.


[Effect of ultrasonic energy on serum thyronine levels]. Changes of total iodine (T.I.) and protein-bound iodine (P.B.I.) content in the blood serum of male rats exposed to ultrasound of different intensity (0.2; 0.6 and 1.0 W/cm2) are described and examined at different time intervals after the exposure. Increased content of T.I. and P.B.I. in the blood serum was observed, chronic group included. The above effect is suggested to result from activated secretion of serotonins to the perypheric blood channel. It is connected with an increased amount of thyreotropic hormone in blood at a decreased content of tyronins in the thyroid gland, and with the effect of discoupling of oxidative phosphorylation induced by ultrasound in the liver mitochondria at a decreased consumption of oxygen.


[Relationship between hydrogen isotope turnover in tropomyosin and the ionization state of side groups]. The influence of side group charge of tropomyosin (TM) on hydrogen-deuterium exchange kinetics in wet TM films is shown to depend solely on the change in alpha-helix conformational motility with the charge. For TM films cast from alkaline solutions the less is the positive charge of basic side groups, the higher is found to be conformational motility of both flexible and rigid alpha-helical regions of TM molecules. For TM films cast from acid solutions the conformational motility of the both types of alpha-helices is shown to be the lowest and independent on the charge.


[Comparison of discrete models of charge transfer in thin membranes. III. Unstable conditions, large polarizing voltages]. The validity of one-jump approach to ion transfer through thin membranes is discussed. The detailed analysis is carried out for the transport scheme with slow conformational processes in the membrane volume. It is shown that there exists some difference between stationary and nonstationary values of the transfer coefficient. The second essential feature of this scheme is the appearance of a slight minimum on the relaxation curve with high voltages in the membrane system.


[Biophysical properties of elastic artery smooth muscle]. The electrical and contractile responses of smooth muscle cells of the rabbit a. pulmonalis and pigeon a. carotis were investigated using extracellular stimulation through sucrose gap and intracellular recording of electrotonic potentials at different distances from the sucrose partitition. Neither spontaneous nor evoked electrical and contractile activities were registered in normal Krebs solution. Current-voltage relations of these muscle cells showed marked rectification. The amplitude of anelectrotonic potentials decreased exponentially with the distance from sucrose partitition, while the time to reach the half-amplitude of the electrotonic potentials was linear function of the distance. These facts indicate that cable equations can be applied to these muscles. The space constant calculated for the rabbit a. pulmonalis was equal to 1.2 mm and that for the pigeon a. carotis--to 1.0 mm. Results of these experiments strongly suggest the existence of low resistance pathways between arterial muscle cells.


[Organization of the background activity of rabbit visual cortex neurons]. Algorithm of calculation of consitent approximation to real entropy and redundancy which helps to understand the connection of two or more related states was realized by means of electronic computer BECM-6. Different laws of changes of information estimates dependeing on the number of approximation and the pitch of temporary quantum of messages corresponded to every type of cell impulsation of rabbits' visual cortex--arrhithimical, grouping and regular. The connections between the laws of impulse succession organization and some of its parameters were revealed. The results of studying the models of casual and deterministic pulse trains were compared.


[Integral characteristics of the eletrical generator of the human heart]. Integral characteristics of the cardiac electrical generator, namely the dipole moment vector, position of the moving electrical center and the complexity parameter (relative quadrupolar potential), are presented for a group of healthy middle-aged men. The characteristics are determined as a result of mathematical processing of electrical potential distribution on the torso surface and geometrical parameters of this surface in a digital computer. The values of the surface potentials and coordinates were obtained by means of experimental measurements using a special automatized measuring system for investigating the cardiac electrical field. Main qualitative properties of the integral characteristics and their possible diagnostic significance are discussed.


[Torsion movements of the human eye. III. Rapid phase of torsion]. Kinematic characteristics of torsional saccades and their peculiarities were investigated. The torsional saccades were found to occur simultaniously in both eyes and had the same size and duration. It was shown that the peak velocity and the duration of the saccades depend on their amplitudes. The number of the saccades decreased and their sizes increased when the head tilts were faster. In the case intervals between the saccades decreased up to 20-30 ms. The likeness of the torsional movements and rotatory nistagmus was discussed.


