Improvement of polymorphonuclear leucocyte oxidative and bactericidal functions in chronic granulomatous disease with 4-amino-4'-hydroxylaminodiphenyl sulphone.

In an effort to restore oxidant-dependent capabilities to chronic granulomatous disease (CGD) polymorphonuclear leucocytes (PMN), we studied a dapsone derivative, 4-amino-4'-hydroxylaminodiphenyl sulphone (DDS-NOH), known to generate H2O2. After incubation of CGD PMN with 0.2 and 1.0 mM DDS-NOH for 30 min, the rate of glucose-1-14C oxidation via hexose monophosphate (HMP) shunt increased 2--4-fold and that of iodination of ingested zymosan particles 1.5--2.7-fold. Both effects could be further enhanced by superoxide dismutase (SOD) but inhibited by catalase. In three patients, 0.2 mM DDS-NOH improved in vitro killing of Staph. aureus. DDS-NOH 0.02 mM induced capping of Concanavalin A (Con A) receptor complexes suggesting interference by the drug with microtubule-associated function. Thus, optimal concentrations of DDS-NOH may be employed as an oxidant to improve metabolic and bactericidal functions of PMN from patients with CGD.


Plasma clearance and renal excretion of erythrocyte cryoprotectant hydroxyethylated amylopectin.

The value of low molecular weight-hydroxyethylated amylopectin (cryo-HES) as an extracellular cryoprotectant has been demonstrated in vitro. It is important that details of the intravascular persistence and urinary excretion be determined to compare with data already available as other grades of HES and with data on transfusion of cryo-HES cryoprotected blood. Following a single 400 ml (14% solution) infusion in man, the intravascular clearance of cryo-HES was well described mathematically by the equation: y = 3.30+6.49e-0.15kappa. The plasma concentration of cryo-HES fell to half its peak value in approximately 9.6 h. Approximately 20% of the total infused cryo-HES was excreted in the urine during the first post-injection hour, and 50% by 72 h. The ESR was not altered significantly by the presence of this material. The present study indicates that cryo-HES is eliminated rapidly and may thus be safe for transfusion to recipients of frozen blood.


Organelle pathology in primary and secondary haemochromatosis with special reference to lysosomal changes.

The organelle pathology of liver biopsy specimens from patients with either primary or secondary haemochromatosis was investigated by analytical subcellular fractionation in combination with enzymic microanalysis. The most striking changes were found in the lysosomes. Increased total activities but decreased latent activities of enzymes selectively localized to the high density population of lysosomes was demonstrated in the iron overloaded biopsies. Depletion of the iron, where possible, by venesection was accompanied by a return to normal of these changes. The other subcellular organelles, plasma membrane, endoplasmic reticulum, biliary canaliculi, mitochondria, peroxisomes and the low density population of lysosomes appear to be relatively unaffected. The minor changes demonstrated are similar to those seen in other forms of chronic liver disease. It is suggested that iron mediated lysosomal disruption is implicated in the pathogenesis of the tissue damage in haemochromatosis.


Non-specific serum iron in thalassaemia: an abnormal serum iron fraction of potential toxicity.

Iron binding in the sera of 35 patients with beta thalassaemia major and intermedia was studied. In patients receiving regular blood transfusions since infancy transferrin was completely saturated and about 2.7--7.1 mumol/l of the serum iron could be removed by dialysis or ultrafiltration in the presence of a chelating agent or by filtration on DEAE-Sephadex-catecholdisulphonic acid columns. In contrast, less than 1.0 mumol/l of transferrin bound iron was removed when subjected to the same procedures. The non-specific iron of thalassaemic sera could no longer be demonstrated after incubation with normal serum. These findings indicate that non-specific iron is a chelatable with normal serum. These findings indicate that non-specific iron is a chelatable compound which is readily available for transferrin binding. In view of the known toxicity of unbound iron, its identification in thalassaemic sera might be of relevance to the pathogenesis of tissue damage and the protective effect of iron chelating therapy in this disease.


Iron deficiency anaemia in newborn sla mice: a genetic defect of placental iron transport.

Newborn mice with X-linked anaemia (gene symbol sla) have lower haemoglobin levels at birth than normal and carrier mice but there is considerable overlap. Serial observations showed that the haemoglobin values of segregating male mice separate into a bimodal distribution of 42 d of age, and 50 d values were used to assign genotypes retrospectively. The anaemia in newborn sla mice is attributable to iron deficiency, since their total body iron is lower than in normal newborn mice, while their birth weights are almost identical. Haemoglobin levels at birth in normal, anaemic and carrier mice are also influenced by the mother's genotype and phenotype, and the haemoglobin value was progressively lower according to the sla gene dose of the mother. Materno-fetal iron transfer was examined by labelling pregnant carrier females with radioiron in various ways. When given as single or intermittent doses by injection no clearcut differences emerged in apparent iron transfer to anaemic as compared to non-anaemic fetuses. However, when radioiron was administered continuously in food a significant reduction in iron transfer to anaemic fetuses was demonstrated. The sla gene is already known to have a major effect in reducing iron transport in the small intestine. The present studies provide evidence of an analogous defect in placental iron transport.


Demonstration of the outer surface of freeze-etched normal human platelets: correlation with buoyant density.

The external surface of the normal human platelet shows clusters of spherical particles in a reticular pattern with smooth and regular surface membrane in adjacent areas. Labelling with cationic ferritin demonstrates a diffuse distribution of ferritin particles which stop at the fracture face of the intramembranous surface. The intramembranous surface is not labelled. Previously discrepant observations on the plasma membrane surface are probably due to different techniques of platelet preparation for freeze-etch electron microscopy. Density-dependent platelet sub-populations do not differ with respect to the ultrastructural features of their external surface and intramembranous portions of the plasma membrane. Based upon the relationship between platelet density and age, it appears that the ultrastructure of the normal platelet plasma membrane does not change with in vivo ageing.


Discrepancy between one-stage and two-stage assay of factor VIII:C.

Two methods (one-stage and two-stage) are commonly used for the assay of factor VIII clotting activity (VIII:C). We present collected data from seven separate studies of VIII:C assay which show that these methods do not give the same result when comparing concentrates and plasma. On average, two-stage assays detect 20% more VIII:C activity in concentrates as compared to plasmas than do one-stage assays.


Vaginal bleeding in childhood: a review of 51 patients.

A search of the medical records at the Hospital for Sick Children, Great Ormond Street and Chelsea Hospital for Women, London, revealed 51 cases of vaginal bleeding in children under the age of 10 occurring in the years 1962 through 1977. The bleeding was caused by some form of precocious puberty in 37 patients (73 per cent). Eight of these patients had cyclical vaginal bleeding in the absence of secondary sexual development or advanced bone age. Fourteen patients (27 per cent) bled because of a local lesion and six of these had a malignant neoplasm of the genital tract.


Tobacco chewing in pregnancy.

The effects of tobacco chewing by pregnant women were investigated. When compared with those who did not do so, tobacco-chewing mothers had a greatly increased stillbirth rate, a low male : female infant sex ratio and a major reduction in birth weight which was due in large part to early delivery.


The acute effect of smoking on intervillous blood flow of the placenta.

Smoking a standard cigarette caused an acute decrease in intervillous placental blood flow. This change normalized within 15 minutes. At the time intervillous blood flow was depressed, heart rate and blood pressure were elevated and remained so throughout the study period. Repeated decreases in the intervillous blood flow could explain growth retardation of the fetus and some other complications of pregnancy in women who smoke.


Effects of maternal cigarette smoking on the fetus and placenta.

In a large, prospective study, mothers who smoked during one pregnancy but not another had smaller infants in the pregnancy in which they smoked, irrespective of birth order and many other factors that affect fetal growth. Light cigarette smokers gained an average 90 g less at term than non-smokers, entirely due to the smaller size of the light smokers' newborn infants. Heavy smokers gained 533 g less than non-smokers, only one-third of which was due to the smaller size of the heavy smokers' newborn infants. As smoking increased, placentas enlarged and developed microscopic lesions characteristic of underperfusion from the uterus. This underperfusion was probably periodic rather than continuous because the smokers' decidua had few of the arterial lesions that are characteristic of chronic low blood flow. Pregnancies were a mean 1.5 days shorter in smokers than in non-smokers, due to more frequent amniotic fluid infections in the smokers.


The plasma half-life of placental hormones.

Blood was collected from 12 women following Caesarean section or normal delivery at term. The decline in plasma concentration of total oestriol, oestriol sulphate, oestriol glucosiduronate, unconjugated oestriol, human placental lactogen and pregnancy specific beta1 glycoprotein following delivery of the placenta was studied for 120 hours. The steroids and human placental lactogen fell very rapidly but pregnancy specific beta1 glycoprotein declined much more slowly. Analysis of the curves of puerperal decline suggests that the oestriol moieties are distributed in many compartments of the mother but that the proteins penetrate only to the plasma and the interstitial fluid.


Blood pressure in the arm and leg in late pregnancy.

Arm and leg blood pressures were measured by an indirect sphygmomanometric method in 12 women in late pregnancy and the results were compared with those in 12 matched non-pregnant women. In most of the pregnant women the diastolic blood pressure measured in the leg fell significantly with a change in posture from the supine to lateral position. In contrast, no significant change was observed in arm blood pressure with postural change after correcting for hydrostatic pressure difference. The interpretation of these findings is discussed with particular reference to methodology of blood pressure measurement.


Plasma prolactin in pregnancy induced hypertension.

In a prospective study, plasma prolactin concentration at 37 to 41 weeks gestation in 45 primigravidae showed a significant correlation with maximal rise in diastolic blood pressure between levels at 7 to 16 weeks and those measured between 28 weeks and delivery.


The effect of low haematocrit on serum human placental lactogen values.

Serial determinations of serum human placental lactogen (HPL) levels in women with haematocrit values of 28 per cent or less between 18 and 30 weeks gestation have been compared with the controls who had haematocrit values of 35 per cent or more. The women with low haematocrit values were found to have significantly raised HPL levels throughout the period of study. The placenta was significantly heavier in the anaemic group (P less than 0.01) but there was no significant difference in birth weights of the infants. It would seem that caution should be exercised in using HPL values as an index of placental function in anaemic women.


The effect of oral salbutamol on serum potassium and blood sugar.

Serum potassium and blood sugar concentrations were measured in six patients treated with oral salbutamol to inhibit premature labour. The fall in serum potassium and rise in blood sugar were variable and not present in every patient. The values returned to the normal range usually within 72 hours of continuous usage of the drug.


Changes in salivary peroxidase and polymorphonuclear neutrophil leucocyte enzyme activities during the menstrual cycle.

