Nasal cancers, symptoms and upper airway function in woodworkers.

In 186 cases of nasal cancer diagnosed over the decade 1965-74, in a population of 2-0 million, 114 of 157 ectodermal tumours were found in men. Adenocarcinoma was found in 17 patients, two women and 15 men; 12 of these had a history of occupational exposure to wood dust in the furniture industry. The period of latency was from 28 to 57 years. Among the remaining 99 tumours in men there was occupational exposure to wood dust in 10 cases. Wood dust concentrations in the breathing zone, respiratory symptoms and upper airway function were studied in 68 workers in the furniture industry. The dust concentrations affecting 63% of the workers were higher than 5 mg/m3 (TLV). Middle ear inflammation and common colds were more frequent at high dust concentrations, and the number of workers with nasal mucostasis was directly proportional to the wood dust concentration.


Heavy metal pollution among autoworkers. I. Lead.

Lead pollution was evaluated in 216 individuals working in 10 garages on the Island of Funen, Denmark and related to data from biochemical and medical examinations. Clinical symptoms were recorded by means of a questionnaire. Increased blood test lead levels were foun in 59% with 9% having above 80 microgram lead/100 ml (3-86mumol/1) whole blood. Mechanics in eight out of ten garages had significantly increased blood lead levels. A decrease in delta-aminolevulinic acid dehydratase (ALAD) activity was associated with increased blood lead levels but the latter were not related to haematological changes, tobacco consumption or to length of service in the trade. Particulate lead air pollution was not the sole cause of increased blood lead levels. Raised lead values were maximal among diesel engine workers who are exposed to high pressure-resistant lubricants containing lead naphthenate. As these workers complained of skin damage, lead absorption may have occurred through the skin. Assay of lead content showed 9290 ppm in gear oil and 1500-3500 ppm in used motor oils. The data are discussed in relation to the occupational risks in auto repair shops.


Simultaneous determination of toluene and xylene metabolites in urine by gas chromatography.

A gas chromatographic method for simultaneous determination of hippuric and o-, m-, and p-methylhippuric acids (metabolites of toluene and xylene) in urine is described. The analytical procedure is based on the extraction of the aromatic metabolites with ethyl acetate containing the internal standard and on a methylation with 3-methyl-1-p-tolyltriazene. With this method, which does not require much time and handling, the different acids can be satisfactorily determined with high sensitivity and specificity. A statistical study shows a good reproducibility for the determination of hippuric and o-, m-, and p-methylhippuric acids. The coefficient of variation for 10 determinations in all cases was less than 5%.


The differential diagnosis of homosexuality.

The treatment of homosexuality presents serious difficulties owing to its multifactorial aetiology and variety of psychopathological views. It is suggested that the combined use of psychiatric and psychoanalytic models can contribute to the establishment of a satisfactory differential diagnosis. Out of the innumerable clinical types seen in practice and described in the literature, three major groups can be isolated. The homosexuality which occurs on the background of hysterical, obsessional and other neurotic personality types and related psychiatric conditions, tends to be linked with latent heterosexuality and responds well to all forms of psychotherapy. On the other hand, true homosexuality is often the major presenting symptom of borderline states, narcissistic disorders, psychopathy and the schizo-affective psychoses and carries an unfavourable prognosis with any form of treatment. Cases of actual bisexuality form the third group with its own specific psychopathology. The homosexual solution is seen as a defence and is regarded as a matter of survival for many individuals. It should be treated with the utmost caution by anyone who attempts to remove it.


Homosexuality in adolescence.

This paper asserts that homosexuality in adolescence should not be regarded in the same way it would be in adults. Different types of homosexuality emerge during adolescence according to the different psychodynamic conditions which occur at different stages of adolescence, particularly the changing relationship to the individual's parents, and these may subside or make an important contribution to the establishment and nature of an individuals' homosexuality in adulthood. The characteristic 'identificatory facility' of adolescence is discussed and it is suggested that this is retained in the make-up of the adult homosexual. Some comments on the clinical assessment of homosexuality in adolescence are briefly made.


Some findings concerning beliefs about alcoholism.