[Resolving capacity of the human auditory analyzer]. Resolution of human auditory analyser to tine-impulse signals in the noise has been studied AX method was put in the basis of the experiment. Signals were the sinusoid intercepts. Relationships between probability of erroneous distinguishing of the signals and untuning, signal/noise and number of periods in the frequency range from 150 to 4800 Hz. It has been shown that the accepting auditory properties are invariants to the time transformations, the wave number of the sinusoid being the determining characteristics of the stimulus. A concept of frequency--time band of the auditory system is introduced. Its width according to the experimental data depends on the signal/noise ratio.


[Decrease in the electrical stability of lipid membranes following UV-irradiation]. The influence of UV-irradiation on the electrical stability of black lipid membranes (BLM) prepared from mitochondrial lipids has been studied. The breakdown voltage of BLM is shown to decrease as a result of photoperoxidation of unsaturated fatty acid chains of phospholipids. It appears reasonable to assume that the UV-radiation induces the dielectric breakdown and alteration of functions of cell membranes (i.e. UV-uncoupling of oxidative phorylation in mitochondria).


[Potential difference across the membrane of subcellular particles. V. Generation of potential differences by mitochondria and submitochondrial particles under anaerobic conditions]. It is shown by the mehtod of penetrating ions that Site O and I of the respiratory chain of submitochondrial particles are able to generate a membrane potential of the normal value under anaerobic conditons. When succinate is an electron donor and ferricyanide-an acceptor (Site II), the oxygen addition sharply increases the membrane potential at pH above 7.5 and does not change or even decreases it in reaction conditions more acid than pH 6.5. The generation of the membrane potential at low pH and in the absence of oxygen is predicted by the chemielectric hypothesis and cannot be explained by the chemiosmotic one. Mitochondria usually generate the membrane potential without O2 at pH 7.5 in the presence of ferricyanide when the substrate concentration exceeds 5 mM.


[Determination of the quantity of useful information in the spike responses of nerve cells]. The new method for quantitative estimation of useful information in the nerve cells responses is suggested. This modification of Charkevich method is based on the calculation of aposterior probabilities of events. The method is applied for the calculation of amount of useful information in the nerve cells responses. It is shown that the quantity of useful information in the nerve cells responses is increased in the course of adaptation processes.


Biofeedback heart rate training during exercise. Eighteen healthy human subjects participated in weekly sessions of five 10-minute trials of walking on a treadmill at 2.5 mph and 6% grade. Eight experimental subjects received beat-to-beat heart rate biofeedback during the exercise and were instructed to try to lower their heart rates; ten control subjects did not receive feedback. By the end of 5 weeks (25 trials), the experimental group showed a significantly lower mean heart rate (96.8 vs. 108.6 bpm), systolic blood pressure (114.0 vs. 131.3 mmHg), and rate--pressure product (11.0 X 10(3) vs. 14.3 X 10(3) bpm-mmHg) during exercise than the control group. These differences were maintained after crossover of the feedback provision for five more weeks.


[Activity of reticulospinal neurons during the scratch reflex]. The activity of reticulo-spinal (RS) neurons was recorded in thalamic and decerebrate cats during actual and "fictition" scratching. Most RS neurons were silent during scratching, but some exhibitied a distinct modulation of their discharge related to the rhythmical activity of motoneurons. They were excited during the flexor phase and inhibited during the extensor phase of the cycle. The pattern of modulation was identical both in actual and in "fictitions" scratching.


Baroreflex sensitivity during operant blood pressure conditioning. Baroreflex sensitivity was measured in baboons operantly conditioned to increase their diastolic blood pressure in daily, 12-hr sessions, by using the extent of increases in interpulse interval per unit of increase in systolic pressure after intravenous phenylephrine injection as an index of baroreflex sensitivity. Following training, baroreflex sensitivity increases averaging 32% were observed before and after the 12-hr conditioning sessions. During the conditioning sessions, however, consistent diastolic blood pressure elevations averaging 17% (14 mmHg) were accompanied by significant decreases in baroreflex sensitivity averaging 44% relative to the increased "before" and "after" sensitivity levels. The results suggest that changes in baroreflex sensitivity participate in operantly conditioned blood pressure changes.