An elevation in salivary peroxidase activity has been found about the time of ovulation in 14 menstrual cycles in a total of six women. This peak coincided with the ovulatory luteinizing hormone (LH) and oestrogen peak in the four cycles in which endocrine studies were performed. Rises in some polymorphonuclear neutrophil leucocyte enzymes were also seen around ovulation. The possible use of changes in salivary peroxidase as a method for detection of ovulation is discussed.


Primary peritoneal pregnancy in association with intrauterine contraceptive devices: two case reports.

Two patients with a primary pelvic peritoneal ectopic pregnancy are reported. Both had been fitted with an intrauterine contraceptive device.


The value of cardiotocography in abruptio placentae. Case report.

The main indication for Caesarean section in cases of placental abruption is fetal distress. We report a patient in whom clinical examination, including auscultation of the fetal heart, suggested a mild haemorrhage, which would normally have been managed conservatively, but continuous fetal heart rate monitoring showed severe asphyxia, necessitating emergency Caesarean section at which a large concealed haemorrhage was found.


Quantitative relationship between cupping of the optic disc and visual field loss in chronic simple glaucoma.

The extent of cupping was measured from optic disc photographs of 300 patients with ocular hypertension and suspected or established chronic simple glaucoma, and their visual fields were recorded. The incidence of field defects increased as the vertical cup:disc ratio increased, a value of 0-70 for this ratio being a particularly useful guide to the presence or absence of field loss. There was a significant correlation between the extent of cupping and the size of the visual field. The vertical cup:disc ratio was the most useful quantitative index of cupping.


The optic disc in glaucoma, III: diffuse optic disc pallor with raised intraocular pressure.

Ten patients (14 eyes) with chronic simple glaucoma are described, each with features atypical for this disease and more suggestive that each had suffered an acute ischaemic optic neuropathy. Diagnosis of an acute ischaemic optic neuropathy in these cases is important, as treatment of ocular hypertension in the contralateral 'normal' eye may prevent a similar attack. Optic nerve disease and visual loss in chronic simple glaucoma probably occur as a result of several different mechanisms. Sudden field loss following an acute ischaemic optic neuropathy may be only one of them.


Ocular hypertension: a comparative follow-up of black and white patients.

Forty-four eyes in 25 black patients are compared with 92 eyes of 47 white patients in a population with ocular hypertension followed up for 1 to 12 years in a glaucoma clinic. The black patients present at a younger age than the whites and their mean initial intraocular pressure is significantly higher. The fact that more black ocular hypertensives developed glaucoma--8 eyes (18.1%) in the black population as compared with 5 eyes (5.4%) in the white population--is attributed to these two differences.


The effect of standardised submaximal work load on intraocular pressure.

A relatively small decrease in intraocular pressure secondary to submaximal work can be demonstrated when factors other than the work load itself are eliminated--diurnal variation, body position, and the effect of repeated examinations. A feedback mechanism that causes a return of IOP towards its normal value after work-induced decreases is postulated.


Effects of cigarette smoking on intraocular pressure and vision.

In a survey of 426 patients of Toronto ophthalmologists the correlations of reported smoking habits with (1) intraocular pressure and (2) visual acuity were examined. Average tonometer readings were closely similar in smokers, ex-smokers, and non-smokers, and multiple regression analysis showed similar age coefficients for the 3 categories. However, in women (but not in men) there was also a significant coefficient related to total cigarette exposure (puff-pack-years). It is suggested that this was due to recent smoking rather than a permanent effect on tonometer pressures, pointing the need to standardise this aspect of ocular pressure measurements. Visual acuity showed similar age coefficients in smokers, ex-smokers, and non-smokers, with no independent effect of exposure to cigarette smoke.


Effect of posture on the intraocular pressure of patients with retinal vein obstruction.

The effect of change of posture on the level of the intraocular pressure was studied in 22 patients who had suffered 'occlusion' of a central retinal vein, 14 patients with occlusion of a tributary of the central vein, and 23 normal subjects. The results showed a significant difference in behaviour between subjects in the control group and both groups of patients with retinal vein occlusions. In general it appears that the normal person maintains a relatively rigid control of ocular tension. The eyes are maintained at similar pressures which vary very little with change in posture. In contrast, the patient with a retinal vein occlusion (whether central or tributary) does not appear to have a similar degree of control. The eyes are not maintained at closely similar pressures, and each eye shows a change of pressure with change of posture which is considerably greater than that shown by the eyes of normal persons. The significance of these results is discussed with particular reference to the clinical association of retinal vein occlusion and chronic simple glaucoma, in which latter condition a similar lack of postural control of the intraocular pressure is found.


Clearance of debris from the iris through the drainage angle of the rabbit's eye.

The fate of particulate material on entering the drainage angle of the rabbit's eye was investigated by transmission electron microscopy. The infiltrate was produced by photocoagulation of the iris. Initially it consisted of plasma, connective tissue and cellular debris, fibrin, and red blood cells. Later, free melanin granules, polymorphs, macrophages, and iris clump cells were all seen to enter the drainage system. Native meshwork cells were found to be active phagocytes and began to degrade various components of the infiltrate within 2 hours of the formation of the iris lesions. Corneal endothelial cells, the cells which cover the iris pillars, and the adventitial cells of the ciliary body were less active but were also phagocytic.


The perimeter as a monitor of glaucomatous changes.

A small group of glaucoma patients with scotomata were compared with normal coevals. Plots were obtained of the error of each measurement as a function of retinal illumination. A statistical analysis shows the limitations of perimetry as a monitor of the progress of glaucoma. The significance of the data is discussed.


Isolation of Chlamydia psittaci from a patient with interstitial keratitis and uveitis associated with otological and cardiovascular lesions.

A case history of a 15-year-old schoolgirl with fluctuating bilateral uveitis, bilateral stromal keratitis with vascularisation, and bilateral deafness associated with tinnitus and balance disturbance is described. Three years from the onset of her clinical signs she died of a sudden cardiac arrest caused by endocarditis associated with valvular and arterial lesions. Chlamydia psittaci was isolated from her conjunctiva. In her blood type-specific antichlamydial antibody at a level of 1/64 against her own isolate was detected. The clinical findings in this patient were suggestive of a Cogan's syndrome. It is highly probable that the chlamydia isolated from the eyes was responsible for her various lesions.


Immunological investigations in post-traumatic granulomatous and non-granulomatous uveitis.

Histopathological and immunological investigations have been performed in 16 cases of post-traumatic granulomatous (10) and non-granulomatous uveitis (6). Most cases of sympathetic ophthalmitis showed evidence of cell mediated immunity to uveoretinal antigens, though it was not possible to make a definitive diagnosis on immunological grounds alone. Three patients with post-traumatic non-granulomatous uveitis showed a positive immunological response to ocular antigens, and 2 of these later developed clinical evidence of sympathetic ophthalmitis, which suggests that post-traumatic non-granulomatous uveitis in such cases may represent a presympathetic (i.e., incipient) or modified stage of the disease.


Canaliculitis with isolation of Pityrosporum pachydermatis.

A case of canaliculitis with obstruction of the lacrimal canaliculus and accumulation of conglomerates is described. Pityrosporum pachydermatis was cultured from the conglomerates. To the best of our knowledge this organism has not been previously implicated in such infections. Cure was obtained by treatment with nystatin administered topically and as an irrigation of the lacrimal pathways. The possibility that the pathogenesis of the canaliculitis and obstruction of the lacrimal pathways lies in decreased function of the lacrimal pump is discussed.


Bilateral acute retinal necrosis.

In this paper 4 patients are described who had bilateral symmetrical confluent retinal swelling followed by apparent necrosis and sloughing of the retina into the vitreous. The disease was accompanied by signs of uveitis and the clinical appearance suggested inflammation rather than infarction as the pathogenic mechanism. No systemic abnormalities were found by which the aetiology could be identified.


Macular abnormalities in the reattached retina.

Sixty-six patients in whom the macula was detached before surgery were observed for at least 1 year after retinal reattachment. Macular abnormalities were recognised clinically in most patients with severely reduced vision. Failure of retinal receptor regeneration or receptor misalignment may account for visual reduction in a minority of patients but may be less important as a cause of reduced visual acuity than was previously supposed. This study confirms that the visual prognosis is related to the duration of the detachment before surgery, and patients with a macular detachment for 2 months or longer are likely to suffer persistently reduced vision.


Placoid pigment epitheliopathy and Harada's disease.

Twenty-six patients are described who suffered from acute bilateral multifocal pigment epithelial disease. In 7 the pattern of disease was indistinguishable from acute posterior multifocal placoid pigment epitheliopathy, while in 8 it was indistinguishable from Harada's disease. In a further 9 cases the pigment epithelial disease was associated with serious detachment of the retina simulating Harada's disease but without systemic symptoms; spontaneous resolution occurred within a few days, and there was no recurrence. One additional case had short-lived disease with detachment initially, but this was followed by severe recurrence, and the last patient had serious detachment in 1 eye but not the other. When seen as a whole these patients appeared to represent a continuous spectrum of disease making it difficult to define boundaries between one condition and another. The difficulties in distinguishing diseases according to morphology alone are emphasised.


Fundus changes in incontinentia pigmenti (Bloch-Sulzberger syndrome): a case report.

A case of incontinentia pigmenti is reported with fundus changes in 1 eye. She had microaneurysms temporal to the macula, with an abnormal branch of inferior temporal vein. There was extensive retinitis proliferans in the upper temporal equatorial region, which showed leakage on fluorescein angiography.


Television slit-lamp biomicroscopy.

The basic requirements for performing television slit-lamp biomicroscopy are outlined, and the methods of demonstrating particular features of ocular anatomy and ocular disease are discussed. The technique has a particular role in teaching in the clinical setting.


B-scan ultrasonography of the anterior segment of the eye.

B-scan ultrasonograms of the anterior segment were performed on selected patients. The difficulties in imaging the anterior segment are discussed and the value of this technique is demonstrated by illustration of B-scans in different pathological conditions. This technique can often provide important information and allow an early and accurate prognosis.


Cine photography and video recording of anterior segment fluorescein angiography.

A description is given of apparatus and technique for carrying out cine photography and video recording of anterior segment fluorescein angiography. We found cine best for single-frame analysis and video tape recording less expensive.


Penetration of tobramycin sulphate into the human eye.

Tobramycin sulphate, a new aminoglycoside antibiotic, was injected intramuscularly (80 and 100 mg) and subconjunctivally (10 mg) into patients, and the concentrations of the drug in serum and aqueous humour were determined 1 hour after the injection by an agar wall diffusion plate method. The intramuscular administration of tobramycin produced insignificant and non-inhibitory concentrations in the aqueous humour, while therapeutically effective levels of the antibiotic appeared in the aqueous after subconjunctival administrationof tobramycin (mean: 18.9 microgram/ml). These data suggest that tobramycin sulphate may be of value in the treatment of ocular infections.


