Patterns of response to bronchodilators in asthma. Twenty-eight asthmatics inhaled an isoprenaline aerosol on several occasions. The dose administered was constant for each subject on each occasion, yet the responses of each subject measured spirometrically differed on most of the separate occasions. The individual response patterns showed the following features. Spontaneous worsening of the asthma was associated with spirometric responses which in some cases increased directly and predictably with the corresponding fall in pre-treatment FEV1. In other cases the increase in response was less regular and in a few it bore no relation whatsoever to the pre-treatment FEV1. In three subjects a response occurred when the pre-treatment FEV1 was greater than the predicted value. There were variations between subjects in the magnitude of increase in spirometric response to inhaled bronchodilator occurring when there was a fall in pre-treatment FEV1. The great majority of subjects never achieved their predicted FEV1 after maximal doses of isoprenaline. The study of individual response patterns is of value in the selection of subjects for comparative trials of different bronchodilator drugs. The present study draws attention to an irreversible component in the reduction of airways calibre that occurs in asthma.


Cytochemical, ultrastructural and immunological studies of circulating Reed-Sternberg cells. Reed-Sternberg (R-S) cells in the circulating blood of a patient with Hodgkin's disease were cytochemically peroxidase and Sudan black negative, devoid of alkaline phosphatase and non-specific esterase, mostly PAS negative but occasionally showing positivity, and nearly always showing moderately strong granular positivity for acid phosphatase. Electron microscopy showed irregular nuclear profiles, conspicuous nucleoli, a moderate development of cytoplasmic organelles but absence of structures resembling monocytic granules. The R-S cells frequently possessed receptors for the Fc region of IgG and were mostly positive for SmIg, but did not form rosettes with sheep or mouse erythrocytes nor have receptors for the Fc region of IgM or the C3 component of complement. The combined results suggest that R-S cells are of B-cell lineage.


Chronic lymphatic leukaemia: cellular effects of glucocorticoids in vitro. Glucocorticoid receptor levels and steroid induced inhibition of nucleic acid precursors have been examined in lymphocytes from 27 patients at different stages of chronic lymphatic leukaemia. No correlation can be found between the level of glucocorticoid receptors and the stage of the disease. On the other hand, a significant difference (P less than 0.02) was found between stage O and stage III/IV patients, in terms of the in vitro effect of dexamethasone on [3H] uridine incorporation.


The effects of glucocorticoids on cultured human endothelial cells. The effects of hydrocortisone, dexamethasone and prednisone on the morphology, replication, DNA synthesis, cell protein content and protein synthesis of cultured, human endothelial cells were evaluated. After culturing the cells with these glucocorticoids for 24-48 h, the cells covered a greater portion of the culture surface area. The mean surface area of the individual endothelial cell treated with glucocorticoids was 1.53 times greater than that of the untreated control endothelial cell. When compared with controls, the endothelial cover provided by the cells treated with glucocorticoids was more extensive and in many instances covered the entire culture surface. The change in morphology was associated with an increase in protein synthesis and protein content of the cells without an increase in DNA synthesis or cellular replication. Dexamethasone was approximately 10-fold more effective than hydrocortisone, while prednisone was the least effective. Aldosterone, DOCA, testosterone, progesterone, oestradiol and oestriol were ineffective. These studies indicate that glucocorticoids can alter the morphology and biochemistry of cultured endothelial cells and may have implications for the effects of steroids in the treatment of thrombocytopenic states and vascular disorders in man.


Myelomatosis and the hyperviscosity syndrome. Eleven patients are described in whom myelomatosis was complicated by the laboratory and clinical features of the hyperviscosity syndrome (HVS). The myeloma type was IgA in nine and IgG3 in two. In those patients with IgA myeloma the HVS was related to the presence of high molecular weight complexes in the serum. Remission of clinical features was obtained in all patients by plasma exchange. Clinical improvement coincided with reduction of whole blood viscosity and in those patients with IgA myeloma, with a parallel reduction of the high molecular weight complexes. The relationship between the IgA complexes and blood viscosity has been examined by physicochemical analysis of purified IgA monomer and polymers and evidence is presented to show that the IgA polymer has a higher intrinsic viscosity and axial ratio than the larger IgM molecule. The significance of these observations is discussed.


The role of adherence to human mononuclear phagocytes in the destruction of red cells sensitized with non-complement binding IgG antibodies. In patients with IgG incomplete non-complement binding warm autoantibodies, the subclass composition of the antibodies was studied in relation to the occurrence of increased haemolysis in vivo and the adherence of the patients red cells to peripheral blood monocytes (PBM) in vitro. The presence of IgG3 autoantibodies was almost always accompanied by haemolytic anaemia, but the presence of IgG1 autoantibodies only in some patients but not in others. IgG2 and IgG4 autoantibodies were not associated with increased red cell destruction. A relation identical to that between subclass composition and increased haemolysis was found between subclass composition and adherence of the patients erythrocytes to PBM and thus a strong correlation between positive adherence in vitro and increased red cell destruction in vivo. These results support an important role of adherence to mononuclear phagocytic cells in the destruction of red cells sensitized with non-complement binding IgG antibodies. Strong indications were found that IgG1 autoantibodies are of two kinds, only one of which causes adherence to phagocytes and thus increased red cell destruction.


An inhibitor of fibrin formation in thromboplastins prepared by saline extraction of human brain. Human brain is a common source of thromboplastin for the prothrombin time, where the end point is the conversion of fibrinogen to fibrin. Experiments showed that human brain also contains a proteolipid which inhibits the conversion of fibrinogen to fibrin. The proteolipid is removed when brain tissue is washed with acetone, but remains as a contaminant when brain is extracted with saline. For this reason prothrombin times on the same plasma are longer when saline extracts, rather than acetone dried preparations, are the source of thromboplastin. The proteolipid explains why the prothrombin time becomes shorter when saline extracts are diluted to standardize their activity against the British comparative thromboplastin.


A case of brief psychotherapy.

A case of brief psychotherapy. The six-month psychotherapeutic treatment of a 29-year-old woman with a hysterical personality is described. Criteria for selection of patients suitable for brief psychotherapy and the various applications of this technique are discussed. Aspects of the psychopathology of the hysterical personality are described and related to that of the patient presented, whose sexualized transference exemplifies the important task of 'focusing' this form of treatment.


Movement therapy with psychotic adolescents. Non-verbal behaviour has long been a focus of attention in the psychiatric literature. Furthermore, most psychiatric hospitals make use of non-verbal therapies in their milieu programmes. These include art, music and dance therapy, and related to the latter, movement therapy. This paper represents the beginning of an attempt to assess more scientifically the effectiveness of movement therapy. It describes the movement therapy programme on our adolescent unit, the evolution of a measuring scale and compares three groups of patients: (A) a group which had received individual therapy; (B) A group which had received group therapy; and (C) a control group. The results tend to confirm the effectiveness of movement therapy for psychotic adolescents.


Ego defences and affects in women with breast symptoms: a preliminary measurement paradigm. Terms derived from psychoanalytic theory such as the concepts of ego defences and affects, have often been regarded as inaccessible to operational analysis and measurement and therefore devoid of empirical meaning. However, these explanatory terms are frequently employed in hypotheses concerning the determinants of behaviour and outcome in naturally occurring illnesses, such as breast cancer as well as other life crises and stress situations. The results of the present study demonstrate not only that it is possible to operationally define and measure the ego defences and affects associated with the crisis induced by finding a breast symptom suggesting cancer and awaiting biopsy, but also that on the basis of such measurement, behaviour related to breast cancer can be predicted and hypotheses concerning the relevance of these variables to aetiology put to the test. The operational definitions and measurement methods described could also be modified for application in other crisis situations to which ego defences and affects are thought to be pertinent and the results of these operations compared empirically to determine the extent of their convergence. the methods can be used by independent observers with different theoretical and professional backgrounds. The delay by women in reporting breast symptoms to their doctors was strongly related to a combination of non-rational, unconscious psychological factors. Those who delayed used the ego defences of denial and suppression, not intellectualization-isolation, and verbally expressed depression but not anxiety while showing behavioural manifestations of anxiety. Conscious factors such as fear and education were unrelated to the length of delay. These findings have important implications for educators and doctors concerned with the early detection of breast cancer.


The reactions to child-bearing and early maternal experience of women with differing marital relationships. The type of marital relationships of 200 women was significantly associated with the attitudes toward child-bearing and their affective reactions, as measured by the application of twelve content analysis scales to their retrospective accounts of a child-bearing year. Positive marital relationships were linked with calmer reactions. Self-oriented relationships were characterized by mutilation anxiety. In ambivalent and negative relationships early separation anxiety and hostility occurred but only the negative group continued in crisis after birth. Women with positive and ambivalent relationships expressed greater maternal warmth.


Self-mutilation.

Self-mutilation. A controlled study of self-mutilators found them to be significantly more introverted, neurotic and hostile. They report excessive physical punishment in childhood, sado-masochistic fantasies and more suicidal attempts.


A six-point scoring system for antenatal cardiotocographs. A six-point scoring system for antenatal cardiotocography based upon baseline fetal heart rate (FHR) ad FHR response to fetal movements and Braxton-Hicks contractions has been described and tested in 89 patients. The 'six-point score' obtained within 24 hours of delivery or death of the fetus (D--1) was shown to be strongly associated both with the one-minute Apgar score and the nutritional status of the infant as reflected by centile birth weight. In 21 patients whose D--1 'six-point score' was 4 or less, analysis ofthe cardiologicographs performed on the previous day (D--2) showed that 14 infants already showed evidence of hypoxia. In the remaining seven patients, however, the previous day's six-point score had been normal (5 or 6); in six of these patients a persistently low daily fetal movement count or placental abruption led to repeat cardiotocography. Hypoxia affected the three components of the score in a progressive manner. Firstly there were alterations in the response of the FHR to Braxton-Hicks contractions followed by changes in the FHR response to fetal movements. Finally, fetal movements ceased and baseline FHR changes occurred mainly in the form of a trachycardia. The method of scoring was simple to use and could standardize reporting.


The site of umbilical cord insertion and birth weight. The site of the insertion of the umbilical cord into the placenta was determined in 940 consecutive term infants. No relation was found between birth weight and whether the cord was inserted centrally, eccentrically or marginally.