Effects of cognitively induced anxiety on hand temperature. This study explored the relationship of cognitively induced anxiety and hand temperature reduction in a nonclinical sample. Forty college students were randomly assigned to either the Anxiety Group or the Pleasant Group. Following a baseline period, individuals in the Anxiety Group discussed with the experimenter topics which were anxiety-producing for them. Individuals in the Pleasant Group discussed topics of a pleasant nature. During these discussions, hand temperatures were recorded from the palmar surface of the third finger on the dominant hand. Data analysis indicated that hand temperature decreases were significant only in subjects discussing anxiety-producing topics.


The use of myoelectric feedback in teaching facial expression to the blind. Five blind subjects were provided with an auditory "mirror" of their facial activity by transducing myoelectric signals from facial muscles into sound. Expressions of happiness, surprise, and anger were defined primarily by involvement of the zygomaticus, the frontalis, and the corrugator, respectively. These muscles were connected through separate voltage-controlled oscilllators to separate loudspeakers, such that each muscle activated a different speaker. Motion pictures taken before and after training were assembled in random order and were shown to preselected judges who attempted to identify the expressions. The judges were correct significantly more often on the posttraining expressions. Appropriateness and adequacy of expressions, as rated by the judges, also improved significantly as a result of training.


Biofeedback in the treatment of long-term temporomandibular joint pain: an outcome study. Eleven patients with long-term pain related to the temporomandibular joint (TMJ) were trained in tension awareness and relaxation using feedback of muscle tension level in the masseter through a digital display. At a follow-up examination 4-15 months after the termination of treatment, 8 of the 11 patients were totally symptom-free or significantly better; 1 patient was slightly better, and there was no effect for 2 patients. Possible reasons for failure and sex differences are discussed.


Self-regulation of hemispheric asymmetry. This study was performed to determine if individuals could demonstrate self-regulation of average EEG power of one hemisphere in comparison to the other. Temporal EEG was recorded from 8 males and 6 females. After a practice session, the subjects were instructed to increase or decrease the practice session, the subjects were instructed to increase or decrease the ratio of left to right temporal EEG. The subjects were given on-line feedback in the form of a graph presented via a computer display screen. The study demonstrated a significant ability of the subjects to change differentially the hemispheric power ratios upon instruction.


Biofeedback, self-control, and self-management. A common goal of biofeedback is self-control of physiologic responses. The conceptions and paradigms of the literature on self-control of motoric and cognitive responses were surveyed to provide a basis for the self-control of physiologic responses. An operational definition of self-control was advanced, and self-control was placed in the framework of a general self-management strategy which included discrimination and maintenance components.


Biofeedback-assisted EMG relaxation for urinary retention and incontinence: a case report. A 27-year-old woman with chronic urinary retention and incontinence since infancy was treated for 8 months with frontal electromyographic relaxation training. Urinary control and sensations of bladder fullness were obtained for the first time in the patient's history. Residual urine readings showed marked improvement. Application of EMG biofeedback to other bladder disorders is discussed.


Technique for in situ perfusion of the guinea pig placenta. A simple technique is described for perfusing the guinea pig placenta in situ through the umbilical circulation. Flow rates, perfusion pressures, and transplacental electrical potential gradients were maintained for the duration of the perfusions (1--2h) at values reported for the exteriorised fetus. Terminally, the placentae were shown to be uniformly perfused and of normal weight.


The effect of prenatal fructose infusions upon metabolic condition of the newborn. Changes of total blood sugars, glucose, fructose, lactate, pyruvate, acid-base balance and free fatty acids were followed in 15 healthy newborns whose mothers received fructose infusions during labour. The results were compared with 20 control newborns and with 10 newborns after prenatal glucose infusions. The levels of total blood sugars during 24 h after infusions remained higher after fructose than in 2 other groups. The values of lactate and pyruvate increased, but the lactate/pyruvate ratio remained unchanged. The free fatty acids were lower than in the control group. Results show that fructose is not a suitable source of energy for the human fetus even under normal conditions.