Reversible dissociation of the alpha beta dimer of tubulin from bovine brain.

The reversible, concentration-dependent dissociation of the alpha beta dimer of bovine brain tubulin (purified by phosphocellulose chromatography) has been demonstrated by equilibrium ultracentrifugation. The dissociation constant is approximately 8 X 10(-7) M at 4.6 degrees C in PM buffer (0.1 M piperazine-N, N'-bis(2-ethanesulfonic acid), 2 mM ethylene glycol bis (beta-aminoethyl ether)-N, N'-tetraacetic acid, 1 mM MgSO4, 0.1 MM guanosine triphosphate, 2mM dithioerythritol, at pH 6.9). This result was confirmed by observation of an appropriate dependence of the sedimentation coefficient of very dilute (is less than 0.5 mg/mL) tubulin on its concentration. Small zone gel filtration experiments on Bio-Gel P-150 also demonstrated an increase in peak elution volume with decreasing column load concentration. Reversibility of the dissociation was demonstrated directly by sedimentation velocity and gel filtration ion experiments on tubulin reconcentrated from dilute solution by pressure ultrafiltration. Control experiments accompanying the sedimentation equilibrium experiments showed that this tubulin retained, under the conditions of the experiments, both its ability to form microtubules and more than 70% of its initial colchicine-binding activity.


Identification of the transferrin receptor of the rabbit reticulocyte.

Reticulocytes were separated on the basis of density by isopycnic centrifugation in dextran gradients. This parameter was shown to correlate with the degree of maturity of the cells. Lactoperoxidase iodination of cells of different densities followed by sodium dodecyl sulfate (NaDodSO4) electrophoresis revealed a 190 000 molecular weight protein which was well labeled in early reticulocyte membranes. Efficiency of labeling decreased as the cells increased in density, and high specific activity iodination of mature erythrocytes did not result in the labeling of any species near this molecular weight. Inclusion of rabbit transferrin prior to the iodination procedure resulted in a specific loss of labeling of this 190 000 molecular weight species. When steps were taken to clear endogenous transferrin from the membranes, the labeling of this species was enhanced. These observations are consistent with the concept that transferrin can block the lactoperoxidase catalyzed iodination of this membrane protein by specifically associating with it. Coomassie blue and periodic acid-Schiff staining of NaDodSO4 gels of these membranes revealed that a glycoprotein present at this molecular weight is lost during the course of reticulocyte maturation. It is concluded from these studies that a glycoprotein of molecular weight 190 000 constitutes the transferrin receptor in the reticulocyte membrane.


Circular dichroism studies on Ca2+-dependent protein modulator oxidized with N-chlorosuccinimide.

The structural features and Ca2+-binding properties of native and N-chlorosuccinimide-oxidized modulator protein were compared by circular dichroism. In the presence of Ca2+,the far-UV spectra of native and oxidized modulator protein are virtually indistinguishable, indicating that oxidation of surface methionine residues does not alter the overall conformation of the molecule. In the absence of Ca2+, however, the circular dichroism spectra of native and oxidized modulator are different with calculated helical contents of 40% and 26%, respectively. As judged by circular dichroism titration studies, the native modulator contains both high-(Kd = 1.9 X 10(-7) M) and low-affinity (Kd = 4 X 10(-4) M) Ca2+-binding sites, whereas the modified modulator appears to possess only low-affinity sites (Kd = 3.8 X 10(-4) M). The reduced secondary structure in Ca2+-free oxidized modulator protein may account for the absence of high affinity Ca2+ binding sites.


Estimation of lateral species separation from phase transitions in nonideal two-dimensional lipid mixtures.

The two-dimensional statistical mechanical equations necessary to analyze the phase diagram of a nonideal lipid mixture are derived. The Prigogine approximation of the combinatorial term is used in the maximum term of the partition function which contains two parameters that describe the degree of nonideality in the liquid crystal and gel phases. These parameters are used to calculate the mole fraction of like-like and like-unlike lipid interfaces which describe the lateral species separation of like lipids in each phase. Data for several temperatures and compositions of the DMPC/DPPC, DPPC/DSPC, and DMPC/DSPC mixtures are analyzed.


Active transport of L-glutamate by membrane vesicles isolated from rat brain.

Membrane vesicles, isolated after osmotic shock of synaptosomal rat brain fractions, actively accumulate L-glutamate. This process requires the presence of external sodium ions and internal potassium ions and is driven by artifically imposed ion gradients as the sole energy source. Either an Na+ gradient (out is greater than in) or a K+ gradient (in is greater than out) or both can be utilized to concentrate L-glutamate inside the vesicles. Transport is enhanced by valinomycin or by external thiocyanate ions and is about 50% inhibited by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone. This transport thus appears to be stimulated by a membrane potential (interior negative). The glutamate transporter, the Km of which has been determined to be 3 micrometer, is specific for L-glutamate. The transport process is unaffected by ouabain but is strongly inhibited by p-hydroxymercuribenzoate as well as by nigericin, which collapses the energizing ion gradients across this membrane. Unlike the sodium dependent, but potassium independent active accumulation of gamma-aminobutyric acid in these vesicles (Kanner, B.I. (1978) Biochemistry 17, 1207) active L-glutamate uptake is not dependent on the presence of small monovalent anions in the external medium. The results provide direct evidence for Na+-coupled electrogenic active L-glutamate transport by rat brain membrane vesicles. The dependence on internal potassium ions is discussed.


Mechanism and kinetics of iron release from ferritin by dihydroflavins and dihydroflavin analogues.

Dihydroflavins reductively release iron rapidly and quantitatively from purified horse spleen or horse heart ferritin. The NAD(P)H:flavin oxidoreductase from Beneckea harveyi is used to generate a constant concentration of dihydroflavin permitting a continuous assay for complete iron release. Sepharose-linked dihydroflavins are not competent to release ferritin iron, demonstrating that the dihydroflavin must pass through the channels of the protein shell prior to iron reduction. Several experiments fail to show any specific flavin binding site, though dihydroflavins do display saturation kinetics with very high apparent Km's. The rates of iron release by a number of dihydroflavin analogues show that the electron transfer is significantly rate determining in iron release by dihydroriboflavin, while diffusion of the dihydroflavin through the protein channel is slow in the release of iron by dihydroFMN. The rate of iron release is also dependent on the initial content of iron, having a maximum at 1200 iron atoms per ferritin.


Charge shift optical probes of membrane potential. Theory.

The chromophores of a series of known and unknown probes of membrane potential are subjected to molecular orbital calculations. These calculations are used to characterize the charge distribution and excitation-induced shift of electron density in the chromophores. This is used to predict or rationalize the magnitude of an electrochromic response to membrane potential. The predictions are consistent with more rigorous calculations on several selected systems as well as with the available experimental data. Emerging from the survey is a variation on previously considered forms of electrochromism involving a simple migration of the charge in an ionic chromophore. The intrinsic amphipathic structures of some of these systems may make them especially well suited for the construction of well oriented, highly responsive probes. A particularly promising charge-shift chromophore is the 4-(p-aminostyryl) pyridinium cation.


Isolation and identification of 5, 6-epoxyretinoic acid: a biologically active metabolite of retinoic acid.

A highly biologically active metabolite of retinoic acid (8III) has been isolated in pure form from intestinal mucosa of vitamin A deficient rats given [3H]retinoic acid. This metabolite has been positively identified as 5, 6-epoxyretinoic acid based on the ultraviolet absorption spectrum and mass spectrum of its methylated derivative. This identification was confirmed by cochromatography of the methylated metabolite and synthetic methyl 5, 6-epoxyretinoate on reverse-phase and straight-phase high-pressure liquid chromatography. The 5, 6-epoxyretinoic acid is a true in vivo generated metabolite of retinoic acid and not an artifact of the isolation procedure. In addition, 5, 8-oxyretinoic acid previously isolated in this laboratory from intestinal mucosa was probably generated from 5, 6-epoxyretinoic acid by the acidic conditions used in the extraction and isolation of the 5, 8-oxyretinoic acid.


Kinetic studies on electron transfer and interaction between nitrogenase components from Azotobacter vinelandii.

Kinetic properties of electron transfer by nitrogenase of Azotobacter vinelandii are dependent on the concentration of the two components of nitrogenase. An excess of the MoFe protein inhibits electron transfer in a distinctive manner, and the inhibition is reversed by increasing levels of reductant. The saturation curve for Fe protein is hyperbolic, indicating that only one Fe protein molecule per MoFe protein is required for full activity in ATP hydrolysis and electron transfer. These results can be interpreted on the basis of a complex between the Fe protein and the MoFe protein that dissociates rapidly during turnover. Both 2:1 and 1:1 complexes (Fe-MoFe) are active. Dithionite appears to be a relatively poor reductant for nitrogenase from Azotobacter vinelandii, whereas azotobacter flavodoxin is much better. In the presence of the flavodoxin it is possible to increase the specific activity of the Fe protein more than 50% relative to its activity with dithionite alone as a reductant; specific activities greater than 3000 nmol of C2H4 formed min(-1) (mg of Fe protein)(-1) have been observed.


Rapid, single-step purification of restriction endonucleases on cibacron blue F3GA-agarose.

After sonication and high-speed centrifugation, crude extracts of B. amyloliquefaciens, P. alcalifaciens, X. holicola, and B. globiggi were adsorbed on the dye Cibacron blue F3GA convalently cross-linked to agarose. The restriction endonucleases BamHI, PalI, XhoI, and BglI together with BglII were isolated by elution of the dye column with linear gradients to 0.5 M NaCl. The enzymes so purified were free of contaminating nucleic acids and other nucleases and were sufficiently concentrated for direct, specific DNA hydrolysis.


Postproline cleaving enzyme: kinetic studies of size and stereospecificity of its active site.

Postproline cleaving enzyme [EC 3.4.21.-] has recently been purified from lamb kidney and tentatively identified as a serine endopeptidase with a high specificity for proline-containing peptides. The interaction of postproline cleaving enzyme with peptide substrates and competitive inhibitors has been studied in an effort to explore the size and stereospecificity of the active site of the protease. The substrates and inhibitors included proline-containing peptide amides, p-nitrophenyl esters, and free acids with increasing numbers of amino acid residues and residues of L and D configuration. Oligopeptides of alanine, which can also be recognized by the protease, were also tested as substrates. This series included Ala3, Ala-D-Ala-Ala, Ala-Ala-D-Ala,Z-(Ala)3, Ala4 through Ala6. The contribution of each of the three amino acid residues flanking the primary specificity site (S1) of postproline enzyme to such kinetic parameters as Km, Kcat, and Kcat/Km in the case of substrates and Ki with inhibitors was determined. The results suggest that postproline cleaving enzyme has an extended substrate binding region in addition to the primary specificity site, S1. It seems to be comprised of three sites located at the amino-terminal site (S1, S2, and S3) and two sites at the carboxyl site from the catalytic point (S1', S2'). High stereospecificity was observed for subsites S1, S2, and S1'.