Observations on maternal and fetal ferritin concentrations at term. The concentration of serum ferritin was measured in 115 paired maternal and cord specimens obtained from patients delivering at term. There was a significantly lower concentration of ferritin in cord serum when the maternal value was 10 microgram/l or less. The maternal ferritin appeared to be lower in mothers who had three or more previous pregnancies or were socially deprived.


The comparative effects of a synthetic and a 'natural' oestrogen on the haemostatic mechanism in patients with primary amenorrhoea. A synthetic and a natural oestrogen were administered alternately for three months to nine women with primary amenorrhoea using a randomized cross-over schedule. Measurements of haemostatic function were performed before and at the end of each treatment period. No significant change in haemostatic function was observed after treatment with the 'natural' oestrogen, oestriol succinate. In contrast, treatment with a synthetic oestrogen, ethinyloestradiol, caused shortening of the prothrombin time and an increase in plasma concentration of factor VII and plasminogen. These data support other observations in suggesting that natural oestrogens may have fewer potentially adverse effects on haemostatic function than synthetic oestrogen.


The effects of long-term therapy with oestriol succinate on the haemostatic mechanism in postmenopausal women. In a previous report we showed that oestriol succinate administered to postmenopausal women caused fewer changes in haemostatic function over a four-month period than ethinyloestradiol. Potential longer-term effects were studied in postmenopausal women treated for osteoporosis with oestriol succinate for up to 12 months. Over this period there was no significant change in concentration of plasma coagulation factors, an increase in plasminogen concentration and euglobulin lysis activity, and an inconsistent increase in platelet sensitivity to aggregation induced by ADP and collagen. The relative lack of effect of oestriol succinate on coagulation function is encouraging with regard to the future incidence of thromboembolic complications of therapy.


Serum FSH, LH and oestrone levels in postmenopausal patients on oestrogen therapy. Serum FSH, LH and oestrone levels were determined in postmenopausal women before and at 1, 3 and 6 months after the onset of cyclical treatment with 0.05 mg of ethinyl oestradiol (n = 19) or 2 mg of oestradiol valerianate (n = 20). Most samples from the women taking oestradiol valerianate were also analyzed for oestradiol. Vaginal smears were obtained from all patients. Although all women were relieved of vasomotor symptoms, the FSH levels usually exceeded the fertile range. Ethinyl oestradiol reduced FSH by 70 to 75 per cent and LH by 30 to 40 per cent while oestrone was increased by 20 to 40 per cent. In the women taking oestradiol valerianate, FSH and LH levels were both reduced by 20 to 25 per cent wheras serum oestradiol increased by 200 to 300 per cent and oestrone increased by 600 to 700 per cent. The mean vaginal maturation value rose equally in the two groups but 40 per cent of the patients in both groups showed no change in the value. There was no correlation between the appearance of vaginal endocrine smears and hormone levels.


The percentage of granulocyte-like cells in human oviduct epithelium. The ratio of granulocyte-like cells to epithelial cells was examined in the fimbriae, ampullary, and isthmic regions of oviducts obtained from normal women of reproductive age, from postpartum women, from menopausal women, from women on combined oral contraceptives and from women with adnexal pathology. Only patients on oral contraceptives had a significantly high percentage of granulocyte-like cells as did one patient with acute salpingitis.


Successful pregnancy in a 44-year-old haemodialysis patient. A 44-year-old patient had been treated by intermittent haemodialysis for almost three years when she presented with a 28-week pregnancy. Successful delivery of a healthy but small infant was achieved by Caesarean section at 36 weeks. The successful outcome of this pregnancy was attributed to close control of the haemoglobin concentration and blood chemistry, and to increased frequency of dialysis. The relative value of various chemical tests of fetal maturity in the presence of maternal renal failure are discussed.


Uterine cirsoid aneurysm.

Uterine cirsoid aneurysm. Cirsoid (or varicose) aneurysm of the uterine vessels causes severe, life-threatening uterine bleeding. Previous trophoblastic neoplasia is a common predisposing factor. Diagnosis is by arteriography and may be missed without it.


The Arden grating test of visual function: a preliminary study of its practicability and application in a rural community in north-west Iran. In a preliminary study involving 75 normal and 10 abnormal eyes the Arden grating acuity test was assessed under difficult conditions of rural field work. It was found to be adequately sensitive and specific for screening out individuals with visual impairment due to refractive errors or ocular disease, and to distinguish between these. Inexperienced paramedical personnel could carry out the test satisfactorily with no difficulty and with a minimum of training. In spite of the unusually distracting conditions under which the test was carried out 95% of the subjects tested performed satisfactorily. The results of this preliminary study suggest that the Arden grating test is a valuable tool for screening large populations in rural areas of developing countries.


An investigation into the variation of human contrast sensitivity with age and ocular pathology. The contrast sensitivities to sine-wave grating stimuli were measured for 36 observers with ages in the range 8 to 67 years. Thirty-one observers had normal, healthy vision while 4 were suffering from diabetic retinopathy and 1 had a early lens opacities and macular degeneration. The younger members of the group with normal vision were less sensitive to low spatial frequencies and the older members were less sensitive to high spatial frequencies than the middle-aged observers. The 5 observers with pathological conditions were less sensitive to most grating stimuli than those with normal vision.


Cortical potentials evoked by a TV pattern reversal stimulus with varying check sizes and stimulus field. With the use of television equipment the influence of check size and stimulus field on the pattern evoked cortical potentials was investigated. Maximum responses were found with 20' and 40' checks. The major part of the responses was initiated from a ring between 1.25 degrees and 2.5 degrees eccentricity, though relatively per stimulus area the response became progressively larger the smaller the stimulus field.


Variation in latency times of visually evoked cortical potentials. Latency times of visually evoked cortical potentials stimulated by reversal of a slow checkerboard pattern are highly dependent on the time needed to accomplish the reversal movement. If, owing to the method, the pattern reversal time is not kept stable, variability of the latency times is unnecessarily high for clinical purposes. This may be the case when television equipment is used.


The electro-oculogram in human retinal detachment. Thirteen patients with retinal detachment and surgical reattachment were studied with preoperative and multiple postoperative electro-oculograms. The light rise was reduced to a mean of 1.17 with detachment but recovered in 77% to a 1.87 ratio, the equivalent of the control eye. This recovery was complete within 66 days in 80% of patients who eventually did return to normal. This rapid recovery parallels the rapid return of anatomical, biochemical, and electrophysiological function demonstrated in experimental detachment.


Long-term follow-up of photocoagulated retinal breaks. 126 cases of retinal breaks treated in our department by photocoagulation during a period of 10 years (1963-1973) were selected for a close annual follow-up. Eliminated from the study were cases of macular holes, breaks which developed in an eye after retinal detachment surgery, and cases in which a close follow-up was impossible for various reasons. The study includes symptomatic retinal breaks in myopic, non-myopic, and aphakic eyes as well as asymptomatic holes which were found in the undetached fellow eye. Four eyes treated by photocoagulation (3.2%) developed a retinal detachment. The causes of failures and complications, such as retinal detachment, vitreous haemorrhage, and macular pucker, are discussed.


Diversion of retinal blood flow by photocoagulation. The normal distribution of the retinal blood flow in the cat eye was modified by photocoagulation to part of the territory supplied by a major arteriole, or by occluding a branch. Volume inflow to the treated territory was reduced, and there was also a reduction of linear flow and, to a less extent, calibre in the parent vessel. Branches of the parent vessel supplying untreated territory showed marginal increases of volume inflow. An  autoregulatory effect appeared to be operative. The relevance of these findings to clinical panretinal photocoagulation is discussed.


Fluorescence in Best's vitelliform dystrophy, lipofuscin, and fundus flavimaculatus. Control photographs, with the Baird Atomic B4 and B5 filters in place prior to fluorescein injection, show exposure of the film corresponding to (1) the small yellow vitelliform lesions at the edge of a disrupted disc, (2) the pseudohpopyon in a vitelliform cyst, (3) orange lipofuscin overlying a malignant melanoma, and (4) some of the flecks in a case of funds flavimaculatus. Because of transmission overlap between the filters, the relative contribtution reflected light and true autofluorescence is difficult to quantitate. Reflectile structures such as the optic nerve or a white scar were essentially unexposed, but minimal fundus detail was seen. Some parallels exist between lipofuscin and the content of a disrupted vitelliform lesion.


Familial bilateral macular colobomata.

Familial bilateral macular colobomata. A mother and daugher had a life-long history of poor vision and photophobia, bilateral macular colobomata, and retinal pigment epithelial abnormalities; psychoelectrophysiological testing indicated extensive loss of cone or cone-rod function. These cases suggest this is a genetically determined condition unrelated to infection.


Constant perfusion for dry eyes and sockets. Several techniques have been used to treat dry eye, to irrigate the eye surface, or to apply drugs in solution. The commonest method is to apply drops. But a technique is needed for giving solutions over long periods either as drops or continuously. The apparatus described here consists of a replaceable plastic tank under mechanical pressure attached to capillary tubing which is kept in contact with the inner canthus of the eye. It was found to alleviate symptoms in severe dry eye conditions not alleviated by other methods.


Inhibition of adenylosuccinase by adenylophosphonopropionate and related compounds. Adenylosuccinase from muscle, liver and yeast is strongly inhibited by the substrate analogue adenylophosphonopropionate (N6-(DL-1-carboxy-2-phosphonoethyl)-adenosine 5'-monophosphate). The inhibition is freely reversible and of the competitive type, with apparent K1 values between 5.4 and 86 nM depending on the source of enzyme. Ratios of Km/K1 with adenylosuccinate as substrate fall in the range of 44 to 1350. Comparison of four carboxyl analogues of adenylosuccinate with the corresponding phosphonate analogues shows that the phosphonates are much better inhibitors. Adenylosuccinate analogues in which the beta-carboxyl is replaced by other functional groups are much poorer inhibitors. The exceptionally high affinity of adenylosuccinase for adenylophosphonopropionate appears to involve the dianion of the phosphonate group.