Development of perirenal adipose tissue in the neonatal lamb: effects of dietary safflower oil. Perirenal adipose tissue development was investigated in lambs from birth until 17 days of age. Changes in the morphology, the lipid, protein, RNA and DNA content, the adipocyte number and size, and the rate of protein, RNA and DNA synthesis of the perirenal fat-pads are reported. During the first 9 days after birth the tissue lost the morphological characteristics of brown adipose tissue and acquired those of white adipose tissue. As there was no change in either the number of adipocytes or the DNA content of the fat-pads between birth and 9 days of age it is concluded that there was a differentiation of brown into white adipocytes. Over 80% of the increase in tissue weight between birth and 17 days of age was due to lipid deposition. Administration of safflower oil to the lambs had no apparent effect on the change from brown to white fat, but it markedly reduced the rate of lipid deposition between days 9 and 17 post partum.


Incorporation in vivo of 1-14C-palmitic acid into placental and fetal liver lipids of the rabbit. The incorporation of free fatty acid into the placental and fetal liver lipids of rabbits was studied after fetal injections of albumin-bound 1-14C-palmitic acid. The fetuses were killed either 5--10 or 10--20 min after the injection. The placentas and livers were extracted for lipids and the specific activities of triglycerides (TG), phospholipids (PL), free fatty acids (FFA), monoglycerides (MG) and diglycerides (DG) measured. The lipids of the liver and placenta took up 17.0 and 3.6% of the dose, respectively, and of that liver TG accounted for 74% and the placental TG 34% of the label in each tissue. Most of the remaining counts were in the PL fraction with the rest more or less evely distributed between the FFA, DG and MG fractions. No activity was recorded in the cholesterol esters. The placental TG, PL, DG and MG specific activities reached the same level as that of the placental FFA, while in the liver these esters had higher specific activities (than the liver FFA). The liver TG, DG and PL had higher specific activities when compared with those of the placenta. The specific activity of the placental FFA was lower at 10--20 min than at 5--10 min; the opposite was seen for the placental TG. No time-related changes were seen in the liver lipids. It is concluded that (i) both placenta and fetal liver incorporate FFA into glycerides and PL; (ii) the liver incorporates FFA more rapidly and to a greater extent than the placenta; (iii) most of the FFA is incorporated into TG and to a lesser extent (PL; (iv) in both organs hydrolysis of PL or TG occurs. These results are discussed with reference to placental transport of FFA and fetal fat metabolism.


Factors affecting materno-fetal transfer of iron in the rat. Materno-fetal transfer of iron was studied in the rat under various conditions affecting maternal iron kinetics. A low maternal plasma iron turnover rate (PITR), whether due to a low serum iron concentration or a reduced plasma volume, was associated with a decrease in the amount of iron transported to the fetuses. Conversely, a high PITR due to iron overload and a high serum iron concentration, produced a marked increase in the amount of iron received by the fetuses. No such increase was observed in fetuses of mothers made anemic by bleeding or phenylhydrazine, in whom both the PITR and erythropoietic activity were increased, persumably due to maternal competition for iron. The amount of iron crossing to the fetuses, therefore, is affected by the interplay of two conflicting factors: the PITR and maternal needs for iron, as dictated by the level of erythropoietic activity.


Symptomatic neonatal plethora.

Symptomatic neonatal plethora. Blood volume and clinical data are reported on 8 premature and 3 full-term infants who presented with symptoms apparently due to polycythemia or hypervolemia. These cases termed 'symptomatic neonatal plethora' were caused by large placental transfusions associated with delayed clamping of the umbilical cord. Tachypnea, mild cyanosis, plethoric skin color, and neurological depression persisted on average for 30 h after birth. Chest X-rays showed mild cardiomegaly, pulmonary congestion and edema, and pleural effusions. Although the course was in general benign, phlebotomy was considered to be indicated in three infants to treat progressive clinical deterioration. The symptomatology and blood volume on these infants were compared with infants in an ongoing study with controlled time of cord clamping. Neonatal plethora must be considered as one cause of 'transient tachypnea of the newborn'.


Phenobarbital-induced brain growth retardation in artificially reared rat pups. Phenobarbital was administered daily to rat pups during the period of their brain growth spurt. A new technique for artificially rearing rat pups, using chronic intragastric feeding cannulas, was used to prevent drug-induced undernutrition. Rats, receiving neonatal phenobarbital, experienced a marked interruption of early brain growth, although their body weights reflected normal growth.


The fibrinolytic pathway in the human fetus. The fibrinolytic pathway and the protease inhibitors in 12 to 24-and 24-to 36-week-old fetuses were examined and compared with full-term newborns. The activity of the fibrinolytic system, as well as the values of the protease inhibitors approach the normal values progressively with the embryonic maturation.