Affinity labeling of histamine N-methyltransferase by 2',3'-dialdehyde derivatives of S-adenosylhomocysteine and S-adenosylmethionine. Kinetics of inactivation.

S-Adenosyl-L-methionine (AdoMet), S-adenosyl-L-homocysteine (L-AdoHcy), and related ribonucleosides have been oxidized with periodic acid to the corresponding 2',3'-dialdehydes. Both AdoMet dialdehyde and L-AdoHcy dialdehyde were observed to rapidly and irreversibly inactivate histamine N-methyltransferase (HMT). Equally active as an irreversible inhibitor was S-adenosyl-D-homocysteine dialdehyde (D-AdoHcy dialdehyde), which is consistent with the known affinity of HMT for S-adenosyl-D-homocysteine (D-AdoHcy). Other analogues of AdoHcy dialdehyde (S-adenosyl-L-cysteine dialdehyde, S-adenosyl-L-homocysteine sulfoxide dialdehyde, and adenosine dialdehyde) also produced irreversible inactivation of HMT, but at predictably slower rates. The corresponding acyclic 2',3'-ribonucleosides, which were obtained by NaBH4 reduction of the ribonucleosides dialdehydes, were found to be very weak, reversible inhibitors of HMT. Kinetic analysis of the inactivation of HMT produced by L-AdoHcy dialdehyde, AdoMet dialdehyde, and D-AdoHcy dialdehyde suggested mechanisms involving the formation of dissociable enzyme-inhibitor complexes prior to irreversible inactivation. Studies using L-[2,8-3H] AdoHcy dialdehyde revealed that incorporation of radioactivity into HMT closely paralleled the loss of enzyme activity. The results of these studies indicate that L-AdoHcy dialdehyde, D-AdoHcy dialdehyde, and AdoMet dialdehyde are affinity labeling reagents for HMT.


Interferon induction and its dependence on the primary and secondary structure of poly(inosinic acid).poly(cytidylic acid).

The synthetic interferon (IF) inducer rIn.rCn was modified by substituting the ribosyl residues with either their corresponding deoxy(dC or dI) or 2'-O-methyl analogues (mC or mI). The polynucleotide duplexes of these analogues are inactive as IF inducers. Circular dichroism (CD) studies revealed that, while the deoxy substitution produces significant changes in the conformation of rIn.rCn, the 2'-O-methyl substitution produces no detectable change. Biological competition experiments indicated that the methylated duplexes mIn.rCn, rIn.mCn, and mIn.mCn all compete with rIn.rCn for IF induction, while the deoxy duplexes dIn.rCn and rIn.dCn do not. These results are consistent with those predicted from the CD data. Copolymer duplexes (mI,rI)n.(mC,rC)n of varying degrees of methylation and residue clustering were also evaluated for IF induction in human fibroblasts. The IF-inducing capabilities of these duplexes correlated highly with the presence of clusters containing six or more consecutive ribosyl residues. These combined observations suggest that interaction of rIn.rCn with the cell in the induction process may occur in a biphasic manner involving first the topological recognition of a large segment of the RNA to allow for proper binding to the putative cellular receptor, followed by recognition of a much smaller region of the RNA corresponding to 6--12 consecutive ribosyl residues (0.5 to 1 helical turn) which is responsible for the triggering of the induction process.


Comparison of DNA polymerase alpha and delta from bone marrow.

DNA polymerases alpha and delta from rabbit bone marrow were purified to specific activities greater than 30 000 nM TMP incorporated (mg of protein)(-1)h(-1). alpha is quantitatively predominant. alpha and delta have the same reaction requirements and are both similarly sensitive to N-ethylmaleimide. The primary functional distinction is the association of 3' to 5' exonuclease activity with delta. Sedimentation coefficients obtained from zone sedimentation in glycerol gradients and Stokes radii values from gel filtration allow the calculation of true molecular weight and frictional ratios. alpha exhibits a bimodal pattern, sedimenting at 6 and 8 S on glycerol gradients and demonstrating components corresponding to 40.5- and 65-A Stokes radii upon gel filtration. The calculated molecular weights of the two forms of alpha are 100 000 and 215 000; the frictional ratios are 1.34 and 1.65. This and other data suggest a possible monomer-dimer relation. In contrast, delta sediments uniformly at 6.5 S and also behaves uniformly upon gel filtration at 4595 A. The molecular weight of delta is distinct at 122 000; its frictional ratio is 1.39. Because of similarities of the DNA polymerizing activities of both forms of alpha and of delta, it is postulated that alpha is derived from delta by structural modification, resulting in a decrease in molecular weight, the tendency to aggregate as dimers, and a concomitant loss of 3' to 5' exonuclease activity.


Binding of hedamycin to deoxyribonucleic acid and chromatin of testis and liver.

The binding of the antibiotic hedamycin to DNA was evaluated by density gradient centrifugation in CsCl to determine the type I binding, which is essentially irreversible at high and low ionic strength. Exhaustive dialysis at low ionic strength was used to determine the sum of type I and type II binding (irreversible at low ionic strength but reversible at high ionic strength). The maximum ratio of hedamycin to DNA nucleotides (rf) is 0.1 for type I and 0.1 for type II binding to free DNA, but these ratios decrease to 0.07--0.08 in chromatin of rat liver and a testis fraction (spermatogonia plus primary spermatocytes). The rf for type I binding of hedamycin to monomeric nucleosomes of this testis fraction is considerably less than maximum binding to polymeric nucleosomes or chromatin, suggesting that hedamycin binds more effectively to "linker" DNA than to DNA attached to the core of the nucleosomes. Hedamycin binding to the chromatin of the spermatid fraction of testis is greatly decreased in comparison with chromatin of early stages; this correlates with the change from nucleohistones to nucleoprotamines at the middle to late spermatid stages of spermiogenesis.


Interaction of calcium and manganese ions with apoconcanavalin A and sugar binding.

The interaction of apoconcanavalin A (apo-Con A) with Mn2+ and Ca2+ was studied at 25 degrees C using fluorescence stopped flow. The reaction was monitored using 4-methylumbelliferyl alpha-D-mannopyranoside whose fluorescence is quenched on binding to the metalloproteins. At pH 5.0 entry of Mn2+ into apo is second-order (rate constant = 1.2 x 103 M(-1) s(-1)); at higher pH the rate constant is greater than 104 M(-1) s(-1). Reaction of excess Ca2+ with Mn(Con A) is pseudo-first-order with kobsd = Kk[Ca2+](1 + K[Ca2+])(-1). This is interpreted as rapid formation of unlocked MnCa(Con A), with a formation constant K = 3.5 x 102 M(-1), which transforms (k = 0.026 s(-1)) to a locked form, indistinguishable from native. At pH 6.4 and 7.2, K greater than or equal to 103 M(-1) and k = 0.043 and 0.050 s(-1), respectively. Ca(Con A) and Mn(Con A) precipitate glycogen and bind to 4-methylumbelliferyl alpha-D-mannopyranoside as effectively as native protein at pH 7.2. Treatment of the Ca or Mn forms with EDTA produces an apo form with a small binding capacity, which it loses slowly.


Conformation as the determinant of saccharide binding in concanavalin A: Ca2+-concanavalin A complexes.

The existence of two conformational states of concanavalin A (Con A) with different metal ion binding properties has been recently demonstrated (Brown, R. D., Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883). Introduction of Mn2+ to the S1 site and Ca2+ to the S2 site of apo-Con A was shown to induce a conformational change in the protein, ascribed to a cis-trans isomerization of a peptide bond in the secondary structure, which results in extremely tight binding of the metal ions. This induced conformation is referred to as "locked" and the initial conformation as "unlocked". The locked ternary complex is identical with the native protein. In the present paper, we report evidence for the formation of a relatively stable, locked, ternary Ca2+-Con A complex that possesses properties similar to those of native Ca2+-Mn2+Con A. The experimental technique involves measurement of the magnetic field and time dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons in solutions of Ca2+-Con A, after the addition of Mn2+ ion which slowly bind to the protein. The kinetic data can be fit by a model for Ca2+ interactions with Con A which indicates that Ca2+, in the absence of Mn2+, can bind at both the S1 and S2 sites of the protein and, furthermore, can induce the protein to undergo the unlocked to locked conformational transition. In terms of this model, the time-dependent binding of the Mn2+ ions is due to replacement of Ca2+ ions at the S1 sites in the locked protein. The off-rate of Ca2+ from the S2 site of the locked ternary Ca2+-Con A complex is much greater than that from the locked Ca2+-Mn2+-Con A complex. From the effects of added alpha-methyl D-mannopyranoside on the rate of replacement of Ca2+ by Mn2+ at the S1 site of the locked ternary Ca2+-Con A complex, it is concluded that the latter complex binds saccharides as strongly as the locked Ca2+-Mn2+-Con A complex. In addition, analysis of the data indicates that apo-Con A in the locked conformation binds alpha -methyl D-mannopyranoside with approximately 7% of the affinity of the fully metallized locked form of the protein. This strong saccharide-binding activity of locked apo-Con A, compared with that of the unlocked apo-Con A, was further demonstrated by equilibration of unlocked apo-Con A with alpha-methyl D-mannopyranoside, which resulted in the formation of the locked apo-Con A-saccharide complex. These results demonstrate that it is the locked conformation of Con A that is primarily responsible for saccharide-binding activity, and that the function of the bound metals is primarily to maintain the protein in the locked conformation.


Selective methyl esterification of erythrocyte membrane proteins by protein methylase II.

Methyl esterification of erythrocyte membrane proteins have been demonstrated by incubating the isolated membrane with purified protein methylase II (S-adenosyl-methionine:protein-carboxyl O-methyltransferase, EC 2.1.1.24) and S-adenosyl-L-[methyl-14C]methionine. Methyl esterification of membrane-bound proteins occurred selectively to proteins corresponding to bands 3 (mol wt 97 000), 4 (mol wt 75 000), and 4.5 (mol wt 48 000) [designated according to Steck, T. L. (1974), J. Cell Biol. 62, 1] as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mild alkali treated depleted vesicles which lacked bands 1, 2, 5, and 6 had a higher methyl accepting capacity; 500 pmol of methyl groups/mg of depleted vesicle proteins vs. 200 pmol of methyl groups/mg of intact membrane proteins. Alkali-extractable membrane components were not methylated.