Interaction of an amine oxide detergent with lecithin vesicles as studied by nuclear magnetic resonance. The interaction of an amine oxide detergent with single bilayer lecithin vesicles was investigated with proton and phosphorus magnetic resonance. The addition of the detergent micelles to vesicles suspensions leads to rapid detergent incorporation into the vesicle bilayer, resulting in a heterogenous vesicle population. Initially, some vesicles take up the equivalent of one detergent micelle, whereas others contain no detergent. Subsequently, the detergent is distributed between the vesicles by vesicle-vesicle collisions. This can be followed by the change in the Pr3+-shifted spectral positions of the detergent and lecithin head groups with time. From the intensity of the head-group signals, it can be concluded that after about 20 h the detergent is almost equally distributed between the outer and inner vesicle membrane monolayers. Vesicles obtained by cosonication of the detergent and lecithin take up metal ions. This ion permeability depends on the vesicle concentration and can be attributed to vesicle-vesicle or vesicle-mixed micelle collisions. Egg lecithin vesicles are stable against the detergent up to molar ratios of detergent to lecithin of 0.2--0.3. At larger ratios mixed micells and multibilayers are formed. Measurements of proton spin-lattice relaxation times confirmed that the internal architecture of the vesicle bilayer is almost unaffected by the incorporated detergent.


Subunit association and heterogeneity of Limulus polyphemus hemocyanin. The molecular weights of the 6S, 24S, 36S, and 60S components of Limulus polyphemus hemocyanin were determined by high speed sedimentation equilibrium to be 69 400, 856 000, 1 690 000, and 3 160 000. The behavior of this hemocyanin appears to be similar to that of other arthropod hemocyanins where the first aggregation step is the formation of a hexamer of the 6S monomer. Here the larger aggregated states (24S, 36S, and 60S) are successive dimers of an unobserved hexamer (16S). The 24S-36S-60S association was found to be heterogeneous, suggesting that 24S components of different composition may be present.


Effects of Hofmeister salts on the self-association of glucagon. The trimerization constants of glucagon at pH 10.6 in 0.76 M K2HPO4 have been calculated from circular dichroism data between 5 and 50 degrees C. The free energy, enthalpy, and entropy of transfer have been evaluated from the current results and published data in 0.20 M phosphate. The free energies of transfer are derived completely from an increase in the entropy of transfer, since the enthalpy of transfer is less favorable at all temperatures. These parameters are compared with those of various model groups and compounds: CH2, peptide, methane, ethane, and the 1--13 N-terminal fragments of ribonuclease. The effects of fluoride and chloride on the self-association of glucagon have been compared with that of phosphate at 25 degrees C. These effects are consistent with the binding of approximately one molecule of salt to the trimer and a systematic decrease in the number of water molecules bound to the trimer compared to the monomer for the series K2HPO4, KF, and KCl.


Thermodynamics of protein cross-links.

Thermodynamics of protein cross-links. The thermal transitions of native lysozyme and a well-characterized cross-linked derivative of lysozyme [Imoto, T., and Rupley, J. A. (1973), J. Mol. Biol. 80, 657] have been studied in 1.94 M guanidine hydrochloride at pH 2. The observed increase in the melting temperature from 32.4 degrees C for native lysozyme to 61.8 degrees C for the cross-linked derivative corresponds to a calculated 5.2 kcal/mol increase in the free energy of denaturation. This free-energy change is attributed to the decreased entropy of the unfolded polypeptide chain following introduction of a cross-link and is shown to compare well with theoretical predictions. The possibility that an introduction of a cross-link could also affect the enthalpy of an unfolded protein was investigated. The heats of reduction of bovine serum albumin and lysozyme by dithioerythritol in 6 M guanidine hydrochloride were determined and compared to that for the model peptide, oxidized glutathione. The near identity of the observed heats was taken as evidence that the introduction of cross-links into a random-coil protein does not, in general, introduce strain.


Preparation of protein conjugates via intermolecular disulfide bond formation. Conjugates of two unlike proteins can be prepared via the intermolecular disulfide interchange reaction, namely, protein A containing thiol groups reacts with protein B containing 4-dithiopyridyl groups to yield a conjugate with the release of 4-thiopyridone. Thiol groups can be introduced into proteins upon amidination with methyl 3-mercaptopropionimidate ester or 2-iminothiolane, and 4-dithiopyridyl groups can be introduced into proteins with these same reagents in the presence of 4,4'-dithiodipyridine. 2-Iminothiolane is stable on storage in contrast to the known lability of imidate esters; therefore 2-iminothiolane is a more convenient reagent for the modification of protein than are the imidate esters. All the reactions can be carried out easily under mild conditions in good yields. Conjugates of bovine plasma albumin with itself, ribonuclease, or a copolymer of D-glutamic acid and D-lysine and of sheep antibody and horseradish peroxidase were prepared with modified proteins containing an average of 1 to 5 thiol or dithiopyridyl groups per mol. These conjugates formed mainly dimers, trimers, and tetramers. The peroxidase labeled antibody retained more than 80% of its enzymatic and antigenic binding activities.


Pressure-induced changes in the nuclear magnetic resonance spectra of a biopolymer in aqueous solution. High resolution proton nuclear magnetic resonance spectra of the poly(amino acid)poly[N5-(3-hydroxypropyl)-L-glutamine], of degrees of polymerization 685 and 137, were measured in a mixed D2O and H2O solvent, at pressures from 1.03 to 1968.5 kg/cm2, and at temperatures of 2 and 10 degrees C. Increasing the pressure appeared to cause an increased mobility of the side chain hydrocarbon residues, and also of the alpha-hydrocarbon residue of the polymer chain. This is interpreted to imply the occurrence of a volume decrease on unfolding of the polymer from a helix to a random coli, with subsequent exposure of hydrophobic groups to the solvent.


Structural similarities and differences among metal ion complexes of phosphoglucomutase by solvent perturbation and ultraviolet difference spectroscopy. Although the binding of bivalent metal-ion activators to phosphoglucomutase produces substantial changes in the near ultraviolet spectrum of the enzyme, the extent to which aromatic residues are exposed to the aqueous environment, as assessed by means of solvent perturbation spectroscopy (using D2O), does not appear to be significantly altered by the binding process. Other ways in which the spectral effects induced by activation might arise are considered by making comparisons with those changes induced by various nonactivating monovalent and bivalent cations. The observed differences are most easily interpreted in terms of an electrostatic perturbation of (at least) two different tryptophan residues. This interpretation is supported by using cationic vs, neutral (zwitterionic) tryptophan in various solvent systems to generate difference spectra that are similar either to the observed metal-ion induced spectral differences or to the differences in the spectral changes produced by various pairs of metal ions. Although a rationale for the striking similarity in the spectral changes produced by Mg2+ and by Li+ (which elicits less than 2 X 10(-8) of the enzymic activity induced by Mg2+) cannot be ascribed to a simple electrostatic effect, alone, the involvement of an additional, negatively charged group in the binding of Mg2+ (but not Li+) could reduce the effective charge of bound Mg2+ to a value close to that of bound Li+.


Over two hundred polypeptides resolved from the human erythrocyte membrane. A modification of O'Farrell's method of two-dimensional polyacrylamide gel electrophoresis has allowed for the resolution of erythrocyte membranes showing up to 200 individual components. Data is presented which indicates that this protein heterogeneity is not produced by artifactual protein-protein aggregation, ednogenous protease activity of secondary charge modification. Similar patterns are obtained when the samples are added to the unpolymerized isoelectric focusing gel, and isolated and stored in protease inhibitor. Individual spots could be eluted off of stained gels, resolubilized under extreme detergent solubilization conditions and run on one-dimensional gels; these run as sodium dodecyl sulfate in the solubilization procedure. The method chosen for solubilization prior to isoelectric focusing appears to cause selective aggregation of all or most of the spectrin and band 3 proteins. This further allows for excellent resolution of more components.


Biochemical aspects of the visual process. XXXVII. Evidence for lateral aggregation of rhodopsin molecules in phospholipase C-treated bovine photoreceptor membranes. Photoreceptor membranes derived from isolated bovine rod outer segments, are subjected to treatment with phospholipase C (Bacillus cereus). This results in varying degrees of hydrolysis of the membrane phospholipids into diglycerides and water soluble phosphate esters without loss of rhodopsin. Electron microscopic observations of thin sections and freeze-fractured preparations indicate extrusion of diglycerides from the membranes and their coalescence to lipid droplets, beginning at 20% hydrolysis of phospholipids. After 90% hydrolysis of phospholipids membranous structures are still present. The rhodopsin is located in these structures, presumably in the form of two-dimensional lateral aggregates. This explains the cross-fracturing of the membranous structures, regularly observed upon freeze-fracturing of the phospholipase-treated photoreceptor membranes.


Biochemical aspects of the visual process. XXXVIII. Effects of lateral aggregation on rhodopsin in phospholipase C-treated photoreceptor membranes. Treatment of bovine rod outer segments with phospholipase C leads to largely lipid-depleted membranous structures. Under these conditions rhodopsin remains spectrally intact, but its thermal stability and regeneration capacity are decreased, whereas upon illumination the metarhodopsin I to II transition is blocked. These observations can be explained on the basis of the previously demonstrated lateral aggregation of rhodopsin molecules which, on the one hand leads to a (partial) shielding of these molecules and, on the other hand, might impose constraints on the flexibility of the molecule to undergo light-induced conformational changes. Upon reconstitution of these lipid-depleted preparations with amphipathic lipids by means of a detergent dialysis procedure, the aggregates are apparently rearranged to lipid bilayer structures with complete recovery of the original rhodopsin properties. Under our conditions the nature of the polar head groups and the fatty acids is not critical in this respect. Simple addition of amphipathic lipids, without the use of detergent, restores the rhodopsin properties only in the case of rod outer segment lipids and of didecanoylphosphatidylcholine, and even then only occasionally. These results are discussed in the light of the strong analogy in properties between phospholipase C-treated rod outer segment membranes and lipid- and detergent-free rhodopsin obtained by affinity chromatography. It is concluded that rhodopsin must be in a freely dispersed state in order to function properly. Apparently, a non-specific lipid bilayer fulfills this condition for the regeneration capacity, whereas normal photolytic behaviour requires, in addition, a minimal membrane fluidity according to the observations of other investigators. Presumably, the uniquely high phospholipid unsaturation of rod outer segment membranes is important for another, as yet unassessed, function of rhodopsin or the photoreceptor membrane.