A study of conduction velocity in nonmyelinated nerve fibers. By treating a nonmyelinated nerve fiber as a continuous cable consisting of three distinct zones (Resting, transitional, and excited), the following mathematical expression was derived: (formula: see text) where v is the conduction velocity, d the diameter of the fiber, R the resistance of the membrane of unit area at the peak of excitation, rho the resistivity of the  medium inside the fiber, and C the capacity of membrane per unit area. The validity of this expression was demonstrated by using squid giant nerve fibers intracellularly perfused with dilute salt solutions. The relationship between these results and previous theories and experiments on conduction velocity is discussed.


Calcium diffusion in transient and steady states in muscle. Rates of diffusion through the extracellular space of thin sheets of myocardium from the right ventricular outflow tract of kittens were estimated at 23 degrees C for 45Ca2+ and an inert reference tracer, [14C]sucrose. The myocardial sheets were mounted in an Ussing chamber and equilibrated with Tyrode solution with varied calcium concentrations, Cao. The tracers were added to one side and their concentrations on the other side measured at 5-15-min intervals for 6 h. The apparent tracer diffusion coefficient for sucrose was 1.11 +/- 0.06 X 10(-6) cm2s-1 (mean +/- SEM, n = 74), 22% of the free diffusion coefficient; the lag time before reaching a steady state provided estimates of the intratissue volume of distribution or diffusion space of 0.41 +/- 0.15 ml/ml tissue (n = 74), a value compatible with expectations for extracellular fluid space. Over the range of Cao from 0.02 to 9.0 mM, the intratissue apparent diffusion coefficient for Ca, DCa, averaged 1.65 +/- 0.10 X 10(-6) cm2s-1, n = 74, which is 21% of the free DoCa, and was not influenced by Cao. Because transsarcolemmal Ca permeation is slow, DCa is the diffusion coefficient in the extracellular region. The paired ratios DCa/Ds averaged 1.32 +/- 0.05 (n = 67) for all levels of Cao but at physiologic or higher Cao averaged 1.45 +/- 0.07 (n = 39), close to the ratio of free diffusion coefficients, 1.53. Equations distinguishing transient from steady state diffusion were fitted to the data, showing that the apparent distribution volume of "binding sites" external to the diffusion pathway diminished at higher Cao in a fashion suggesting that a least two different Ca2+ binding sites were present.


An osmometer model for changes in the buoyant density of chromaffin granules. We present a model for the structure of isolated bovine adrenal medulla chromaffin granules derived from the dependence of granule density on the osmotic pressure of the suspension medium at 2 degrees C. The granule consists of a flexible, inelastic membrane bounding an osmotically active core. The core consists of a solution space and a separate, nonosmotic phase. Since the granule behaves like a "perfect" osmometer over a wide range of osmolarities, we conclude that (a) within these limits, the core consists of a constant amount of condensed material and a constant number of particles in solution, (b) from the constraints of the osmometer model, the osmolality inside the granule must equal the osmolality outside. Therefore the high concentrations of catecholamines (greater than 0.7 M) and ATP (greater than 0.18 M) measured biochemically cannot be dissolved in the core solution as separate molecules, but must be condensed into larger aggregates. These results are supported by electron micrographic examination of the effect of osmotic pressure changes on granule morphology.


Hydrodynamic theory of swimming of flagellated microorganisms. A theory of the type commonly used in polymer hydrodynamics is developed to calculate swimming properties of flagellated microorganisms. The overall shape of the particle is modeled as an array of spherical beads which act, at the same time, as frictional elements. The fluid velocity field is obtained as a function of the forces acting at each bead through Oseen-type, hydrodynamic interaction tensors. From the force and torque equilibrium conditions, such quantities as swimming velocity, angular velocity, and efficiency can be calculated. Application is made to a spherical body propelled by a helical flagellum. A recent theory by Lighthill, and earlier formulations based on tangential and normal frictional coefficients of a curved cylinder, CT and CN, are analyzed along with our theory. Although all the theories predict similar qualitative characteristics, such as optimal efficiency and the effect of fluid viscosity, they lead to rather different numerical values. In agreement with Lighthill, we found the formalisms based on CN and CT coefficients to be somewhat inaccurate, and head-flagellum interactions are shown to play an important role.