Conformational preferences of amino acids in globular proteins.

In a previous paper [Levitt, M., and Greer, J. (1977), J. Mol. Biol. 114, 181--239], an objective compilation of the secondary-structure regions in more than 50 different globular proteins was produced automatically. In the present paper, these assignments of secondary structure are analyzed to give the frequency of occurrence of the 20 naturally occurring amino acids in alpha helix, beta sheet, and reverse-turn secondary structure. Nineteen of these amino acids have a weak but statistically signficant preference for only on type of secondary structure. These preferences correlate well with the chemical structure of the particular amino acids giving a more objective classification of the conformational properties of amino acids than available before.


13C Nuclear magnetic resonance studies to the binding of isocyanides to various hemoglobins and myoglobins.

Interactions between ethyl and isopropyl isocyanides and various hemoglobins and myoglobins have been studied by 13C nuclear magnetic resonance. The results indicate that the chemical shift of the bound isocyanide depends on the structure of the hemoglobin subunit or myoglobin. The resonances exhibited by isocyanides bound to myoglobin are sensitive to pH in contrast to the situation with rabbit and human hemoglobins. beta subunits of opossum, rabbit, and human hemoglobins show a significantly greater preferential affinity for CO relative to EIC than do alpha subunits which have allowed the assignment of resonances. Rabbit, human, and opossum hemoglobin subunits bind ethyl isocyanide without observable preferences and an excess of DPG does not appear to affect this random order of ligation. In contrast, an excess of IHP seems to cause preferential ligation of the alpha subunits in these hemoglobins. The results have been used to gain insights into the differing characteristics of the ligand binding pockets of these various hemoglobins.


An IgG2a-producing variant of an IgG2b-producing mouse myeloma cell line. Structural studies on the Fc region of parent and variant heavy chains.

The IgG2b-producing MPC 11 mouse myeloma cell line has yielded a number of variants which synthesize heavy chains characteristic of a different immunoglobulin subclass, IgG2a, as previously shown by serology, peptide maps, and assembly profiles. We have studied the Fc regions of the IgG2a protein synthesized by one variant, ICR 9.9.2.1, and of the IgG2b protein synthesized by MPC 11 and compared them to MOPC 173, an IgG2a protein of known sequence. We analyzed the Fc regions of the three immunoglobulins by several analytical techniques, such as immunoelectrophoresis of papain digests, NaDodSo4-polyacrylamide gel electrophoresis analyses of Fc CNBr fragments, and comparative ion-exchange chromatography of radiolabeled tryptic and chymotryptic Fc peptides. In addition, Fc CNBr fragments of the variant gamma2a and MPC 11 gamma2b molecules were isolated and subjected to amino acid analysis and partial sequence determination. From these data, we concluded that the Fc fragment of ICR 9.9.2.1 is most probably identical to that of MOPC 173 and different from the parental gamma2b Fc fragment. A number of residue positions which discriminate between gamma2b and gamma2a sequences are described. In two of three segments sequenced, gamma2b and gamma2a molecules share more identical residues than either shares with another mouse subclass, gamma1.


Glutamate metabolism in relation to glutamate transport in kidney cortex mitochondria of rabbit.

1. The metabolism of glutamate was followed by measurements of phosphoenolpyruvate production, aspartate synthesis and ammonia release, whereas the transport of glutamate across the inner membrane of kidney cortex mitochondria was studied using an oxygen electrode and the swelling technique. 2. When added separately, avenaciolide and aminooxyacetate only partially inhibited both State 3 and uncoupled respiration of the mitochondria, as studied in the presence of glutamate as substrate. In contrast, the addition of both inhibitors to the reaction medium resulted in an almost complete inhibition of glutamate oxidation. 3. Swelling of kidney mitochondria in an isosmotic solution of ammonium glutamate was accelerated by uncoupler and inhibited by avenaciolide, while the swelling of mitochondria in potassium glutamate was stimulated by valinomycin and inhibited by uncoupler. 4. When glutamate was used as the sole substrate, inhibition of aspartate formation by aminooxyacetate resulted in a stimulation of both ammonia release and phosphoenolpyruvate production. In contrast, with glutamate plus malate as substrate an elevation of the rate of glutamate deamination on the addition of aminooxyacetate was accompanied by an inhibition of phosphoenolpyruvate synthesis in both State 3 and uncoupled conditions. 5. In the presence of valinomycin to induce K+-permeability a marked enhancement of glutamate deamination was accompanied by a significant inhibition of glutamate transamination. 6. Based on the presented results it was concluded that in rabbit renal mitochondria utilizing glutamate as substrate the rate of ammonia production, phosphoenolpyruvate formation and aspartate synthesis vary in response to different metabolic conditions, in which both the glutamate--H+ symport and the glutamate--aspartate exchange systems are functioning to different extents.


Turnover kinetics of photosystem I measured by the electrochromic effect in Chlorella.

The rise kinetics of the absorption changes induced at 515 nm and 480 nm by a flash were studied using two types of xenon flashes of different durations. The 'slow' rise of the absorption change (t 1/2 = 15--20 microseconds) observed by Cox and Delosme (1978 C.R. Acad. Sci. (Paris) Sér. D 282, 775--778) and Joliot was found to be due to double hits occurring in the reaction centers of System I during the flash. The turnover kinetics of the reaction centers of System I after a short flash were studied by a double flash method. They are in agreement with a second order reaction between P+-700 and its electron donor.


Flash kinetic study of the last steps in the photoinduced reaction cycle of bacteriorhodopsin.

The reaction cycle of light adapted bacteriorhodopsin (BR) in aqueous purple membrane suspensions was studied by laser flash photolysis at different temperatures (2--49 degrees C) and pH values (3--10). The activation energy for several reaction steps was determined at pH 7.6. The kinetics of O-bacteriorhodopsin (one of the last intermediates in the cycle) were analyzed in some detail and it was found that the simple consecutive reaction scheme M-BR leads to O-BR leads to BR may explain the kinetics of O-bacteriorhodopsin as measured at 680 nm. Since the pH change in neutral aqueous suspensions of purple membrane follows similar kinetics as O-bacteriorhodopsin it is suggested that protons are released during the reaction M-BR leads to O-BR and taken up again during the reacton O-BR leads to BR. Another long-lived intermediate, which absorbs to a greater extent than bacteriorhodopsin at 570 nm and less than bacteriorhodopsin at 420 nm, was identified with the strongly fluorescing species, pseudo- or P-bacteriorhodopsin. The decay of P-bacteriorhodopsin in bacteriohodopsin had an activation energy of only approx. 1.2 kcal/mol, which suggests that the last step of the photocycle is a relaxation around a single bond. At pH 9--10, the simple first-order kinetics of all the intermediates were changed into a kinetics consisting of two first-order decays. This change of kinetics was accompanied by a drastic decrease in the rotational diffusion relaxation time. To explain the results obtained in this work and those of others, a model involving proton uptake and release by the Schiff base nitrogen combined with an isomerization reaction is finally proposed.


Evidence that the intermediate electron acceptor, A2, in photosystem I is a bound iron-sulfur protein.

Absorption changes accompanying the formation of light-induced P-700+ were investigated in a highly enriched Photosystem I preparation where an intermediate electron acceptor preceding P-430 could be detected. In an enriched Photosystem I particle, light-induced reversible absorption changes observed at 700 nm in the presence of dithionite resembled those previously seen at 703 nm and 820 nm [9], thus indicating the presence of a backreaction between P700+ and A-2. After this same Photosystem I particle was treated to denature the bound iron-sulfur centers, the photochemical changes that could be attributed to P-700 in equilibrium A2 were completely lost. These results provide evidence that the intermediate electron acceptor, A2, is a bound iron-sulfur protein. Additional studies in the 400--500 nm region with Photosystem I particles prepared by sonication indicate that the spectrum of A2 is different from that of P-430.


The multifarious couplings of energy transduction.

I have attempted to show in this article that biological coupling is of great complexity resembling that in an electronics circuit with many different forms of feed-back control. Major features of the biological apparatus are (i) the control of diffusion (ii) the structural organisation (iii) the simultaneous use of electronic and electrolytic devices. It is important in the analysis of the coupling to discover the regions of space which can equilibrate. I have put the argument for localised reactions. Chemiosmosis, which is not so very different from these schemes, demands more general equilibration. The great uncertainties lie in the energy distribution in the system and the mechanics of ATP formation. Other couplings may be understood more easily e.g. electron/electron, proton/proton, proton/electron, proton/cation, proton/anion.


Water binding and phase structures for different Acholeplasma laidlawii membrane lipids studied by deuteron nuclear magnetic resonance and x-ray diffraction.

Water binding capability and phase structures for different lipid species extracted from Acholeplasma laidlawii A membranes have been studied using deuteron nuclear magnetic resonance and low-angle X-ray diffraction. The dominating membrane lipids are monoglucosyldiglyceride and diglucosyldiglyceride and each of them takes up limited amounts of water (bound plus trapped), i.e., up to 13% (w/w), whereas the phospholipids and phosphoglycolipids have larger hydration capacities. Addition of magnesium and calcium ions, but not sodium ions, to the diglucosyldiglyceride increases the hydration capability. This increase is accompanied by the formation of a metastable liquid crystalline phase and a hysteresis effect for the transition temperature. Large differences in water deuteron quadrupole splitting were observed between mono- and diglucosyldiglyceride. Both 2H nuclear magnetic resonance and low-angle X-ray diffraction studies on lipids containing biosynthetically incorporated omega-d3-palmitic acid clearly indicate the existence of a reverse hexagonal phase structure for the monoglucosyldiglyceride and lamellar structures for the diglucosyldiglyceride and the other membrane lipids. The low hydration capability of the large diglucosyldiglyceride polar head is discussed in terms of polar head configuration. Both mono- and diglucosyldiglyceride have several physical properties similar to those of phosphatidylethanolamine.


A differential interaction of daunomycin, adriamycin and their derivatives with human erythrocytes and phospholipid bilayers.