Mobilization of iron from the plasma membrane of the murine reticulocyte. The role of ferritin. 1. Plasma membranes prepared by pre-incumbation of mouse reticulocytes with 125I, 59Fe-labeled murine transferrin were able to release 59Fe in preference to 125I when incubated in the presence of murine reticulocyte cytosol, demonstrating that the latter mobilized iron which had been dissociated from transferrin. 2. 59Fe in cytosol was associated with at least two components in addition to hemoglobin, a high molecular weight component, identified as ferritin by specific immunoprecipitation, and an as yet unidentified, low molecular weight component of approx 17 00. 3. Ferritin itself, in the absence of added cytosol, was abloe to mobilize 59Fe from s9Fe-labeled reticulocyte plasma membranes. 4. Lysates of reticulocytes synthesized 59Fe-labeled heme when incubated with 59Fe-labeled ferritin. 5. These findings reflect a pathway of iron uptake and incorporation into heme in which ferritin plays an active role.


Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane. After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A2 cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B (1976) Biochim. Biophys. Acta 436, 353--365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys Acta 323, 178--193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of less than 10(6) dalton. Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents. It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.


Effects of bile salts of human erythrocytes. Plasma membrane vesiculation, phospholipid solubilization and their possible relationships to bile secretion. Glycocholate removed approximately 25% of the membrane acetylcholinesterase and 10% of the membrane phospholipid from intact human erythrocytes prior to the onset of cell lysis. At low concentrations (up to 6 mM), glycocholate caused human erythrocytes to become echinocytic and to pinch off microvesicles, whereas at higher concentrations glycocholate also specifically released components from the outer leaflet of the plasma membrane in a 'soluble' form (as defined by their presence in a 150 00 X g/60 min supernatant) and caused the cells to become stomatocytic. Whilst the phospholipdi profile of the 'soluble' material differed from that of the whole membrane, the profile of the microvesicle fraction was similar. The microvesicles were depleted in several membrane proteins with respect to phospholipids. These observations are discussed in relation to the possible role of bile salts in the origins of biliary phospholipid and protein.


Loss of resealing ability in erythrocyte membranes. Effect of divalent cations and spectrin release. Washed human erythrocyte membranes can recover impermeability to macromolecules upon warming in solutions of sufficient ionic strength. This ability is rapidly lost from most ghost preparations in dilute salt solution at temperatures of 15 degrees C of higher. Divalent cations both reseal ghosts in the absence of high ionic strength and prevent loss of resealing ability. The effective concentrations are 40 micron for Ca2+ and 200 micron for Mg2+. The loss of resealing ability is associated with the release of spectrin polypeptides from the inner surface of the membrane. In ghost preparations that have not become irreversibly leaky, or in the presence of Ca2+, loss of spectrin does not occur. These results suggest that an intact spectrin netwoek is required for resealing to macromolecules, and divalent cations stabilize this network. In light of this information, the effect of temperature on resealing kinetics is described.


Oxidized cholesterol bilayers. Dependence of electrical properties on degree of oxidation and aging. Black lipid membranes made from oxidized cholesterol were examined for their specific resistance, capacitance, and physical stability, as a function of cholesterol oxidation time and of age. Membranes formed from cholesterol oxidized in n-octane were not physically stable even after 7 h of oxidation unless they were aged for at least a month. Membranes formed from cholesterol oxidized in decane and tetradecane (1 : 1) were stable immediately after 2--6 h of oxidation. Oxidation times outside this range produced unstable membranes. After 1 month storage, membranes from cholesterol solutions oxidized in decane and tetradecane from 0.75--3 h were stable. After 11 months, only the 0.75 oxidation time produced stable membranes. Storage in nitrogen retarded the aging process. After initial forming of the membrane, total membrane area and capacity increased and then stabilized, although specific capacity and resistance did not change, indicating inherent stability in the bilayer's intrinsic electrical properties. Bilayers formed soon after cholesterol oxidation had membrane capacity which ranged from 0.42 to 0.55 muF/cm2. Specific membrane resistance ranged initially from 2 . 10(6) to 37 . 10(6) omega/cm2 in 0.2 M NaCl with lower resistances in the more oxidized membranes. With aging, membrane capacity decreased gradually over 11 months to values approaching 0.1 muF/cm2 indicating membrane thickening. Membrane resistance ordinarily decreases with storage time. The rate of these changes with age is dependent on the extent of initial cholesterol oxidation and subsequent oxidation, with long term stability best in the least oxidized membranes.


The myoglobin of an echidna (Tachyglossus aculeatus aculeatus). The skeletal myoglobin of the echidna was composed of 153 residues. Its primary amino acid sequence has been determined and compared with that of platypus. Nine differences were found. The number of differences from known marsupial and eutherian myoglobin is about twice as much.


The role of gamma-carboxyglutamyl residues in the positive cooperative binding of Ca2+ to blood coagulation factor X. 1. The calcium binding properties of factor X and its analogous decarboxyprotein have been compared with the aid of flow rate dialysis and ultraviolet difference spectroscopy. 2. Factor X binds approx. 20 mol of calcium per mol of protein. The first four sites exhibit positive cooperativity. 3. Changes in the ultraviolet difference spectrum when Ca2+ is bound suggest a conformational change. 4. In decarboxyfactor X low affinity of Ca2+ and no ligand-induced conformational change was observed. It is concluded that the presence of gamma-carboxyglutamate residues is a prerequisite for positive cooperative Ca2+ binding.


The effect of gamma-carboxyglutamate residues on the enzymatic properties of the activated blood clotting factor X. I. Activity towards synthetic substrates. The esterolytic and amidolytic properties of activated blood coagulation factor X (factor Xa) and the analogous decarboxy species were compared in order to find out if the gamma-carboxyglutamic acid residues influence the function of the active centre. It was found that the two proteins (1) showed similar kinetic parameters when titrated with p-nitrophenyl-p'-guanidinobenzoate hydrochloride (2) had a similar Km and kcat for various synthetic chromogenic tri- and tetrapeptides and (3) were inhibited in the same way by benzamidine. Further it was observed that (4) Ca2+ inactivates factor Xa, but has no influence on the amidase activity of decarbyxyfactor Xa (5) factor V prevents Ca2+-induced inactivation of factor Xa but does not influence the amidase activity of both factor Xa and decarboxyfactor Xa. We conclude that the interaction of the gamma-carboxyglutamic acid residues with Ca2+ in factor X has no measurable influence on the properties of the active site per se.


Molecular data on urinary glycoproteins with human leucocyte antigen (HLA) activity. Two glycoproteins characterized by their serological activities (HLA-A9 and HLA-B12), their isoelectric points and their molecular weights were purified from urine from a patient suffering from tubular proteinuria (cystinosis). Their physicochemical properties as well as an important increase of their specific activities during the different purification steps suggested that they behave as human leucocyte antigens (HLA) which had been excreted into urine. Their amino acid compositions and N-terminal sequences were different to those described for HLA solubilized from cultured human lymphoblast cell lines. The N-terminal sequences of the two serologically active glycoproteins were identical to the N-terminal sequence of another recently purified human urinary glycoprotein called human complex-forming glycoprotein. The relationship between HLA, human complex-forming glycoprotein and the serologically active urinary glycoproteins is discussed.


Sepharose-linked concanavalin A in the purification and characterization of glycoprotein hormones of the bovine pituitary. Affinity chromatography on concanavalin A-Sepharose is a time saving step in both large and small scale isolations of the bovine pituitary glycoprotein hormones. After ion-exchange chromatography, the final yield of purified lutropin is 40-50% of material in starting concentrates and of purified thyrotropin is approximately 20%. The final products have the same electrophoretic and immunological properties and amino acid compositions as previous preparations. Less than 3% of the immunoreactive lutropin, follitropin and thyrotropin are present as non-glycosylated forms in either crude pituitary extracts or concentrates. Thyrotropin and follitropin elute from the immobilized lectin as a single fraction, whereas lutropin separates into two glycosylated fractions. Gel filtration of both crude extracts and the glycoprotein fractions shows that less than 5% of the immunoreactivity of the hormones is present as material of apparently high molecular weight. Substantial alpha subunit immunoreactivity, however, is in three fractions (as found by others in human pituitary extracts) corresponding to "high molecular weight material" (7%), intact hormones (46%) and free subunit (47%).


Complexes of chromogranin A and dopamine beta-hydroxylase among the chromogranins of the bovine adrenal medulla. 1. The core proteins of chromaffin granules have been examined by polyacrylamide gel electrophoresis and crossed immunoelectrophoresis against monospecific antisera. 2. Dopamine beta-hydroxylase (dopamine beta-monooxygenase, EC 1.14.17.1) appeared as the major immunogen of the core proteins and accounted for 4 and 8% by weight of the crude lysate and membrane-containing fractions, respectively. 3. The non-ionic detergent, Berol, solubilized dopamine beta-hydroxylase from the membranes in a form which was immunologically identical but of lower relative mobility by crossed immunoelectrophoresis. In the absence of detergent a difference in relative mobility was also noted between the purified enzyme and that contaminated by chromogranin A. These observations suggest that several molecular forms of dopamine beta-hydroxylase may occur which differ in size and/or charge due to interactions with the contaminants under the experimental conditions. 4. The main chromogranin in the crude lysate was absent from electropherograms of the acidic chromogranins (95--96% of total protein in lysate). These were obtained free of dopamine beta-hydroxylase by concanavalin A adsorption at high ionic strength or by acidification in 2 M acetic acid. The main band reappeared upon recombination with dopamine beta-hydroxylase, indicating the presence of some dopamine beta-hydroxylase, possibly as dimers, in this main, chromogranin A band. A protein concentration-dependent aggregate of dopamine beta-hydroxylase-free chromogranin A was detected, with a relative mobility slightly faster than the main band of the crude lysate.