Effects of internal and external cations and of ATP on sodium-calcium and calcium-calcium exchange in squid axons. Calcium-45 efflux was measured in squid axons whose internal solute concentration was controlled by internal dialysis. Most of the Ca efflux requires either external Na (Na-Ca exchange) or external Ca plus in alkali metal ion (Ca-Ca exchange; cf. Blaustein & Russell, 1975). Both Na-Ca and Ca-Ca exchange are apparently mediated by a single mechanism because both are inhibited by Sr and Mn, and because addition of Na to an external medium optimal for Ca-Ca exchange inhibits Ca efflux. The transport involves simultaneous (as opposed to sequential) ion counterflow because the fractional saturation by internal Ca (Cai) does not affect the external Na (Nao) activation kinetics; also, Nao promotes Ca efflux whether or not an alkali metal ion is present inside, whereas Ca-Ca exchange requires alkali metal ions both internally and externally (i.e., internal and external sites must be appropriately loaded simultaneously). ATP increases the affinity of the transport mechanism for both Cai and Nao, but it does not affect the maximal transport rate at saturating [Ca2+]i and [Na+]o; this suggest that ATP may be acting as a catalyst of modulator, and not as an energy source. Hill plots of the Nao activation data yield slopes congruent to 3 for both ATP-depleted and ATP-fueled axons, compatible with a 3 Na+-for-1 Ca2+ exchange. With this stoichiometry, the Na electrochemical gradient alone could provide sufficient energy to maintain ionized [Ca2+]i in the physiological range (about 10(-7) M).


Analysis of various indefinite self-associations. Two methods have been developed for the analysis of four types of indefinite self-associations. Unlike previous treatments by others, the procedures can be applied to nonideal cases. The two methods were first tested with simulated data, and it was found that one could indeed distinguish between the four types of indefinite self-associations. For a more realistic test, sedimentation equilibrium experiments were performed on solutions of beta-lactoglobulin A at 16 degrees C in 0.15 ionic strength acetate buffer, pH 4.65. The self-association of the beta-lactoglobulin A was best described by either method as a sequential indefinite self-association having two equilibrium constants and one second virial coefficient.


Theory of the delocalized binding of Mg(II) to DNA: preliminary analysis for low binding levels. A simple theoretical equation for the binding of Mg2+ to DNA in the presence of excess 1:1 salt is derived from a model that does not specify discrete binding sites but rather allows the associated metal ions to move freely near the surface of the DNA polyion. Use of a numerical value for the free volume, determined uniquely, in a separate communication, by a free energy minimization, leads to predicted values for the Mg2+ binding constant that are in essential agreement with measured values taken from the literature.


On the binding of Mg2+ and Mn2+ to tRNA. In this paper previous binding studies of Mg2+ and Mn2+ ions to tRNA's are reconsidered. Binding data of some representative examples are interpreted including interactions between charges located on the macroion. Both curved and bell-shaped Scatchard plots can be accounted for quantitatively if corrections are made for electrostatic interactions and, if necessary, for the effect of conformational changes on these interactions. It appears that there is no need to invoke more than one class of binding sites on tRNA's, meaning that the experimental binding data can be described using the same intrinsic pK value for all phosphate groups.


The effect of carbon monoxide on haem-facilitated oxygen diffusion. The aim of this paper is to quantify the effect of small quantities of carbon monoxide on the facilitated diffusion of oxygen by haemoglobin in the steady state. It is the first phase in the study of a mathematical model for carbon monoxide poisoning. Here we extend the Wyman model for facilitated diffusion to the case in which there are two ligands. The equations are solved using an asymptotic technique developed by Murray. We obtain accurate analytic approximations for the biologically important quantities of the problem for various percentages of carbon monoxide. These are the concentrations of free oxygen, haemoglobin, oxyhaemoglobin and carboxyhaemoglobin, and hence the saturation of the protein and the facilitated oxygen flux. The major effect of very small quantities of carbon monoxide on the oxygen flux is shown.