Drug-membrane association of daunomycin, adriamycin and three of its derivatives, adriamycin-14-octanoate (AD-14-OCTA), adriamycin-14-acetate (AD-14-ACE) and N-trifluoroacetyladriamycin-14-valerate (AD32), was studied using phospholipid bilayers and human erythrocytes. The various drugs exhibited a differential affinity to membrane-lipid domains. Lipid-incorporated drugs exhibit a marked change in the shape of the emission spectrum which was utilized for the evaluation of the apparent dielectric constant, epsilon, of the environment surrounding the anthracycline moiety, as well as for the determination ofthe partitioning constant. By measuring the fluorescence polarization and the fluorescence lifetime of the incorporated drugs, rotational relaxation times of 4--8 ns were derived. These parameters provide a supportive evidence of the association of the fluorophore of the drugs with membrane-lipid domains. The anthracycline derivatives interact to a different degree with dipalmitoyl phosphatidylcholine and phosphatidylserine as reflected by changes in their thermotropic properties assessed by differential scanning calorimetry. Daunomycin was the most effective in decreasing the temperature of the phase transition and brought about a comparable reduction in the enthalpy of melting as AD32 and AD-14-OCTA. Adariamycin was the least potent of the series. AD-14-ACE and AD32 protected erythrocytes against hypotonic lysis, adriamycin and daunomycin had no significant effect on the susceptibility to hypotonic lysis, whereas AD-14-OCTA proved to be hemolytic even at low concentration (approx. 10(-7M).


Shape and stability changes in human erythrocyte membranes induced by metal cations.

Asymmetric human erythrocyte ghost membranes behave as bilayer couple and exhibit a radius of curvature preference depending on the state of expansion or contraction of each side of the bilayer. The inside-out preference in the absence of added metal cations is gradually reduced as the K+ concentration is raised to 200 mM until a slight right-side-out preference may be exhibited Divalent cations (denoted 72+; Ca2+, Mg2+ and Mn2+) induce inside-out curvature at very low concentrations, right-side-out curvature at intermediate concentrations, and inside-out curvature again at high ones. This "triphasic" response is attributed to changes in the packing of acidic phospholipid (PL-) pairs in the A-face as a function of M2+ : PL- binding stoichiometry: 0 : 1 (PL- electrostatic repulsion and A-face expansion), 1 : 2 (PL2M crossbridging an contraction), and 1 : 1 (PLM+ repulsion and expansion). Generally increasing parent vesicle size is associated with higher cation concentrations. This is distinguished from the internal and external membrane blebbing preferred at different concentrations in accord with sidedness preference. Parent vesicle size was interpreted to be most closely associated with cation stabilisation (resistance to fragmentation) of the membrane, while sidedness and size (radius of curvature) of blebs were most closely correlated with packing of lipid molecules in the bilayer.


Acetylcholinesterase: a probe for the study of antiarrhythmic drug-membrane interactions.

Structural consequences of antiarrhythmic drug interaction with erythrocyte membranes were analyzed in terms of resulting changes in the activity of membrane-associated acetylcholinesterase. When enzyme inhibitory effects of drugs were compared at concentrations producing an equivalent degree of erythrocyte antihemolysis, a number of distinct groupings emerged, indicating that the molecular consequences of drug-membrane interaction are not identical for all agents examined. Differences in drug-induced acetylcholinesterase inhibition in intact erythrocytes, erythrocyte membranes and a brain synaptic membrane preparation emphaized the role of membrane structural organization in determining the functional consequences of antiarrhythmic interaction in any given system. While the inhibitory actions of lidocaine, D-600 and bretylium in intact red cells were not altered by an increased transmembrane chloride gradient, enhanced enzyme inhibition by quinidine and propranolol was observed under these conditions. The diverse perturbational actions of these membrane-stabilizing antiarrhythmics observed here may be indicative of a corresponding degree of complexity in the mechanisms whereby substances modify the potential-dependent properties of excitable tissues.


Free-calcium and tension responses in single barnacle muscle fibres following the application of L-glutamate.

The effect of the putative transmitter, L-glutamate, on free intracellular Ca2+, tension and membrane potential in single muscle fibres from the barnacle Balanus nubilus has been investigated. External application of L-glutamate (0.1--10 mM) resulted in a transient increase in free intracellular Ca2+, monitored by the Ca2+-activated protein aequorin. This increase in free intracellular Ca2+ was associated with membrane depolarization and force development, and was followed by a period of 'desensitization' in which the preparation was unresponsive to L-glutamate. This could be reversed by removing L-glutamate from the external saline. External application of a number of closely related compounds, including D-glutamate and L-aspartate, were ineffective for initiating the transient light response. The L-glutamate response was virtually abolished in Na-free (Li) medium and completely abolished in Ca-free (Na) medium. The responses to L-glutamate were not reduced in Mg-free medium. The fibre's response to 1mM L-glutamate was also inhibited by D-600 (10 micrometer) or by La3+ (1mM), suggesting that Ca was directly involved in the underlying ionic conductance changes brought about by this putative excitatory transmitter.


Effect of magnesium on calcium efflux in dialyzed squid axon.

The Ca efflux mechanism located in the axolemma of the tropical squid Doritheutis plei is shown to be affected by the concentration of intracellular Mg (Mgi). The removal of all of the Mg from the experimental preparation causes an increase in Ca efflux. This effect seems to be more pronounced at low levels of internal ionized calcium and high levels of internal Na.


A new assay system of phospholipid exchange activities using concanavalin A in the separation of donor and acceptor liposomes.

A new assay system of phospholipid exchange activities is described. The exchange activities were quantitated by measuring the stimulation of phospholipid transfer between two separate populations of liposomes, which contained, as the major constituents, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, and cholesterol in molar ratios of 6 :2 : 1: 1: 5. One population of the liposomes was made reactive to concanavalin A by the incorporation of 1.8 mol% alpha-D-mannosyl-(1 leads to 3)-alpha-D-mannosyl-sn-1, 2-diglyceride from Micrococcus lysodeikticus. The concanavalin A-reactive liposomes, a phospholipid donor, were doubly labelled with [6-3H] galactosylglucosyl ceramide and that class of 32P-labelled phospholipids whose exchange was being measured. The 3H-labelled glycolipid served as a non-exchangeable reference marker. The other population of the liposomes, a phospholipid acceptor, was concanavalin A nonreactive. These two populations of liposomes were incubated with the cytosol protein of rat liver in a total volume of 0.2 ml. After the incubation, two different procedures were used to separate the two liposomal populations. In one procedure concanavalin A was added to agglutinate the reactive liposomes; the flocculated lectin . liposome complex was separated from the non-reactive liposomes by brief centrifugation. In the other procedure the reactive liposomes were trapped by binding to concanavalin A covalently coupled to Sepharose 2B; the complex was separated from the non-reactive liposomes by filtration through a filter paper under suction. In both assay procedures the amount of phospholipid transferred from the donor to the acceptor liposomes was calculated from the decrease of 32P/3H ratio of the concanavalin A-reactive liposomes during the incubation. By the assasy system it is possible to determine phosphatidylcholine and phosphatidylinositol exchange activities in 100 micrograms of rat liver cytosol protein.


Sequential preparation of rat liver microsomal and Golgi membranes.

A new procedure for the preparation of microsomes, microsomal subfractions and Golgi membranes from the same piece of rat liver has been developed. The smallest amount of liver with which the preparation can be performed is about 1 g (wet weight). 35% of the total activity of marker enzymes for the endoplasmic reticulum was recovered in the microsomal fraction. This recovery is approximately the same as that obtained in our laboratories using other procedures. Golgi membranes, mitochondria, lysosomes and plasma membranes represent less than 13% of the microsomal protein as calculated on the basis of marker enzymes. Golgi membranes must be prepared in two steps to achieve a reasonable recovery and thus a representative sample containing both very low density lipoprotein-rich Golgi vesicles and the heavier cisternal elements. The recovery of UDP-galactosyltransferase activity in the Golgi fraction from the livers of alcohol-treated animals is around 30% of the total activity in the total particulate fraction.


Carnitine-induced uptake of L-cartinine into cells from an established cell line from human heart (CCL 27).

L-Carnitine is actively transported into Girardi human heart cells, an established cell line from human heart. The present study was undertaken to investigate the effect of different concentrations of L-Carnitine in the growth medium on the rate of uptake of L-[3H] carnitine. Increasing the concentration of L-Carnitine from 2 to 100 mumol/l in the growth medium of cells, increased the rate of uptake of L-[3H] carnitine by about 50%. The maximal effect was reached after approx. 72 h incubation. The increase in rate seemed to be caused by synthesis of increased number of carriers, as judged by the increase of V with unchanged apparent Km for the transport process. This effect of L-carnitine could be inhibited by cycloheximide, indicating the dependence on intact protein synthesis. The morphology of the cells was studied by electron microscopy. No myofilaments were found, thus the cells are dedifferentiated and no longer typical muscular cells.


Calorimetric studies of the structural transitions of the human erythrocyte membrane. Studies of the B and C transitions.

Differential scanning calorimetry has been used to study several structural transitions of the human erythrocyte membrane. Earlier studies have shown that one of these transitions (the A transition) is due to the thermal unfolding of spectrin on the membrane. In this paper, it is shown that two of the other transitions (B and C) exhibit a high sensitivity to a local anesthetic, benzyl alcohol. Increasing the ionic strength of the suspending medium results in a splitting of the B transition into two indepent transitions (B1 and B2). It is found that one of these (B2) is associated with titrating groups, since the midpoint for the transitions shifts by about 20 degrees C, with an apparent pK near 7.5 Extensive bilateral proteolysis by papain causes a drastic decrease in the size of all transitions except the C transition, which remains unaltered. On the other hand, treatment with phospholipase by A2 largely affects the C transition, causing its disappearance. Because of the lack of sensitivity to proteolysis and the high sensitivity to phospholipase, it appears that the C transition has a large extent of 'lipid involvement'. It might result from the melting of a small fraction of phospholipid which exists in a crystalline state under physiological conditions. Alternatively, the C transition could arise from changes in protein-lipid interactions or from lipid-dependent changes in protein-protein interactions, providing one assumes that only protease-resistant portions of membrane proteins are participating.


The effects of anion transport inhibitors on structural transitions in erythrocyte membranes.

Red blood cell membranes have been labeled with several covalent and noncovalent inhibitors of anion transport and their heat capacity profiles determined as a function of temperature. Covalent inhibitors include the amino reactive agents 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, pyridoxal phosphate and 1-fluoro-2,4-dinitro benzene. The non-covalent inhibitors include several well known local anesthetics. The study was underataken in order to identify regions of the membrane involved in anion transport. Covalent modification in all case resulted in a large upward shift of the C transition, which is beleived to involve a localized phospholipid region. Evidence is presented which indicates that Band III protein and this phospholipid region are in close physical proximity on the membrane. Addition of non-covalent inhibitors affects the membrane in either or both of two ways. In some cases, a lowering and broadening of the C transition occurs; in others the B1 and B2 transitions are altered. These latter transitions are beleived to involve both phospholipid and protein, including Band III. These results may indicate that the non-covalent inhibitors produce their inhibitory effect on anion transport at least in part by interacting with membrane phospholipid.


Cellular uptake of lithium via amino acid transport system A.