The beliefs that alcoholics hold about alcoholism and themselves, and the beliefs that hospital staff and other personnel hold about alcoholism, were investigated using methods derived from the repertory grid, with anonymous facial photographs as elements. Data are presented as to the degree of association of various characteristics with alcoholism for the various groups. Differences between groups, and overall changes during group psychotherapy for patients were small. The relative contribution of personal and shared aspects of belief was measured. It was found that the agreement between groups was high and closely similar to that predictable from consistencies within groups. It is concluded that there may be a very general stereotype of alcoholism, consistent with a personality disorder concept which is implicitly held even in the face of nominal acceptance of other concepts. The self constructs of alcoholics were found to be complex. In a small follow-up study of patients, there was found to be a relationship of poor outcome with tightness of construing for key constructs after psychotherapy. The implications of the findings are discussed briefly.


Self-esteem and psychiatric syndromes.

The purposes of the study were (1) to dichotomize each of the personal illness classes into those expected and those not expected to retain a relatively high degree of anxiety-depression; (2) to seek confirmation of this division by means of the combined DSSI sets of Anxiety and Depression; (3) to examine their respective scores on the Personality Deviance Scales; and (4) to examine the relationship between Anxiety-Depression and intropunitiveness. It was found that within each of the four classes the predicted 'affective' group scored significantly higher on DSSI Anxiety-Depression than did the 'non-affective' group. On intropunitiveness, the 'affective' group scored significantly higher within three classes and almost so in the forth. Although anxiety-depression and intropunitiveness both discriminate between the 'affective' and 'non-affective' groups their conceptual distinction is empirically based.


Hostility, personality and depression.

The study describes findings on two samples of depressives and normals matched for age, sex, social class and religious affiliation. The following observations on the psychopathology of depression were made by considering the relationships between personality, various aspects of hostility, and the degree of depression: (1) depression, guilt and anxiety are linked together; (2) the extent to which anxiety is somatized is not dependent on the way hostility is handled; also, somatic symptoms occur independently of hysterical symptoms in depressive illness; (3) obsessional symptoms are likely to occur together with hysterical symptoms and to be associated with hostility-aggression during depressive illness; (4) hysterical tendencies may accentuate intropunitiveness.


Personality correlates of study difficulty and academic performance in university students. I. The Middlesex Hospital Questionnaire and Dynamic Personality Inventory.

The personality correlates of study difficulty and academic performance in university students have been measured in terms of psychiatric symptomatology and premorbid characteristics. The UCLSQ, MHQ and DPI were administered to two groups of male and female students presenting at an health centre: one (n = 72) of patients seeking psychological help, the other (n = 73) a control group. Significant differences between the two groups were formed on the MHQ, confirming previous results; and these were supplemented by differences on the DPI. Motivational and psychoneurotic components of study difficulty were significantly related to MHQ scores in both groups. DPI scores were more closely related to study difficulty in the patients than controls; and served particularly to characterize their sylbism and work satisfaction. The DPI added to the picture of a subgroup of students observed both in an earlier and the present study: academically successful patients scoring high on phobic anxiety.


Personality correlates of study difficulty and academic performance in university students. II. Conscience and self-esteem.

Scales measuring self-esteem and conscience have been included in a further study of work-related symptoms in students. Two groups were tested: a group of patients attending the UCL Student Health Centre for psychological reasons, and a group of normal controls. Conscience and self-esteem were inversely related to one another. Both tests were reliable and differentiated between the groups. The patients tend to have low self-esteem, high conscience, and to score higher than controls both on neurotic symptoms as such as measured by a personality inventory (MHQ) and as assessed on work-related symptoms measured by our study difficulty questionnaire (UCLSQ). In terms of academic performance, an interesting subgroup of students was delineated who are rated highly by their tutors but who appear subjectively troubled with phobic anxiety, low self-esteem and a harsh conscience structure. The conscience and self-esteem scales, in view of their brevity, acceptable reliability, and in terms of the relationships discussed in this paper, will be retained for further research.


Fetal heart rate patterns and uterine activity after segmental epidural analgesia.