On the analysis of chemical relaxation amplitudes. The calculation of chemical relaxation amplitudes for a reaction mechanism involving an arbitrary number of thermodynamically independent steps is discussed. A formalism is developed which considerably reduces the computational labor required to derive expressions relating experimental amplitudes to thermodynamic functions and specific signal changes of elementary steps. The treatment includes the case of strong kinetic coupling between all reactions. The results are of particular interest in regard to the numerical fitting of amplitude data to an assumed mechanism.


Limiting laws and counterion condensation in polyelectrolyte solutions. IV. The approach to the limit and the extraordinary stability of the charge fraction. The limiting laws for polyelectrolyte solutions developed in previous papers of this series have been amply confirmed by measurement. A surprising result of the accumulated data is that the limiting polyelectrolyte charge fraction (fraction of fixed charges uncompensated by condensed counterions in the limit of zero concentration), persists up to concentrations of 0.1 M or even higher. Here the theory is extended in a simple manner to finite concentrations, and the stability of the charge fraction is found to be firmly based on consequences of the long-range polyelectrolyte field. The associated counterions are assumed to translate freely in a region centered on the contour axis of the polyion. The numerical value of the free volume is determined self-consistently from the axial charge density of the polyelectrolyte and is used as the general framework within which specific binding effects are treated.


Mössbauer and susceptibility experiments on different compounds of Fe3+-myoglobin. The static magnetic susceptibilities of different ferric high spin and low spin compounds of myoglobin (Mb(H2O), Mb(H2O) frozen under high pressure, MbF, MbCN) were measured in the temperature region between 4.2 K and 130 K. Mössbauer absorption experiments on Mb(H2O) and MbF were performed at different temperatures between 4.2 K and 180 K and in small magnetizing fields H less than or equal to 1 kOe. The evaluation of our experimental data was performed with a Hamiltonian describing of 3d-configuration of the ferric iron by taking into account the Coulomb repulsion of the five electrons within the 3d-shell, the crystal electric field of C2v-symmetry, and the spine-orbit coupling. The Hamiltonian contains the splitting energies of the five antibonding d-orbitals (dxy, dx2, dy2, dx2-y2, dz2) as parameters. The values of these energies were obtained by a least squares fitting procedure using our magnetic susceptibility data together with the g-factors taken from the literature. In the case of MbF the energy difference between the two lowest Kramers doublets was also determined from present Mössbauer data. The results of the susceptibility and the Mössbauer data are in good agreement. The splitting energies of the 3d-orbitals can be correlated to the distances between the iron and its nearest neighbours. The different positions of the iron in the compounds investigated are discussed.


Investigation of two deoxygenated haemoglobin-haptoglobin complexes by Mössbauer spectroscopy. Haemoglobin Haptoglobin complexes formed when [Hp+]/[Hb]= 1/1 and [Hp]/[Hb] =2/1 were investigated by 57Fe Mössbauer spectroscopy. Both samples gave a spectrum consisting of a single quadrupole doublet. The temperature dependence of the quadrupole splitting was also identical for both samples. This proves that in both samples the nearest neighbour environment of the iron atom must be the same. A comparison with earlier investigations on myoglobin and haemoglobin indicates that the electronic structure of iron in the HbHp-complexes is similar to that in myoglobin.


Application of radiationless energy transfer for distance measurements across membranes. Transition Probabilities for radiationless energy transfer within several assemblies of donors and acceptors at stationary distances. For several 2-or 3-dimensional configurations of stationary donors and acceptors on or near a spherical membrane shell the transition probabilities for radiationless energy transfer are calculated, using Förster's approximation obtained for Coulombic dipole-dipole interaction of the transition moments. It turns out that the difference in the refractive indices for membrane (lipid) and bulk phase (water) has only a small influence on the transition probabilities. Furthermore, the curvature of biological cell surfaces can be neglected, but affects the energy transfer across small vesicles. The ratio thickness/radius of small vesicles can be determined by measuring fluorescence quenching of excited donors by acceptors on the other side of the membrane.