Thermodynamic studies of the interaction of alpha-chymotrypsin with water. I. Determination of the isosteric enthalpies and entropies of water binding to the native enzyme. The enthalpies (deltaH) and entropies (deltaS) of the interaction of water with alpha-chymotrypsin were evaluated from multitemperature sorption isotherms in the temperature range 283--313 K, determined in a fully automatized, computer controlled sorption apparatus. The temperature dependence of deltaH and deltaS shows a marked anomaly in the temperature range 295--298 K. The experimental results are interpreted by a phase transition of the enzyme protein, and by the existence of a low- and high-temperature conformer of alpha-chymotrypsin below and above the transition region. The two conformers differ significantly in their water binding energetics, as proved by two F-tests based on analyses of variance. The deltaH and deltaS versus water content functions of the high and low temperature conformer show markedly different anomalies at and below 70 mol H2O per mol protein. This water content corresponds closely to the monolayer volume vm, (as defined by Brunauer, Emmett and Teller). The protein surface covered by one water-monolayer, agrees well with the polar and charged surface area of chymotrypsin, computed from X-ray data. The experimental results suggest that the interaction of the first water-monolayer with the protein surface induces major conformational changes. The energetic contributions of these structural changes dominate the deltaH and deltaS terms below vm, giving rise to the anomalies observed. Above this water content, their influence is drastically reduced, and the isosteric quantities are predominantly determined by the water binding process per se. This process is possibly related to the pronounced enthalphy-entropy compensation pattern exhibited by the deltaH and deltaS terms. A more detailed analysis and discussion of this compensation effect will be given.


The reaction of tryptophan with cystine during acid hydrolysis of proteins. Formation of tryptathionine as a transient intermediate in a model system. Under the conditions commonly used to hydrolyze proteins with 6 M HCl, tryptophan reacted with cystine to give a transient intermediate, which was isolated and identified as 2-(2-amino-2-carboxyethylthio)tryptophan (tryptathionine) by NMR studies, etc. Studies on the formation and degradation of the above compound showed that beta-3-oxoindolylalanine and cysteine, which were previously reported to be the main degradation products of tryptophan and cystine, respectively, are formed by the hydrolysis of this intermediate during the course of the reaction.


Pseudo-dynamic contact surface areas: estimation of apolar bonding. A new Monte Carlo based algorithm has been written for the computation of pseudo-dynamic contact surface areas. The linear correlation of this contact area with solute transfer free energies (water leads to organic liquid) is established for apolar amino acid side chains. The slope of these linear plots, deltaGosp, is a unitary free energy which has potential use in the estimation of apolar bond free energies in proteins. The magnitude of deltaGosp is dependent upon the nature of the organic solvent involved in the transfer process, varying from 86 to 130 cal/A2. Analogues linear correlations with the same range of deltaGosp values are observed for inhibitors of protein-catalyzed reactions.


Structural stability of glycophorin. Effects of heat and guanidine . HCl. The effects of guanidine hydrochloride and high temperature on human glycophorin and sialic acid-free glycophorin were monitored by circular dichroism, viscosity, and fluorescence of 1-anilino-8-naphthalane sulfonate (ANS). The following observations were made: 1. Glycophorin and its sialic acid-free counterpart are unusually stable to both guanidine . HCl and heat. 2. CD and viscosity measurements indicate that guanidine . HCl neither causes a cooperative unfolding nor generates a random coil. 3. The ANS binding site is much more sensitive to guanidine . HCl than the ellipticity at 220 nm (theta 220). 4. The effect of temperature on CD is reversible whereas the effect of guanidine . HCl is not. 5. The carbohydrate moiety influences the viscosity, and also contributes to the changes in theta 220 when solutions of glycophorin are heated. These unusual properties indicate a complex mechanism of unfolding for this structurally stable macromolecule.


Effect of salt concentration on immunoglobulin G structure. An effect of salt concentration on the human myeloma immunoglobulin G structure was studied by means of circular dichroism, thermal perturbation difference spectroscopy and isoelectric focusing in a pH gradient created by a concentration gradient of glucose in borate buffer solution. Immunoglobulin G (K) Iva showed a significant shift of isoelectric point to the alkaline region as a result of the increase in salt concentration. The difference spectra indicated a change in the exposure of tyrosine residues as a result of increase in salt concentration. No changes in the circular dichroic spectra with salt concentration were observed between 205 and 250 nm. Spectral changes observed for the undigested immunoglobulin G molecule are more marked than those observed for the isolated Fab fragments.


Quantitation of submicrogram quantities of protein by an improved protein-dye binding assay. A quantitative assay method for protein is described which is based on the color change occurring in Coomassie Brilliant Blue G-250 when it binds to protein. This modification of two similar procedures further increases the sensitivity, simplicity, and stability of the protein-dye binding assay over those of other commonly used assays for protein.


Conformation of oxytocin studied by laser Raman spectroscopy. The peptide backbone conformation and salient structural details of oxytocin were examined by laser Raman spectroscopy. Spectra were obtained in the solid phase, water, 2H2O, and dimethyl sulfoxide solutions. A distinct Amide I band was obtained at 1663 cm-1 for aqueous and deuterated samples and 1666 cm-1 for the solid sample. A relatively high frequency Amide III band at 1260 cm-1 was obtained. It is concluded that these Amide I and III bands arise from the "beta-turn"-like conformation of oxytocin. The tyrosine side chain, according to the I850 cm-1/I830 cm-1 intensity ratio, is exposed to the solvent. The S-S stretching vibration at 512 cm-1 indicates the conformation of C-C-S-S-C-C in the disulfide bridge of oxytocin in the ring is gauche-gauche-gauche.


Infrared stretching frequencies of CO in carbomonoxyhemoglobin from trout. The infrared spectra of the carbomonoxy derivatives of the hemoglobin components I and IV from trout have been measured in the CO stretching frequency region using a high resolution infrared spectrometer. The CO stretching frequency of Hb I CO is very close to that of carbomonoxy human hemoglobin and is pH-independent. In contrast, the CO stretching frequency of Hb IV CO is higher and shows a small but significant pH dependence in the range 6.2-7.8. These results point to a decreased strength of the iron-CO bond in Hb IV CO at low pH, in agreement with the conclusions drawn from the reported difference spectra of Hb IV CO as a function of pH.


Ultraviolet flow dichroism of brain microtubule. Ultraviolet flow dichroism of brain microtubule was measured. Large positive dichroisms were observed at 255 nm, 285 nm and 292 nm. The dichroism at about 255 nm is due to bound GDP or GTP. The dichroism at 285 nm is due to tyrosine and tryptophan residues, and that at 292 nm is due to the tryptophan residues of the protein. These results show that the electronic transition moments of nucleotides and aromatic groups are both nearly parallel to the polymer axis.


[Study of the relationship between secondary DNA strucure and base sequence by the technic of theoretical conformational analysis]. An investigation of double-stranded polynucleotide conformations using the algorithm for exclusion of redundant dihedral angles and flexible sugar unit has been performed. The fragment of DNA including three nucleotide pairs was taken into account for polynucleotides poly[d(A--T)].poly[d(T--A)], poly[d(G--C)].poly[d(C--G)], poly(dA).poly(dT) and poly(dG).poly(dC). The resulting structures being energetically optimal have helical parameters corresponding to X-ray experimental values for A- and B-forms. It was revealed that B-form is the most energetically favorable structure, therefore the earlier conception that water is of importance for B-form stabilisation may be revised. Relative stabilities of B- and A-forms depend upon base sequence because unequal contributions of the stacking and backbone energy in whole potential energy of the structrues with different sequences.


[Effect of ultrasound on porphyrin biosynthesis. I. Changes in the levels of proto-, copro- and uroporphyrins after a single exposure to ultrasound]. Abdominal region of white rat males was exposed to ultrasound with the intensity of 0.2, 0.6 and 1.0 Wt/cm2. 1, 24 and 48 hours after sonication proto-, copro- and uroporphyrines were determined in erythrocytes and excrements, copro- and uroporphyrines in urea. Single ultrasonic exposure does not affect the biosynthesis of uro- and coproporphyrines in gialoplasm, however, it results in the change of their excretion with excrements and urea. Biosynthesis of protoporphyrine IX which is the final stage of haemoglobin biosynthesis is changed under the effect of ultrasonic energy.


[Calorimetric study of the fusion of the liquid-crystalline structure of poly-gamma-benzyl-L-glutamate solutions]. A calorimetric study has been carried out of the specific features of melting of liquid-crystalline structure in the system PBG--dioxan depending on the conditions of its formation on the one hand, and on destruction rate on the other. It has been found that an increase of the temperature of solution crystallization as well as an increase in crystallization time shifts the conformation transition to the region of high temperatures, the value of the temperature shift decreasing with the rise of multiplication of heating for all the concentrations studied. By means of kinetic studies a sharp increase of heat effect with the rise of the velocity of heating the system has been found. A change in the scanning rate does not displace the beginning of the conformation transition, while Tmax and Tult is increased with an increase of Vheat. The kinetic regularities obtained allowed to suggest the presence of two processes proceeding when the system studied is passing the interval of conformation transition--the melting processes and subsequent aggregation of PBG molecules. They allowed also to separate these two processes.


[Heat denaturation of alpha-tropomyosin and its fragments]. The reasons for three heterogeneity of tropomyosin melting curves are considered. It is shown that this phenomenon is due to the molecular heterogeneity of the preparation. Different states of the SH-groups as well as the different stability of molecule regions. The melting curves of alpha-tropomyosin and two of its fragments are obtained. The thermodynamic parameters stabilizing their helical structure are determined. The existence of a thermodynamical transition at 31 degrees C is shown for alpha-tropomyosin leading to the loss of the ability of the molecule to form supra-molecular structures.


[Study of the conformational mobility of globular proteins in aqueous solutions according to their proton relaxation in a rotating system of coordinates]. Measurements of the nuclear magnetization decay in the rotating frame for protons of SA and RNAase proteins in aqueous solutions indicate the dispersion of the relaxation rate for SA protons within the region of correlation frequencies of 10(5)--10(6) s-1. These frequencies are much lower than frequencies of rotational diffusion movements of the SA macromolecules in aqueous solutions. These must be ascribed to internal movement within protein globules due to which the distances between interacting magnetic moments are changed. This conclusion gives direct evidence in favour of existence of conformational mobility in most of the protein globule volume.


[Qualitative analysis of a mathematical model of a open enzyme reaction]. A general case of the set of two differential equations, describing an open reaction v1 leads to S v reversible E P v2 leads to, has been considered. The requirements to the character of the functions v1([S]), v2([P]) and v([S], [P]) were formulated for the case of existence and absence of alternative steady states and sustained oscillations. The formulae were derived to determine the slope of the unstable portion of the quasi-steady state characteristic. The generalized model of Monod, Wyman and Changeux has been considered as an example of v([S], [P]). It has been shown that with monotonically decreasing v1 and monotonically increasing v2, the alternative steady states and oscillations are possible only in the presence of substrate inhibition or product activation. However, under the joint action of substrate inhibition and product activation, the system will exhibit bistability rather than an oscillatory behavior. In the case of an irreversible two-substrate reaction which can be described by a similar mathematical model, inhibition by the first and second substrate is equivalent to substrate inhibition and product activation.