The effects of low-dose segmental epidural analgesia on fetal heart rate (FHR) patterns, uterine activity, and some clinical features were examined in a series of 105 normal parturients. The aim of the analgesic technique was to relieve pain during the first stage of labour by blocking the sensory nerve fibres at the spinal level of T 10 to T12, with either 0-5 per cent bupivacaine or bupivacaine with adrenaline 1 : 200 000, the dose varying from 10 to 25 mg. Obvious pathological changes (late decelerations, prolonged fetal bradycardia, variable deceleration, or loss of beat to beat variation) in FHR patterns within the first 30 minutes after the beginning of analgesia were noted in 9 per cent of the patients. Addition of adrenaline to the analgesic agent seemed to reduce uterine activity for 60 minutes after the analgesia and this was reflected in a longer interval between the block and delivery. A 25 per cent fall in systolic blood pressure was recorded in 11 per cent of the patients. Nine patients required vacuum extractions. Our results show that the epidural technique employed has distinct advantages over the previous methods, which require larger doses of the analgesic, agent, and is therefore safer for the fetus; the addition of adrenaline to the analgesic agent is contraindicated.


Fetal heart rate responses to fetal movements.

A classification of fetal heart rate (FHR) responses to fetal movements is based on the study of 210 hours of antepartum cardiotocographic tracings in clinically normal pregnant patients between 30 and 42 weeks gestation. The physiological mechanism underlying the FHR responses to fetal movement are discussed.


Metabolic effects of salbutamol infusion during premature labour.

Plasma electrolytes, glucose and insulin concentrations were measured and serial electrocardiograms performed during the intravenous infusion of salbutamol to five women in premature labour. The plasma potassium (mean +/- standard error) fell from 3-5 +/- 0-1 to 2-7 +/- 0-1 mmol/l, and the glucose and insulin rose by 4-2 +/- 0-7 mmol/l (75 +/- 13 mg/100 ml) and 26 +/- 5 mU/l respectively. The maximum fall in potassium, and the rise in glucose and insulin occurred two hours after starting the infusion. No changes in other electrolytes were found. Electrocardiograms showed no signs of hypokalaemia. Highest pulse rates (148 +/- 4 beats per minute) occurred at two hours but no arrhythmias were observed.


Effect of labour on the lecithin/sphingomyelin ratio in serial samples of amniotic fluid.

Serial samples of amniotic fluid were taken from 48 patients during labour. The lecithin/sphingomyelin (L/S) ratio was estimated and found to rise in 52 per cent, remain almost unchanged in 33 per cent and fall in 15 per cent. The rate of rise in the L/S ratio seemed inversely related to the length of labour.


The physiological macrocytosis of pregnancy.

Sixty-four healthy women were followed during normal pregnancies in which they took supplements of iron or iron+folate or an inactive placebo. The red cell size increased in those patients receiving iron alone or iron+folate. The group on the inactive placebo who received no supplemental iron had red cells of constant size and a falling serum iron. Nineteen women not included in the trial developed macrocytosis during pregnancy:marrow samples from these showed normoblastic erythropoiesis in 18 and minor megaloblastic changes in one. Macrocytosis developing during pregnancy is a physiological change in the majority of cases.


Outpatient termination of pregnancy.

Termination of pregnancy was performed under local anaesthesia as an outpatient procedure on 251 patients. The gestational ages ranged from 5 to 12 weeks. Either the portable Karman curette equipment or the syringe kits were used. The incidence of complications was low. Only 6 per cent of patients would have preferred general anaesthesia. The optimal gestational age at which to terminate pregnancies appeared to be between 6 and 10 weeks. Ninety-one per cent of patients used effective contraception after the termination.


Evacuation of retained products of conception in a treatment room and without general anaesthesia.

One hundred patients with an incomplete aboriton, 14 patients with retained products of conception after a second trimester termination, 5 patients with missed abortion and 2 patients with secondary postpartum haemorrhages (making a total of 121 patients) had a uterine evacuation in a treatment room using the portable Karman curette equipment. No general anaesthesia was used but patients were given intravenous pethidine or papavaretum (Omnopon) and diazepam. Four patients found the procedure painful but only two of them would have preferred general anaesthesia. There were no immediate complications but three patients required re-evacuation of the uterus.


Colposcopic evaluation of pregnant patients with abnormal cervical smears.