An error in interpretation of double-reciprocal plots and Scatchard plots in the studies of binding of fluorescent probes to proteins, and alternative proposals for determining binding parameters. One of the objects of experiments in which fluorochrome is added to suspensions of cell membranes is to determine the parameters n and Kp, the capacity of unit mass of protein to bind fluorochrome and the dissociation constant, respectively. Currently, these are estimated from Scatchard plots, construction of which first requires that observed fluorescence intensity be converted to moles of bound fluorochrome. This in turn is said to be possible by analysis of the intercept of a plot of reciprocal fluorescence intensity against reciprocal protein concentration. However, analysis of the classical mass action equilibrium equation, upon which the foregoing procedures are said to be based, reveals that the intercept of the double-reciprocal plot always underestimates the desired value. The error is formalized and shown to increase without bound with fluorochrome concentration. The error in turn leads to erroneous assessment of n and Kp. Alternative methods for calculating the desired parameters are proposed, based on direct plots of fluorescence intensity.


Kinetics of reactivation of rabbit muscle aldolase after denaturation and dissociation in various solvent media. The denaturation of aldolase from rabbit muscle in various solvents leads to significant qualitative and quantitative differences with respect to the structural disintegration of the enzyme. The differences refer to the quaternary structure and to the conformation which is changed only slightly in MgCl2 while in guanidine-HCl or urea at pH approximately 2 the molecule is close to the state of the random coil. Using the enzymic activity as a quantitative measure for the refolding process, the reaction order and the rate constants of the processes of structure formation (vi leads to N*) are found to be identical. This observation suggests a common intermediate D in the process of renaturation after denaturation and dissociation in the different solvent media. D may be considered an intermediate state with a defined number of nucleation centers whose rapid formation is predetermined by the aminoacid sequence. As taken from the first order kinetics in the given range of enzyme concentration, transconformation reactions are rate limiting in the obligatory pathway of refolding. At low enzyme concentrations second order steps gain importance which indicates that the enzymic activity is significantly modified by the formation of the native quaternary structure.


Studies of nonlinear electrical effects of model membranes. The structure of lipid bilayer membranes in investigated by analysis of the harmonics generated by electrical ac excitation with dc bias. For the detection of nonlinear effects this method is complementary to current relaxation experiments and may be suitable for gating current measurements at nerve membranes.


Symbiosis and the evolution of prokaryotes. It is postulated, with support from kinetic modelling, that a succession of symbioses was the major process of evolution during the early stages of life. The process became less effective with the passage of time, while evolution by the natural selection of variants became more effective. The postulate may contribute usefully to discussions on the evolution of biochemical complexity and the structure of cells.


The functional approach in biology.

The functional approach in biology. The concepts of structural and functional approaches are analysed. The existence of a logical limit to the domain of applicability of the structural approach is indicated. Some sources of possible failure of the structural method in biology are pointed out. Two fundamental characteristics of biological systems, inductive development and inductive functioning, necessitating the functional approach are discussed.


Electric field control of lipase membrane activity. Lipase (EC 3.1.1.3., from Pseudomonas sp.) was entrapped in collagen membrane containing liquid crystal (4-methoxybenzilidene-4'-n-butylaniline). The activity of the lipase-liquid crystal membrane at an applied voltage of 4 V was 3.4 compared to a membrane tested without imposition of an external electric field. A linear relationship was observed between the activity of the lipase-liquid crystal membrane and the current. The apparent Michaelis constant (K'm) of the lipase-liquid crystal membrane under electric field was identical to that of the membrane under ordinary condition. Activation of the lipase-liquid crystal membrane was observed repeatedly, i.e., activation in the presence of an electric field and reversion to a basal level upon removal of the field occurred cyclically. Activity control of immobilized enzymes is desirable for switching devices of a bioreactor. Possible mechanisms of the lipase activation by electric field are discussed.


Source of hydrogen peroxide and of chemiluminescence observed in activated human platelet preparations. Human platelet suspensions can be observed to produce small amounts of H2O2 (0.04 nmoles H2O2/min/2.5 X 10(5) cells/cu mm) and measurable chemiluminescence when exposed to target particles for phagocytosis, such as latex spherules. Both H2O2 production and chemiluminescence are characteristic of phagocytosing polymorphonuclear leukocytes (PMN) and analysis of the purified platelets indicates contamination by PMN at the level of 0.2%. The amount of H2O2 produced and the chemiluminescence observed can be duplicated by adding latex spheres to a preparation of PMN at a concentration equivalent to the contaminant in the platelet preparations. We conclude that the H2O2 produced and chemiluminescence observed from activated platelets is due to the presence of small amounts of contaminating PMN. These studies emphasize the importance of controlling for PMN contamination in studies of platelet biochemistry and physiology.