[Study of the aqueous phase transition in a collagen--water system by the calorimetric and NMR methods]. Heat capacity and line width of proton magnetic resonance of collagen were studied by means of low-temperature calorimatry and high resolution NMR spectroscopy at different values of moisture in the temperature range of ice-water phase transition. Peculiarities of water phase transition in the presence of biopolymers were revealed. It was shown that a definite part of H2O in the "hydration layers" of the macromolecules (0.35 g of H2O/g of protein) is not transformed into ice even at a temperature decrease up to 4 K. The NMR data show that the total mobility of the bound water molecules remains high enough up to the water concentration at which the inner hydration layer of the macromolecule is formed. After the formation of hydrogen-bound water chains in the hydrate layers of collagen the mobility of the bound water molecules decreases.


[Membrane mechanisms of activating depolarized smooth muscle contraction (guinea pig small intestine) during exposure to physiologically active compounds]. The experiments were performed on the depolarized smooth muscle of taenia coli with the use of double sucrose-gap arrangement. Muscle contractions were induced by 1) application of hystamin or bradikynin; 2) rectangular long-lasting (10--20 s) pulses of hyperpolarizing current--the strong contraction appeared in response to the switching off the current (off-response). Both the on- and off-responses to the hyperpolarizing current recorded before, during and after hystamine (or bradikynin)-induced contration were, as a rule, very similar. Treatment of smooth muscle with local anaesthetics (procaine, trimecaine, QX-572) removed hystamine- and bradikynin-induced contrations and only decreased off-responses. The analysis of the data obtained suggested the existence of the independent electrically and chemically excitable systems (channels?) OF Ca2+ ion transport in the membrane of smooth muscle cells.


[Electrodiffusion model of a nerve impulse]. Electrodiffusion equations are deduced which describe the formation of the action potential in the axone. It is suggested that the membrane dividing internal and external axone electrolytes after being stimulated with an electric impulse returns after some time to the initial "closed" state. It is shown that the overshut of the action potential takes place due to non-linear profile distortion of the shock wave of electrical field tension vector created by the movement of sodium and potassium ions through the membrane of the axone.


[Basis for application of the Monod formula to the process of biological purification of flocs of active sludge]. Monod's empyrical relationship for the growth of active silt biomass is explained by diffusion of the limiting substrate inside active silt flakes. The Michaelis constant is determined not by the bacteria species which are a part of the flakes, but by flake dimensions. Calculations have shown that the concentration of solved oxygen in the mixer-tank which does not limit the total process of biological purification can be maintained at the level of 1 mg/l. In the plug flow-tank the concentration of solved oxygen must be considerably higher.


[Thermal fluctuations in membranes and the problem of mechanoreception]. In order to estimate thermal fluctuations in erythrocyte and mechanoreceptor membranes the transverse fluctuations of plane and spherical bilayer membranes and the fluctuations of the surface of a part of such a membrane, possessing disc shape of the radius R are calculated. The obtained values of the transverse fluctuations are two orders smaller than in the paper [5]. Total plane fluctuations of the disc with R=5.10(-7) cm are some orders higher than the threshold values epsilonpi of relative deformation in mechanoreceptor membranes, but their value in the frequency range of 0 dividied by 20 kHz is of the same order as epsilonpi. The estimates of fluctuations are also produced for Pacinian corpuscle membrane and for the globular protein molecule. The conditions necessary for high sensitivity of mechanoreceptor membranes are the large value of Young modulus E and low membrane viscosity eta.


[Modeling atrial and ventricular contractility of the heart muscle. I. Modeling single contractions]. A system of differential equations describing myocardium contractions in isometric and isotonic regimes has been obtained. On the basis of these equations functioning of myocardium was modelled on an electronic computer. An effect has been revealed of the coefficients of rheological equations and activation function on isometric and isotonic contraction of the force/rate ratio. A good agreement between the results of modelling and the experimental data has been observed.


[Modification of a model of a cell population inhibiting its mitotic activity]. A series of mathematical models of cell population is analysed. The mitotic activity of stem cells of this population is controlled by population density. The effect of different factors on autooscillation regimes has been studied. The effect of reversible stages of cell differentiation is analysed, as well as that of cell losses at each stage of differentiation. The behaviour of cell population under the conditions of a limited supply of the limiting substrate was analysed. The effect of cell doubling at each differentiation stage on the stability of the population was studied too.


[Torsion movements of the human eye. IV. Passive body movements, effect of skeletal muscle reflexes, opticokinetic torsion tracking]. A comparative study of torsional movement of the eye in passive and active tilting of the head and body of the object was carried out. Similarity of torsional movement of the eyes in passive and active movements was shown. It was found by the method of exclusion and selective stimulation of vestibular, cervikal, lumbar optokinetic reflexes, that neither the cervikal, nor lumbar reflexes elicited spontaneous torsional movements of the eyes and had no influence on them. A direct study (coinciding with rotation direction of the stimulus of head rotation) and the reverse (noncoinciding) torsional tracing of a rotating disc and tracing without head movements was investigated. During direct tracing depression of saccades and extention of the slow phase of torsion was found; during the reverse one--a decrease of the eye drist and increase of the amplitude and number of saccades. Phenomena of a seeming acceleration and deceleration of disc rotation etc. have been observed. It was found that with torsional saccades vision was retained. The presence of optokinetic control of phases of torsional eye movements formation has been recorded. Tracing without rotation of the head was accompanied by torsional nistagmus. Possible causes of incomplete stabilisation and optokinetic torsional tracing are discussed.


[Model studies of lateral inhibition as a mechanism of detecting motion. II. Modeling feedback inhibition]. A program is worked out which imitates non-stationary lateral inhibition in two-dimensional retina with physiologically real characteristics. Experiments with moving and flashing stimuli of different size are carried out. It is shown that lateral inhibition may serve as a mechanism which forms selective sensitivity of the elements involved to the movement of the stimuli. Recurrent inhibition proves to be more effective than the direct one providing that a longer delay in time exists. The results obtained are explained by the peculiarities of recurrent inhibition as the feedback system.


Iron-binding ligands in the catalytic site of protocatechuate 3,4-dioxygenase. The tryptophan fluorescence maximum for holoprotocatechuate 3,4-dioxygenase(holo PCD) is blue-shifted slightly (3 nm) from that of the apoenzyme. In the preparation of apoenzyme, increases in tryptophan fluorescence intensity coincided with decreases in enzyme activity and decreases in iron content. The tryptophan emission intensity of reconstituted enzyme having full enzyme activity was about 90% of that of the holoenzyme. Although apo PCD has similar molecular weight, amino acid content and essentially the same gross quaternary conformation as holo PCD, the absence of iron in apo PCD causes the changes in emission intensity of tryptophan. Findings indicate that some tryptophan residues may be (or may be near) the iron-binding ligands in the catalytic site of protocatechuate 3,4-dioxygenase.


[Suppression of singlet oxygen luminescence by porphyrins and metalloporphyrins]. The luminescene of 1O2 (1270 nm) has been observed upon illumination of air saturated solutions of different porphyrins and their complexes with Zn in CCl4. In solutions of Co-, Cu-, Ni- and Fe-porphyrins this luminescence has not been revealed. All the porphyrins studied have shown to quench 1O2, the rate constants of the "physical" and "chemical" quenching being measured. The physical way of quenching is found to be much more effective. The quenching activity of the pigments depends greatly on the presence and nature of the central metall atom incorporated into porphyrin (H2 less than Cu less than Zn less than Co approximately Ni approximately Fe) increases with hydrogenation of the semiisolated double bonds (porphyrins are less active than chlorins and bacteriochlorins).


Biological function of metallothionein-IV. Biosynthesis and degradation of liver and kidney metallothionein in rats fed diets containing zinc or cadmium. The half-lives of liver and kidney metallothionein of rats fed diets containing either 2,000 ppm Zn or 100 ppm Cd were determined by a single injection of 14C-cystine. This protein was purified by Sephadex G-75 and DEAE cellulose column chromatography. Half-lives of liver metallothionein in rats fed the basal (18 ppm Zn), high Zn (2,000 ppm Zn), high Zn then basal, and high Cd (100 ppm) diet were, respectively, 3.4, 2.7 1.3, and 3.3 days, and that of kidney metallothionein were, respectively, 2.0, 2.2, 1.4, and 3.6 days. Thus, Cd-thionein has a longer half-life than Zn-thionein in both liver and kidney. The major species of metallothionein, which were separated on DEAE cellulose, had similar half-lives. Metallothionein, a protein having high content of metals and relatively short half-life, is possibly involved in cellular regulatory function.


The relationship of some copper (II) complexes of facultative tetrathioethers to the coordination environment in the "blue" copper proteins. The facultative potentially tetradentate thioether ligands 1,2-bis(methylthioethylthio)ethane (2,2,2), 1,3-bis(2-methylthioethylthio)propane (2,3,2) and 1,2-bis(3-methylthiopropylthio)ethane (3,2,3) react with copper(II) salts to form Cu2(2,2,2)Cl4, Cu3(ligand)X6 (ligand = 2,3,2 and 3,2,3 X = Cl; ligand = 2,2,2 2,3,2 and 3,2,3 X = Br), and Cu(ligand)(ClO4)2. The stoichiometry and structures of these complexes are discussed in terms of the steric demands of the ligand and the nature of the halide. The [Cu(2,3,2)(ClO4)] ClO4 and [Cu(3,2,3)(ClO4) [ClO4 complexes have electronic spectra which exhibit the intense 600 nm band characteristic of the "blue" copper proteins. In fact, the spectrum of [Cu(2,3,2)(ClO4)]ClO4 is very similar to that of pseudomonas aeroginosa azurin.


[Formation kinetics of the AT-specific complex: DNA-distamycin A]. The kinetics of DNA-distamycin A complex formation and dissociation was studied by means of the stop-flow method. It has been found that the complex formation has at least five steps, the formation of AT-specific complex is limited by the dissociation of less specific intermediate complexes, then follows the statistical rearrangement of the ligand molecules and "fixation" of the specific complex with the longest lifetime and the biggest number of the additional H-bounds with the matrix. The energy of a single specific H-bond between the ligand molecule and AT-pair is equal to--1.05 kcal/mol.