Colposcopic examination and biopsy were used to assess 123 pregnant patients presenting with abnormal cervical smears. Eighty-seven per cent were 30 years of age or less and 95 (77 per cent) had had one or no previous children. Two patients were found to have microinvasive carcinoma and, in an additional 95 patients, either severe dysplasia or carcinoma in situ was present. Fifty-five patients (45 per cent) had subsequent conization or hysterectomy and in no instance was the histological diagnosis more serious than that anticipated from the colposcopic evaluation. Only three patients (1-6 per cent) had a cone biopsy during pregnancy; only one minor complication occurred. Colposcopic examination is the choice method of evaluating patients with abnormal cervical smears in pregnancy.


Maternal plasma testosterone levels in molar pregnancy.

Plasma testosterone levels in 14 patients with intact molar pregnancy were found to be elevated compared to patients with normal pregnancies of corresponding gestation. The plasma testosterone concentration ranged from 2-0 to 8-4 ng/ml with a mean +/- SE of 5-2 +/- 0-5 ng/ml. After evacuation of the uterus, there was a significant fall in plasma testosterone levels which ranged from 1-6 to 5-9 ng/ml with a mean +/- SE of 3-2 +/-0-3 ng/ml. The trophoblast might have been the source of extra testosterone in molar pregnancy.


Carbohydrate tolerance in the puerperium during suppression of lactation with bromocriptine and oestrogens.

The influence of bromocriptine on carbohydrate tolerance in the puerperium was examined and its effects compared with those of an oestrogenic compound. Sixteen patients were studied in each group. The controls consisted of 16 women who were breast-feeding. Compared with the controls, no significant difference was found in the oral glucose tolerance test in patients who had oestrogen, but blood glucose values in women receiving bromocriptine were significantly lower. Inhibition of lactation with bromocriptine may obscure abnormal glucose tolerance.


Tuberculosis of the female genital tract-- a changing picture.

Twenty-one patients with tuberculosis of the female genital tract are presented, and the clinical features, method of diagnosis and treatment are discussed. The condition affected an older group of patients than described earlier, and ofter took the form of chronic infection with abscess formation.


Perforation of the sigmoid colon during pregnancy: a rare complication of endometriosis. Case report.

Perforation of the sigmoid colon in a 28-year-old woman during the 37th week of pregnancy is reported. There was almost complete replacement of the bowel wall by endometriotic tissue showing extensive decidualization of its stroma. This appears to be a very rare complication of endometriosis and the effects of pregnancy upon endometriosis are briefly reviewed.


Pregnancy following bilateral uterine artery ligation.

A successful pregnancy following bilateral uterine artery ligation performed in an attempt to relieve dysfunctional menorrhagia is reported. Serial biparietal diameters showed a normal growth curve and the newborn infant weighed 2640 g. The patient was normotensive throughtout the pregnancy. Prior to uterine artery ligation, she had had four pregnancies but there were no surviving children. It is suggested that uterine artery ligation may have a limited place in the management of patients with dysfunctional menorrhagia.


Circulating levels of pregnancy-specific beta1-glycoprotein and human placental lactogen in the third trimester of pregnancy: their relationship to parity, birth weight, and placental weight.

Circulating levels of pregnancy-specific beta1-glycoprotein (SP1) and human placental lactogen (HPL) were measured in 153 normal subjects during the third trimester of pregnancy. The levels of SP1 and HPL showed a skewed distribution and rose progressively to reach a plateau by the 36th week. The normal range was calculated by ranking the data and division into centiles as well as by logarithmic transformation of the values. A low but significant correlation was observed between SP1 and HPL levels, SP1 and birth weight. There was also a correlation between HPL levels and placental weight.


Plasma oestriol and placental proteins: a cross-sectional study at 38 weeks gestation.

Blood was collected from 53 normal women in the 38th week of pregnancy. The plasma concentration of unconjugated oestriol, total oestriol and oestriol sulphate was measured and also human placental lactogen and pregnancy specific beta1 glycoprotein. The normal spread of the two proteins was found to be slightly smaller than that of the oestrogens. The concentrations of unconjugated and of total oestriol were related; otherwise these substances varied independently of one another. The concentration of these substances was not related to other obstetric parameters.


Fetal growth retardation and the arteries of the placental bed.