We now add to the agencies by which cells take up lithium the process of cotransport with neutral amino acids via System A. In the Ehrlich cell various natural and synthetic amino acids, depending on their structure, can cause substantial accelerations of Li+ uptake over a considerable range of levels of Na+, Li+ and H+. Half the maximal augmentation of uptake, namely 1.2 mequiv. Li/kg cell water per 15 min, was obtained for 5.4 mM alanine in a double-reciprocal plot. Alanine also stimulated the exodus of Li+ from the Ehrlich cell. The  human red blood cell, lacking System A as it does, becomes an imperfect model for studying cellular uptake of Li+. Until the Li+ dependence of amino acid uptake in the reticulocyte is known, reticulocytosis can be suspected of contributing to the interpersonal variations seen in Li+-for-Na+ exchange.


Accumulation of small fragments of DNA in isolated HeLa cell nuclei due to transient incorporation of dUMP.

[3H]dUMP was incorporated into DNA of isolated S-phase HeLa S3 cell nuclei during DNA synthesis. The incorporated radioactivity was made acid soluble during a chase with excess TTP. A partially purified DNA polymerase alpha incorporated [3H]dUMP into activated salmon sperm DNA. The incorporation rate was equal to the incorporation of [3H]TMP, and the radioactivity incorporated was not made acid soluble during a chase. The nuclei thus have the ability to remove misincorporated uracil. From cytosol we have partially purified an enzyme (80 times purification) that splits the N-glycosidic bond between uracil and deoxyribose in dUMP-containing DNA. This uracil-N-glycosidase has a molecular weight of about 50 000. It does not accept dUTP or RNA as substrates. Pulse labelling of isolated nuclei with radioactive deoxyribonucleoside triphosphates in the presence of dUTP lead to a large accumulation of label in small DNA fragments. The size of these fragments was about 80 nucleotides in a 60 s pulse and no increase in size was observed with increasing pulse length. The corresponding value for control experiments with no dUTP, was 200 nucleotides and the fragments increased in size with increasing pulse length. About 90% of the radioactivity was found in the small fragments after a 3 min pulse when the concentration of dUTP in the test mixture was 100 micrometer and no exogenous TTP was present. In control experiments with no dUTP present, only 14% of the radioactivity was found in small DNA pieces. When test mixture containing dUTP was preincubated with cytosol for 60 s before adding the isolated nuclei, the small fragments increased in size to that of DNA fragments found in control incubations; also the relative amount of label bound to the fragments returned to the levels found in the controls. Increasing the TTP concentration from 5 micrometer to 1.88 mM in the absence of exogenous dUTP had no effect on the size of the DNA fragments.


In vitro HeLa cell DNA synthesis. II. Partial characterization of soluble factors stimulating nuclear dna synthesis.

When HeLa cells are lysed in hypotonic buffer and the lysate is fractionated by centrifugation into particulate and soluble components, the particulate component (nuclei) is deficient compared to unfractionated lysate in the extent to which it can carry out in vitro DNA synthesis. In addition, rapidly labeled short DNA chains (Okazaki pieces) accumulate in purified nuclei, and are chased into higher molecular weight DNA to a lesser degree than in unfractinated lysate. When purified nuclei were reconstituted with soluble component, the capacity of the nuclei for in vitro DNA synthesis was fully restored, as was the capacity of the nuclei for conversion of Okazaki pieces to higher molecular weight DNA. This suggests that the soluble component contains a factor or factors necessary for normal DNA replication. The major incorporation-stimulating activity was partially characterized and partially purified from the soluble component. It is heat labile, non-dialyzable, partially recoverable in the supernatant after pH 5 precipitation, found mainly in a 55--85% saturated (NH4)2SO4 fraction, and is included on Sephadex G-100. After passage through Sephadex G-100, the activity displays increased instability to storage at either 4 degrees C or --70 degrees C. Part of the activity does not bind to phosphocellulose at pH 7.2 and low salt; no additional activity can be recovered in a 0.5 M KC1 wash of the phosphocellulose column. The Okazaki-piece-joining activity was found, along with the bulk of the incorporation-stimulating activity, in the 55--85% (NH4)2SO4 fraction. These findings provide some of the groundwork for future attempts to completely purify and characterize those activities in the soluble component of cell lysates which are involved in DNA replication.


Cellular polyamine depletion reduces DNA synthesis in isolated lymphocyte nuclei.

The accumulation of putrescine, spermidine and spermine in activated bovine lymphocytes was blocked by the combined action of two inhibitors of polyamine biosynthesis, methylglyoxal bis(guanylhydrazone) (MGBG) and alpha-methylornithine. Lymphocytes were cultured under three conditions: (1) alpha-methylornithine alone, (2) MGBG alone, or (3) alpha-methylornithine plus MGBG. DNA synthesis in nuclei isolated from these cells was reduced from control rates by approx. 10, 55 and 75%, respectively. In each case, the degree of inhibition was similar to that observed with the intact cells. Stimulation of nuclear DNA synthesis with the postnuclear supernatant fraction was not affected by polyamine depletion of the cells. Several experiments indicate that the reduced rate of in vitro DNA synthesis was caused by the lack of polyamines and not by alternate effects of the drugs. No inhibition was observed (1) when spermidine was added to inhibited cultures 12 h before harvest and nuclear isolation, (2) when the drugs were added after polyamines had accumulated, and (3) when the drugs were added directly to the in vitro assay. In addition, the degree of inhibition of in vitro DNA synthesis correlated with the degree of polyamine deficiency. These in vitro studies confirm the results obtained with whole cells and support the hypothesis that DNA synthesis is one cellular site of action of the naturally occurring polyamines.


Purification and properties of DNA polymerase-beta from guinea pig liver.

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) has been purified over 100 000-fold from a whole cell extract of guinea pig liver. The enzyme yields a single stainable band when subjected to non-denaturing polyacrylamide gel electrophoresis, and this band corresponds to the DNA polymerase activity when a sister gel is sliced and assayed. The final fraction has a specific activity of 21 000 units/mg; this value can be increased significantly by addition of various components, including glycols, polyamines or any of several protein factors which can be purified from the crude extract. The DNA polymerase-beta lacks detectable exonuclease or endonuclease activity, has an alkaline pH optimum and has a requirement for all four deoxyribonucleoside triphosphates, a divalent cation and a primer-template for maximal activity. While activated DNA is the preferred primer-template, the enzyme is capable of utilizing native and denatured DNA as well as several synthetic polynucleotides as primer-templates. The latter are especially effective when manganese is the divalent cation. Magnesium, at 10 mM, is the preferred divalent cation when activated DNA is used. Manganese, and to a lesser extent cobalt, can substitute for magnesium while zinc and calcium cannot. The beta-polymerase has a half-life of 10 min at 40 degrees C and this is increased in the presence of either DNA or NaCl. The enzyme is stimulated by glycols, polyamines and NaCal or KCl, and is inhibited by several known inhibitors of DNA polymerase activity including o-phenanthroline, heparin, organic solvents and sulfhydryl blocking agents. Guinea pig liver DNA polymerase-beta is remarkably similar to the rat Novikoff hepatoma beta-polymerase with respect to its isoelectric point of 8.4 and its molecular weight of 32 000 as determined by sucrose gradient centrifugation under high or low salt conditions or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This similarity is further extended to the removal, at the final step in purification, of a protein capable of stimulating the homogeneous enzyme. Removal of this protein could explain the lower molecular weight of the guinea pig and other rodent-derived beta-polymerases, when compared to the beta-polymerases from other systems.


A study of an endogenous nucleolytic reaction and of the action micrococcal nuclease and DNAase I on a salt-soluble, compact form of chromatin.

The endogenous nucleolytic reaction occurring in rabbit thymus nuclear lysates has been studied at extended incubation times (up to 4 h). Production of nucleosomal polymers containing multiples of 205 base pairs of DNA was observed. The stability of the bands and the low release (1%) of acid-soluble nucleotides indicated there was only a small fraction of sensitive DNA between the subunits. The salt-soluble chromatin formed in the endogenous reaction at short incubation times (14--24 min) and purified over Sephadex G-200 has been treated with micrococcal nuclease and DNAase I. With micrococcal nuclease, nucleosomal polymers containing multiples of 201 base pairs of DNA were formed. Extensive digestion reaveled a core subunit containing 145 base pairs of DNA. With DNAase I only random degradation was observed and nucleosomal complexes were not produced.


DNA-dependent RNA polymerase II from Acanthamoeba castellanii. Comparison of the catalytic properties and subunit architectures of the trophozoite and cyst enzymes.

The actively growing cells (trophozoites) of the amoeba Acanthamoeba castellanii were found to contain three or perhaps four different forms of class II DNA-dependent RNA polymerase (EC 2.7.7.6). The chromatographic and catalytic properties of all forms of the Acanthamoeba class II polymerases suggest them to be cognates of the class II polymerases previously reported. The predominant form was purified to near homogeneity and its subunit composition determined. Nine different polypeptides were found associated with the purified enzyme: 21 000; 185 000; 140 000; 70 000; 35 000; 21 000; 19 000; 18 500 and  16 200. These polypeptides were interpreted in terms of two class II RNA polymerases which differ in the molecular weight of their largest subunit. When A. castellanii is transferred to a medium lacking nutrients, the cells undergo cellular differentiation resulting in the formation of metabolically inactive cells (cyst formation). During this process there are significant changes in the RNA sequences transcribed. In contrast to this, we find that the chromatographic and catalytic properties of all of the class II RNA polymerases remain unchanged. Further, the subunit architecture of the predominant form(s) of polymerase II is unaltered. These findings suggest that although new RNA sequences are transcribed during encystment their appearance is not a consequence of extensive alterations in the subunit composition of the major class II RNA polymerase.


Evidence for the existence of two forms of RNA polymerases I and II in insect wing epidermis.

DNA-dependent RNA polymerases were solubilized from developing wings of the oak silkmoth, Antheraea pernyi, and partially purified by ion-exchange chromatography and sucrose gradient sedimentation. Four enzyme species were resolved on the basis of chromatographic behavior, divalent cation requirements, ionic strength optima, template preference and alpha-amanitin sensitivity. Each class (i.e. RNA polymerase I and II) was present in two forms termed IA, IB and IIA, IIB on the basis of their elution pattern from the column. Both class I enzymes were sensitive to high concentrations of alpha-amanitin but this may be due to general toxicity rather than specific inhibition. The intraclass variants did not differ significantly in enzymatic properties although form IIB was more sensitive to alpha-amanitin (50% inhibition at 2 . 10(-9) M) than form IIA (3 . 10(-8)M).


Stabilization of adenovirus nuclear RNA by intercalating drugs.