[Free radical lipid oxidation and several ways of regulating it with ascorbic acid]. The correlation between the content of primary molecular product of free radical fat acyls oxidation--hydrolipid peroxidation and ascorbic acid amount in guinea-pig tissue was presented by means of polarographic method. The data obtained led to the following conclusion: the importance of ascorbic acid as prooxidant in vivo is rather great and its deficiency is the reason for an increase of its content in the primary products of lipid free radical oxidation, which probably plays the main role in C-avitaminosis pathogenesis.


Activity of glutamate and malate dehydrogenases in liver and kidneys of rats subjected to multiple exposures of mercuric chloride and sodium selenite. Rats were subjected for 2 weeks to separate and combined exposures to mercuric chloride and sodium selenite at doses of 0.5 mg Hg/kg and 0.5 mg Se/kg. The content of mercury, selenium and protein as well as the activities of glutamate dehydrogenase (GLDH) and malate dehydrogenase (MDH) were determined in homogenates, mitochondria and intramitochondrial structures of the exposed animals. It was found that both separate and combined exposures of rats to mercuric chloride and sodium selenite inhibited GLDH activity and did not affect MDH activity in the examined organs. Mercury-selenium interaction brought about a decrease in the content of mercury in the intramitochondrial structures of kidneys and an increased accumulation of both elements in the outer and inner membranes of liver mitochondria. The biochemical mechanism of mercury-selenium interaction is discussed.


Selenium in human nutrition: dietary intakes and effects of supplementation. The dietary selenium intakes of a young couple residing in Southern California were determined to be 107 and 99 micrograms/day for the husband and the wife, respectively, on the basis of a 30 day study. For other young adult Californians, the selenium intakes were estimated from 90 to 168 micrograms/day. The highest intakes were observed in individuals subsisting on diets rich in whole wheat grain cereal products and seafoods. The selenium concentrations in whole blood of the subjects under study correlated with the dietary selenium intakes directly (P less than 0.001). The administration of 150 micrograms of selenium/day in the form of commercially available supplements increases the blood selenium concentrations. After 3 weeks of supplementation, the selenium concentrations in whole blood of our subjects reached 0.21 micrograms/ml. Prolonged supplementation at higher Se dosage levels causes further increases of the blood concentrations: Two individuals who had been ingesting 350 and 600 micrograms/day for 18 months exhibited blood selenium levels of 0.35 and 0.62 micrograms/ml. The blood selenium concentration of all subjects declined slowly after cessation of supplementation. Selenium uptake from the supplements was not affected by the joint administration of zinc supplements at 15 mg zinc/day. Glutathione peroxidase blood levels did not correlate with blood Se concentrations.


Interactions of cis- and trans-platinum(II) complexes with dehydrogenase enzymes in the presence of different mono- and polynucleotides: evidence for a ternary complex. The inhibition of several dehydrogenase enzymes by cis- and trans-Pt(NH3)2Cl2 have been measured in the presence of baker yeast ribonucleic acid (RNA), calf thymus and salmon sperm deoxyribonuclic acid (DNA) and several mononucleotides (AMP and ATP). The binding constants for the interaction of the platinum complexes to the nucleotides have been calculated and a comparison of those values to the previously calculated platinum complex-enzyme binding constants strongly suggest that platinum compounds are more tightly bound to the enzymes. The binding of the platinum complexes to most of the enzymes was decreased in the presence of any nucleotide, yet it was observed that when using rabbit muscle (M4) lactate dehydrogenase the mononucleotides reduced the binding to a lesser degree while the polynucleotides actually enhanced the platinum-enzyme interaction. The implications of these interactions are discussed.


A proposed mechanism relating the antitumor behavior of cis-platinum amine complexes to their inhibition of a model enzyme. The twenty-four hour inhibition of m-malate dehydrogenase (E.C. 1.1.1.37) by various complexes of cis-platinum(II) and cis-platinum(IV) was measured as a function of the platinum concentration. It was observed that increased alkylation of the amine groups of Pt(II) and to a lesser degree of Pt(IV) decreased the activity consistently. It was also observed that the Pt(IV) analogues inhibit the enzyme to about an order of magnitude greater than the Pt(II) complexes. These phenomena will be interpreted.


A fluorescence study of the binding of calcium and terbium ions to angiotensin. The interactions of angiotensin II and a synthetic analogue, [Asn1, Val5] angiotensin II, with Ca2+ and Tb3+ have been monitored using the intrinsic fluorescence of the tyrosine residue at position 4 in both molecules. The data indicate that angiotensin II binds both metals with a dissociation constant of approximately 1 X 10(-4) M-1, while no significant binding was observed with the amide analogue. This suggests that the side chain carboxyl group of aspartic acid forms part of the binding site. Since the value of the dissociation constant suggests chelation of the metals by the hormone, the terminal carboxyl group of the peptide is also probably involved in metal binding. The fact that energy transfer was observed between Tb3+ and the tyrosine of angiotensin places the hydroxl or carbonyl group of the tyrosine close to the metal binding site.


[Extraction and purification of tissue phosphopeptides]. We describe a method of extraction and partial purification of phosphopeptides isolated from pig brain or from the electrical organ of Torpedo marmorata. The extraction of the phosphopeptides was achieved by alcoholic 0,04 N potassium hydroxyde solution or by 10(-1) M KCL containing 10(-3) M EDTA and 10(-4) M DTT. After having tried various fractionation methods like ion exchange chromatography or gel filtration we chose chromatography on DEAE Sephadex followed by purification of the isolated fractions by Sephadex G 25 filtration. The most important phosphate fractions (one was purified to about 90 per cent) were characterized by the determination of the N/P ratio which was different from one phosphopeptide to another. The amino acid composition showed a high glycin, serine and "acid" amino acid content. The presence of phosphoserine was shown by electrophoresis and chromatography of a partial hydrolysate of in vivo 32P labelled phosphopeptides isolated from rat liver. The polyanionic structure of these phosphopeptides allow them to act as real ion exchangers which might be involved during active transport mechanisms in cellular membranes.


Glycosyl transfers from UDP-sugars to lipids of plant membranes: identification and specificity of the transferases. Microsome preparations extracted from wheat roots or sycamore cell suspensions catalyzed the transfer of sugar from nucleotide-sugars to endogenous lipidic acceptors. The nature of the products biosynthesized from UDP-Glc, GDP-Glc, UDP-Gal, UDP-Xyl or UDP-Arab was examined. Sterylglycosides were obtained from UDP-Gglc, GDP-Glc or UDP-Xyl. Galactosyldiglycerides were synthesized from UDP-Gal. When UDP-Glc or UDP-Gal was used as a substrate, a membrane-bound 4-epimerase interconverted the epimeric nucleotide-sugars, thereby allowing the simultaneous biosynthesis of galactosyldiglycerides and sterylglucosides. The biosynthesis of free and acylated sterylglucosides from UDP-Glc, without interference of other glycosyl transfer reactions, was obtained by the omission of Mg++ ions from the incubation medium. The biosynthesis of galactosyldiglycerides from UDP-Gal without interference of other transfer reactions was obtained when digitonin was added to the incubation medium of sycamore microsomes.


[Demonstration, kinetic study and solubilization of particular adenylate cyclase from Nocardia restricta]. An adenylate cyclase (EC r.6.1.1.) was found in cell-free extracts of several Nocardia species. The enzyme from Nocardia restricta has been specially studied. It is a membrane enzyme which exhibits a strong specific activity, one hundred times greater than that of mammals. It has an optimal pH of 8.5 in Tris buffer and an absolute requirement for divalent ions, Mg++ or Mn++ (Mn++ ions are the most efficient). The kinetic properties of this adenylate cyclase are similar to those that could be expected of an allosteric enzyme having, as a substrate, the ATP-Mg++ complex and, as an activator, free Mn++ ions. Ca++ ions are activators: they set up the maximum velocity without modification of the KM. GTP is a competitive inhibitor (KI = 5.10(-5) M). Fluoride ions have no detectable effect on activity. Non-ionic detergents, Lubrol WX and Triton X 100, are inhibitors of the enzyme which has been partially solubilized by repeated freezing and thawing, following by brief ultra-sonic treatment. Catalytic sites are not modified after the solubilization, but cooperative effects between moles of substrate ATP-Mn++ are diminished: the KM becomes smaller and the sigmoidal shape of the curve v = f (ATP-Mn++) is attenuated.


[Fatty acid distribution of the normal sciatic nerve lipids in the rat]. We isolated and purified the main lipids of the rat sciatic nerve. After methanolysis, fatty acids were isolated and purified by thin layer chromatography, and studied by gas chromatography. C 16 and C 18 fatty acids are the most abundant. Among the long-chain fatty acids, arachidonic acid (20:4) is present in some lipids; highly desaturated fatty acids in C 22 and C 24 are also present. In general, the fatty acids are highly saturated; cholesterol esters and ethanolamine phosphoglyceride fatty acids are highly unsaturated.


[Kinetic and spectral parameters of the amines oxidative N-dealkylation with participation of the liver microsomal cytochrome P-450. Amines oxidation with organic hydroperoxides]. Kinetics of demethylation of a number of amines involving hepatic microsomal cytochrome P-450 and organic hydroperoxides (tret-butyl- and cumylhydroperoxide) have been investigated. Decomposition rate constants for the substrate-cytochrome P-450-ROOH complexes have been determined in a generalized form. Activation parameters, deltaH* and deltaS*, are calculated for decomposition of the complexes. There is a linear relation between deltaH* and deltaS*: deltaH*=18.7 kcal + 333 degrees K deltaS*. Compensation relationship is characterized by the value of alpha=333 degrees K/Taverage=1.11. The nature of the limiting step in the cytochrome P-450-NADPH-O2-system and the cytochrome P-450-ROOH-system is discussed.


[Interconversions of ubiquinone and ubichromenol in rat liver under the effect of alpha-tocopherol]. Incubation of ground liver preparation isolated from vitamin E-deficient rats resulted in correlated in time changes of ubiquinone and ubichromenol contents and their ration in tissues. A suggestion is made on the possibility of ubiquinone and ubichromenol interconversions and of the existence of a system controlling (depending on a functional state of the organism) a certain ratio of the quinones contents in animal tissues. Possible biochemical role of ubichromenol in animals is discussed.