The morphology of the placental bed spiral arteries was studied in 68 pregnancies complicated by fetal growth retardation and in 40 pregnancies with a normally grown fetus. When the birth weight was normal the extent and depth of physiological vascular changes were normal except in those pregnancies complicated by pre-eclampsia. When the birth weight was low and the mothers were normotensive the extent and depth of physiological vascular changes were either normal or restricted, and in all patients with hypertension and a baby with low birth weight the physiological changes were restricted to the decidual segments of the spiral (uteroplacental) arteries. Acute atherosis was only found in pregnancies complicated by hypertension, particularly if there was proteinuria. We do not believe that there exists an arteriopathy which is common to hypertensive and normotensive pregnancies complicated by fetal growth retardation.


The effect of intrauterine fetal transfusion and a beta-sympathomimetic substance on the lecithin/sphingomyelin ratio in human amniotic fluid.

The apparently low incidence of respiratory distress syndrome in babies born prematurely after intrauterine transfusion and the simultaneous prophylactic administration of intravenous isoxsuprine (a beta-sympathomimetic agent) was investigated by serial study of amniotic fluid lecithin/sphingomyelin ratios. The ratio rose very significantly after intrauterine transfusions and significantly (but less markedly) after the administration of intravenous isoxsuprine alone.


Hepatitis B antigen-positive obstetric patients.

In a period of six years (1971--77) 9400 sera taken at booking from antenatal patients were examined for hepatitis B antigen (HBsAg), and 40 patients were positive. These 40 patients had 45 pregnancies and were treated with special precautions so far as venepuncture and the management of delivery were concerned. No member of the hospital staff dealing with the patients developed hepatitis B. All infants born to these mothers were normal, two infants were HBsAg-positive at 7 months and 2 years, but are clinically well. No obstetric problems attributable to the HBsAg-positive state were encountered.


Intracranial birth trauma in vaginal breech delivery: the continued importance of injury to the occipital bone.

Between 1966 and 1976, postmortem examinations revealed significant intracranial birth trauma in 17 infants delivered breech first and in ten infants delivered head first; separation of the squamous and lateral parts of the occipital bone (occipital osteodiastasis) was found in five of the infants who died after breech delivery. The finding of the lesions in these infants is attributed to the adoption, since 1971, of a postmortem technique involving dissection of the suboccipital region before opening the skull. Review of earlier necropsy reports suggests that the lesion was sometimes missed. Analysis of the fresh stillbirths and neonatal deaths occurring in the 477 vaginal breech deliveries at the Hammersmith Hospital (1966 to 1976) showed that all neonatal deaths in infants weighing more than 3000 g at birth associated with intracranial birth trauma: 2 out of 3 had occipital osteodiastasis which seems to be more common than is generally recognized and is only found with careful autopsy technique.


Relationships between spermatogenesis and serum hormone levels in subfertile men.

The measurements of serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone were related to the sperm concentration and testicular biopsy in 137 subfertile men. The biopsies were classified into four histological groups: normal, focal tubular atrophy, maturation arrest and Sertoli-cell-only syndrome. A positive correlation was found between sperm concentration and the histological grade of spermatogenesis. FSH was directly related to the spermatogenesis and the values were significantly raised in maturation arrest and Sertoli-cell-only syndrome. This relationship was present, but to a much lesser extent, for LH. Serum testosterone levels remained unchanged and no relationship between testosterone and either sperm concentration or testicular biopsy was observed. These data indicate the value of FSH in the investigation of the subfertile male, but suggest that the routine measurement of testosterone is unnecessary.


Venous distensibility during the menstrual cycle.

Changes in the distensibility of dorsal hand veins during the menstrual cycle were measured in 19 healthy young women using a new optical method relying upon a pressure-diameter principle. Significantly greater venous distensibility was found during the first half compared with the second half of the menstrual cycle. The possible causes for this finding, particularly the role of oestrogen, are discussed.


The bacteriology of the cervix and uterus.

Fifty hysterectomy specimens were studied by a multiple biopsy technique to determine the normal bacteriological flora of the uterine cavity and cervical canal. All the uterine cavities and 23 of the cervical canals were sterile. The lower half of the other 27 cervical canals contained organisms similar to those found in the vaginal vault.


Amniotic fluid testosterone and fetal sex determination in the first half of pregnancy.