The effect of the intercalating drugs proflavine, ethidium bromide and daunomycin on the rate of degradation of newly synthesized adenovirus RNA was examined. As shown previously for heterogeneous nuclear RNA (HnRNA) and 45 S precursor ribosomal RNA (pre-rRNA), proflavine immediately stabilizes newly synthesized adenovirus RNA, while ethidium bromide stabilizes the RNA after a 30--60 min lag period. In contrast to its effect on HnRNA and pre-rRNA, daunomycin also stabilizes newly synthesized adenovirus RNA. These results are consistent with the hypothesis that the processing of late adenovirus nuclear RNA involves cleavage of base-paired regions by specific cellular nucleases.


Protein biosynthesis in a cell-free system from bovine retina.

A cell-free extract active in the incorporation of [3H]leucine into protein has been prepared from bovine retina. This system may be useful in identifying early precursors and transported forms of the visual  pigment apoprotein. The system completed a minimum of 50% of the preexisting nascent polypeptide chains. 20% of the labelled product accumulated in the soluble fraction of the extract. Gel electrophoresis revealed that this fraction contained six major labelled polypeptide species, each of which correspond to a major unlabelled soluble polypeptide. Some completed polypeptides were retained in the particulate fraction of the extract; several of these were resolved on gel electrophoresis and were of very high specific radioactivity. An antiserum to rod outer segment disc proteins precipitated several labelled species from each fraction. The most interesting of these is a polypeptide of molecular weight 41 000 which was found only in the particulate fraction.


Ursodeoxycholic acid. Effects on sterol metabolism in rats.

Sterol balance studies using isotopic and chromatographic techniques were performed in rats fed diets supplemented with ursodeoxycholic acid. Compared to controls, ursodeoxycholic acid dramatically altered sterol metabolism. Ursodeoxycholic acid was absorbed and circulated in the enterohepatic circulation. The biliary bile acid composition was significantly altered with ursodeoxycholic acid the predominant biliary bile acid (67%). Cholesterol absorption was depressed by 34%; bile acid synthesis was depressed by 30%; however, cholesterol balance was significantly increased. It is apparent that the effects of ursodeoxycholic acid on sterol metabolism are different in several respects from chenodeoxycholic acid.


Regulation of lipoprotein lipase immunological study of adipose tissue.

An antibody to purified rat heart lipoprotein lipase was used to determine the relative specific activities of adipose tissue lipoprotein lipase from fed and fasted rats. The antibody was immobilized by coupling it to a Sepharose gel. This antibody bound approx. 80% of the lipoprotein lipase activity of extracts of rat adipose tissue. When the extracts were separated by gel chromatography into two lipase activity fractions (lipoprotein lipase "a" and lipoprotein lipase "b") and these fractions incubated with the antibody, only 10% of the lipoprotein lipase "a" activity was bound by the highest antibody concentration employed, whereas 93% of the lipoprotein lipase "b" was bound by the same amount of antibody. Increasing amounts of antibody incubated with extracts of adipose tissue of fed or fasted rats yielded similar titration curves. When a constant amount of antibody was incubated with increasing amounts of the adipose extracts, no significant difference was noted between extracts from fed and fasted animals. The data indicate that the high lipoprotein lipase activity of adipose tissue of fed rats, compared with that of rats fasted overnight, results from the presence of more lipoprotein lipase protein.


Retention of acyl groups in LM cell fibroblasts with altered phospholipid composition.

Incorporation and loss of membrane lipids were measured in murine fibroblasts grown in suspension culture with [2-14C]acetate in a chemically defined medium devoid of serum or lipid. Greater than 98 and 94% of the 14C present in the phospholipids and neutral glycerides respectively was found in the fatty acyl groups. During a 3-day growth period LM fibroblasts lost 65% of their 14C-labelled acyl groups from the phospholipids and 36% from the neutral lipids. Selective retention of 14C in individual isolated phospholipid and neutral lipid species occurred only in phosphatidylethanolamine. Retention of acyl groups in the phospholipids or neutral glycerides was not a property of LM suspension cultured fibroblasts. Supplementation of the LM cells with choline analogues, such as N,N'-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine instead of choline, decreased the loss of 14C-labelled fatty acids from phospholipid and neutral lipid. Ethanolamine supplementation resulted in increased loss of 14C from desmesterol as well as decreased incorporation of 14C into desmosterol. Thus, polar head group manipulation affected acyl group composition, acyl group retention, and sterol metabolism in suspension cultured LM cells.


Conjugation of 15-keto-prostaglandins by glutathione S-transferases.

Incubation of 15-keto[3H]prostaglandin F2alpha with glutathione (GSH) produced a metabolite of 15-keto-prostaglandin F2alpha which was not extractable from aqueous solution and thus termed 'water-soluble metabolite'. The addition of cytosol of guinea pig liver to the incubation mixture increased the formation of water soluble metabolite of 15-keto-prostaglandin F2alpha 3-fold. The conversion of 15-keto-prostaglandin F2alpha to water soluble metabolite in both the presence and absence of enzyme was linear during 10 min of incubation and required 2.5 mM GSH for maximal activity. Liver and kidney cytosol possess about 70 and 25 times, respectively, as much activity as compared to lung cytosol. Chromatographic analysis of the water soluble metabolite obtained from incubation of either 15-keto[3H]prostaglandin F2alpha and GSH or [3H]GSH and 15-keto-prostaglandin F2alpha showed that the water-soluble metabolite was an adduct of 15-keto-prostaglandin F2alpha and GSH. The addition of prostaglandin A1, a substrate of GSH S-transferases, to the incubation mixture competitively inhibited the formation of the water-soluble metabolite of 15-keto[3H]prostaglandin F2alpha. Presumably, 15-keto-prostaglandin F2alpha and other 15-keto-prostaglandins are converted to GSH conjugates by GSH S-transferases. This indicates that 15-keto-metabolites produced by prostaglandin dehydrogenase may be further metabolized to GSH conjugates.


Stimulation of hydroxymethylglutaryl-CoA reductase activity after a single dose of the porphyrogenic chemical, allylisopropylacetamide.

Hydroxymethylglutaryl-CoA reductase activity in fasting rats was almost doubled 12 h following a single dose (400 mg/kg) of the porphyrogenic chemical allylisopropylacetamide. This doubling effect of the drug on enzyme activity was observed during the basal period, as well as at midnight, when it is maximal in the diurnal rhythm. Cycloheximide, whether given alone or simultaneously with the drug, reduced the enzyme activity to very low levels. Actinomycin D administered alone prevented the normal rise in reductase activity which occurs at night; however, when administered with the drug, it inhibited but did not completely suppress the inducing effect of allylisopropylacetamide. These data suggest a requirement for protein synthesis to observe the stimulating effect of allylisopropylacetamide on hydroxymethylglutaryl-CoA reductase activity. A modification in the reductase assay is also described wherein the formation of mevalonolactone from mevalonic acid, the end-product of the assay, is maximized with 3 M HCl. Using this procedure, consistent yields of mevalonolactone in the 90% range are observed since the use of 3M HCl prevents dehydration of mevalonic acid to delta3, 4-methyl-delta-valerolactone.


Thiamine-induced alteration in sterol composition of Saccharomyces carlsbergensis 4228.

The cells of Saccharomyces carlsbergensis 4228 grown with added thiamine in a vitamin B-6-free medium did not contain ergosterol and zymosterol (the predominant sterols in cells grown without thiamine). Instead, significant amounts of squalene, lanosterol and unidentified sterols accumulated in the thiamine-grown cells. Of the unidentified sterols, the most predominant was delta5,7-ergostadien-3beta-ol at 26.0% of the total sterol. The accumulation of this sterol in the place or ergosterol (delta5,7,22-ergostatrien-3beta-ol) indicates that the desaturation at C-22 of sterol structure is completely blocked in the thiamine-grown cells. On the basis of chromatographic behaviours, the structures of other unidentified sterols were assumed to be 4alpha-methyl-delta8,24(25)-cholestadien-3beta-ol, 4alpha-methyl-delta 8,24(28)-ergostadien-3beta-ol, delta8,24(28)-ergostadien-3beta-ol and delta5,7,24(28)-ergostatrien-3beta-ol. The accumulation of 4alpha-methyl sterols, in addition to that of a large amount of lanosterol (48.4% of total sterol), suggests that the demethylation processes from lanosterol to zymosterol was partially depressed in the thiamine-grown cells.


Thermodynamic studies on the binding of adenosine diphosphate and calcium to beef cardiac myosin.

Thermodynamic quantities for the binding of MgADP, CaADP and Ca2+ to purified beef cardiac myosin have been determined by flow calorimetry at 25 degrees C and by equilibrium dialysis at 4 degrees C in 0.5 M KCl, 20 mM tris-HCl (pH 7.5). About 1.65 +/- 0.15 mol MgADP and 1.9 +/- 0.1 mol CaADP were bound per mol myosin. Free energies of binding of MgADP and CaADP were -6.7 and -5.7 kcal/mol, respectively. Enthalpies for binding of MgADP and CaADP were about -12.5 and -19.0 kcal/mol, respectively. Furthermore, there were 1.8 +/- 0.2 mol high affinity Ca2+ binding sites per mol myosin with an affinity constant of about 10(5) M-1. The enthalpy of Ca2+ binding was about zero. It is concluded that CaADP binds to cardiac myosin with a much greater negative enthalpy than MgADP. Also, the free energy of MgADP binding to cardiac myosin is similar to values reported for skeletal myosin. However, the enthalpy of binding is much less negative than the value obtained for skeletal myosin by Kodama and Woledge (J. Biol. Chem. (1976) 251, 7499--7503). The latter results suggest a subtle difference in the nucleotide binding sites of these myosins.


The PO protein. The major glycoprotein of peripheral nerve myelin.

A glycoprotein, referred to as PO protein, was isolated from rabbit sciatic nerve myelin by gel filtration on Agarose 0.5 m in dodecyl sulfate. The purified myelin was first defatted and extracted at pH 2. The water-soluble proteins such as myelin basic protein and P2 protein were extracted leaving a glycoprotein-rich residue, from which the PO protein was isolated. The purified protein showed a single band on gel electrophoresis in dodecyl sulfate when stained with Coomassie Blue of periodic acid-Schiff reagent. The carbohydrate, comprising 6.3% by weight, appears to exist as a nonasaccharide unit having 3 mannose, 3 N-acetylglucosamine, 1 sialic acid, 1 galactose and 1 fucose residue. The polypeptide moiety has a high content of non-polar amino acids. A single amino acid, isoleucine, was found at the NH2-terminal end by dansyl and Edman procedures. The PO protein is the major protein of peripheral nerve myelin.