[Changes in peroxidation under conditions of 3,4-benzypyrene-induced carcinogenesis]. The activities of enzymatic systems generating and destroying peroxides and the lipid peroxide content in neoplastic rat liver and 3,4-benzpyrene-induced sarcoma were studied. The tumour was characterized by high activity of glutathione peroxidase and low activity of catalase. No urate- and glycolate oxidases or ascorbat dependent peroxidation of lipids and lipid peroxides were found in the tumour. In the liver of neoplastic animals the activities of glutathione peroxidase and NADPH-dependent system of microsomal phospholipid peroxidation and the lipd peroxides content were increased, whereas the activities of catalase and urate oxidase were decreased.


[On the impossibility of the incorporation of 5-methylcytosine and its nucleosides into higher plant DNA]. No radioactivity was detected in 5-methylcytosine isolated from wheat DNA after incubation of wheat seedlings with 3H-labelled 5-methylcytosine, 5-methylcytidine and 5-methyldeoxycytidine. No label from 3H-5-methylcytosine was found in DNA of seedlings. After incubation of seedlings with 3H-labelled nucleosides of 5-methylcytosine, radioactivity was discovered only in thymine of DNA. Thus 5-methylcytosine and its nucleosides can not be used in plants as direct precursors of 5-methyl cytosine residues in DNA, but nucleosides of 5-methylcytosine may be deaminated to thymidine (or deoxythymidine) and subsequently incorporated into DNA.


[Estimation of mescaline and pellotine in Lophophora coulter plants (Cactaceae) by means of the oscillographic polarography]. Oscillographic polarography has been applied for the mescaline and pellotine estimation. These alkaloids produce in 0.5 N NaOH electrolyte a sharp peak within the cathode region of the oscillogram, each of them showing different potential. It makes possible to estimate them at a concentration of 5.10(-6) g/ml. All the forms of Lophophora williamsii were found to contain mescaline and lower content of pellothine, L. jourdaniana--to have equal content of both alkaloide, L. diffusa and L. fricii--to contain pellotine and only traces of mescaline. Plants grown in the greenhouse accumulated the same amount of alkaloids as native plants. Grafting on roodstock which does not produce essential amount of the alkaloids, does not affect the ability of Lophophora to synthesize mescaline and pellotine.


[Removal of surface-active substances from proteins by reversible immobilization on water-insoluble carrier]. Techniques for removing of surface-active substances from cysteine and cystine-containing proteins preliminarily immobilized by thiol-disulfide exchange on water-insoluble carrier, followed by protein transfer into solution, as examplified in sodium dodecyl sulfate, cetyltrimethylammonium bromide and bovine serum albumin are described. This procedure may be used to remove any sort of material non-covalently bound to protein.


[Oxidation of 4-N-(aniline)-5-methoxy-1,2-dioxybenzene via the mitochondrial respiratory chain]. A possibility of efficient oxidation of 4-N-(aniline)-5-methoxy-1,2-dioxybenzene (AMOBQH2) via the mitochondrial respiratory chain is demonstrated. The AMOBQH2 oxidation is accompanied by oxidative phosphorylation of ADP. The site of AMOBQH2 interaction is located in the region of cytochrome b, between rotenone and antimycin block. The bimolecular rate constant of AMOBQH2 oxidation using a steady-state approach is found to be 7.8.10(5) M(-1) S(-1). Differentiation of the two Amobqh2 oxidation pathways (mitochondrial one and that of auto-oxidation) and modelling of the auto-oxidation are carried out. Biochemical estimates of biological activities of AMOBQH2 analogues are discussed and proposed.


[Isoenzyme system of lactate dehydrogenase in human cells and tissues]. Comparative investigation of total activity and molar ratio of M-polypeptide chains (MM) of lactate dehydrogenase in diploid human cells with normal karyotype is carried out. It is found that the activity values in the passages studied (from 8 to 22) varies considerably, the variation coefficient Wn being 47.5%. Molar ratio of M-polypeptide chains of lactate dehydrogenase turned to be more stable (Wn=9.1%). Comparison of cell lines from different tissues and of respective initial tissues for MM values has revealed an increase of the content of lactate dehydrogenase M-polypeptide chains in embryonic cell lines with respect to initial tissues. Nevertheless, reliable differences in MM values between some cell lines of different tissue origin retain through 9--20 passages. A mechanism of lactate dehydrogenase biosynthesis and a system controlling the ratio of its subunits are discussed.


[Incorporation of radioactive protein and RNA precursors into animal liver polysomes under hydroxythiamine injection]. Effect of hydroxythiamine, B1 antivitamin, on the incorporation of labelled protein precursor (14C-hydrolysate of chlorella protein, 14C-leucine) and 14C-orotic acid into polysomes and RNA fractions of rat and mouse liver is investigated. Single subcutaneous injection of hydroxythiamine (400 mg/kg) is found to inhibit in 24 hours the synthesis of 28S and 18S rRNAs, polysome profile being unchanged and the incorporation of the label into protein being increased. Hydroxythiamine is proposed to induce the synthesis of TDP-dependent enzyme proteins responsing the decrease of their activity.


[The aminoacyl phosphatidyl glycerols of the plastid membrane system in the process of chloroplast biogenesis]. The composition of aminoacyl phosphatidyl glycerols in maize plastids at different stages of chloroplast differentiation has been studied. In the course of incubation of 14C amino acids or 14CO2 with maize and bean seedlings in vivo the 14C amino acids were incorporated preferably into the acid phospholipid fraction, forming O-esters of amino acids with phosphatidylglycerols. The rate of lipoamino acid compounds formation increased with the chloroplast differentiation and reached its maximum in the seedlings containing chloroplasts with a developed lamellar system. Changes in the amino acid composition of 14C aminoacyl phosphatidyl glycerols were observed at all stages of chloroplast ultrastructure development.


[Heterogeneity of leghemoglobin from yellow lupine nodules]. A possibility is demonstrated to separate summary lupine leghemoglobins (which are salted out within 55--90% of ammonium sulphate saturation) into Lb I and Lb II components by means of ionic exchange chromatography on DEAE-cellulose. Lb I is eluted at lower ionic strength buffer than LbII, and differs from the latter in the form and the size of crystals. Both components have the same electrophoretic mobility and contain N-terminal glycine. LbII and LbI precipitate under gradual salting out within 55--75% and 78--90% of saturation respectively.


[Inhibition of acetylcholinesterase hydrolysis of cationic substrates by the reaction product]. The kinetics of acetylcholinesterase-catalyzed hydrolysis of the two cationic substrates (I and II in Russian text) was analyzed by means of the integrated Michaelis equation (3). The constants kII, kcat Km and the enzyme-product complex dissociation constant Ki were determined. (Table 1). It was shown that acetylcholine (II) binds to to the enzyme active center more effectively than the alcohol product of its hydrolysis. In case of the pipecholine derivative (I) reversed situation occurs. The different dependence of the ester substrate and appropriate alcohol binding effectiveness upon the reagent structure indicates the dissimilar location of the molecules in the active center of acetylcholinesterase. Some structural implications of the enzyme active center were discussed.


[Analysis of brain chromatin subunit organization]. Autodigestion of chromosomal DNA does not take place during the brain nuclei incubation in the presence of Ca2+ and Mg2+. The kinetic of chromatin digestion in brain and liver nuclei by staphylococcal nuclease and the formation of DNP-fragments suggest that subnucleosomes are generated in both cases by digesting of monomer specific sites. This monomer contains 185--200 DNA base pairs and the most starting DNA going throughout it. However the quantity of nuclease-resistant DNA in brain chromatin is more and the rate of subnucleosome formation is less than in liver chromatin. Redigestion of isolated monomers of brain chromatin results in the appearance of subnucleosomes similar to those which are formed under limited digestion of nuclear chromatin. The incubation of brain nuclei in the presence of Ca, Mg-dependent endonuclease prepared from liver nuclei results in the appearance of fragment. DNA-spectra of these fragments are similar to those prepared under digestion of liver chromatin in situ. These data suggest definite resemblance of subunit organization in brain and liver chromatin.


[Skeletal muscle troponin and phosphorylation: a site of troponin T, that is phosphorylated by specific protein kinase]. A procedure is described of the isolation of protein kinase, which phosphorylates isolated troponin T with a rate, 5--30 fold exceeding the phosphorylation rate of other substrates (phosvitine, caseine, protamine sulphate, H1, H2A, H2b, H3, H4 histones). Troponin T-specific protein kinase transfers 0.85--0.95 moles of P per 1 mol of dephosphorylated troponin T. It phosphorylates only N-terminal acetylated serine residue, i. e. the site of troponin T structure, which is normally phosphorylated, when the whole troponin complex is isolated from skeletal muscles. Protein kinase is incapable to phosphorylate N-terminal serine residue in a mixture of triptic peptides of troponon T.


[Inhibition of the phosphorylase kinase activity by ATP analogs and their binding to the enzyme subunits]. The interaction between phosphorylase kinase (EC 2.7.1.38), isolated from rabbit skeletal muscles, and the ATP analogs with the modified triphosphate fragment: adenosine-5'-chloromethane pyrophosphonate (1), adenosine-5'-chloroethyl phosphate (2), adenosine-5'-bromethane pyrophosphonate (3), adenosine-5'-bromoethane phosphonate (4), adenosine-5'-chloroacetylaminomethane phosphonate (5), adenosine-5'-chloroacetylaminomethane pyrophosphonate (6) and adenosine-5'-chloromethane phosphonate (7), was studied. The compounds 1, 2 and 3 irreversibly inhibit the enzyme activity. In the presence of ATP the rate of inactivation is decreased. The radioactive compounds 1, 2 and 3 are stoicheometrically incorporated into the beta- and gamma-subunits of phosphorylase kinase. A correlation is shown to exist between the degree of the beta-subunit modification by compound 1 and the enzyme inactivation. The compounds 4, 5 and 6 inhibit the enzyme reversibly: in the presence of ATP complete protection of the enzyme activity is observed. The compound 7 does not affect the kinase activity; however, it binds itself to the beta-subunit of the enzyme. The binding of analogs 1 and 7 to the beta-subunit occurs at different sites. The data obtained are indicative of the catalytic role of the beta-subunit of phosphorylase kinase.