The concentration of freely extractable testosterone in amniotic fluid was measured in 54 patients whose gestational ages ranged from 10 to 20 weeks; 31 of the fetuses were male and 23 were female. The mean amniotic fluid testosterone concentration was significantly higher with a male fetus than with a female fetus. On only 7 out of 31 occasions was the presence of a male fetus associated with amniotic fluid testosterone values below 100 pg/ml, whereas the values associated with a female fetus were never higher than 90 pg/ml. After 14 weeks gestation no amniotic fluid testosterone level was lower than 120 pg/ml in the presence of a male fetus.


Urokinase in the management of vitreous hemorrhage.

Urokinase is a plasminogen activator of human origin which breaks up the fibrin base of blood clots. When given as an intravitreal injection it produces hypopyon and glaucoma, both of which transient. In a series of 27 patients (34 eyes) with unresolved vitreous haemorrhage, this simple and relatively atraumatic treatment has produced marked objective improvement in 10, and greatly improved the life styles of a further 9. This series brings the total of reported cases to 93. When compared with recent American reports of surgical vitrectomy for vitreous haemorrhage, intravitreal urokinase appears to have a higher success rate, with a lower complication rate both in the short and long term. This study suggests that, despite the high cost of the purified enzyme, urokinase should be come the first line of attack in vitreous haemorrhage, vitrectomy being reserved for those patients who fail to respond.


Normal response to pilocarpine and phenylephrine.

Fifty-eight eyes from 58 patients in which there was no evidence of glaucoma were provoked with pilocarpine and phenylephrine drops. The result was a significant reduction in intraocular pressure and a significant increase in outflow facility. The 58 eyes were randomised and 19 submitted to a 'dummy' provocative test. There was no significant change in either pressure or outflow facility. The effect of the pilocarpine/phenylephrine provocative test in normal eyes is to produce a response that is the opposite of a positive provocative test in eyes at risk of developing closed-angle glaucoma.


Association between acute glaucoma and the weather and sunspot activity.

The dates of presentation of 191 cases of acute closed-angle glaucoma which presented at the Birmingham and Midland Eye Hospital between 1971 and 1974 were analysed and the seasonal incidence of acute glaucoma confirmed (P less than 0-001). These data were compared with meteorological data for air temperature, rainfall, hours of sunshine, atmospheric pressure, cloud amount, terrestrial magnetic field level, and sunspot numbers. A statistically significant direct association (P less than 0-05) was found with hours of sunshine and an inverse association (P greater than 0-05) with cloud amount. A statistically significant inverse association (P less than 0-05) was found with sunspot count, and this assumed high significance (P less than 0-0005) on consideration of sunspot activity in the preceding period. The literature is reviewed and the significance of these findings discussed.


Partial angle closure.

During the course of negative provocative test for closed-angle glaucoma using pilocarpine and phenylephrine 60% of eyes develop significant reductions in outflow facility at some stage during the test. It is shown that these reductions can be explained by postulating the presence of partial-angle closure since: (1) A random sample (6) of 53 eyes showing an abnormal response subsequently had a peripheral iridectomy. On reprovoking they then behaved as normal eyes with a uniform increase in outflow. (2) Fifty-eight eyes that had a peripheral iridectomy for closed-angle glaucoma (spontaneous or induced) responded to provocative testing as do normal eyes.


Patient compliance in glaucoma.

Of a randomly selected sample of 40 patients with chronic simple glaucoma 11 were identified as having failed to comply adequately with medical advice. Noncompliers were more likely: to be men, to have had no other medical disorder but glaucoma, not to rank glaucoma as most troubling if they had another illness, to have experienced side effects from the treatment, and not to have appreciated the association between glaucoma and blindness. Detailed clinical study revealed that several interrelated psychosocial factors contributed to noncompliance.


Trabeculectomy: long-term follow-up.

The postoperative course of 309 eyes subjected to trabeculectomy has been studied over a 7-year period. The results have been compiled with regard to effectiveness to maintain a normal range of intraocular pressure with and without postoperative medication. The figures have been assessed in toto and for 3 groups of glaucoma, namely, open angle, chronic angle-closure, and acute angle-closure. Results support those of other reports to show that trabeculectomy gives satisfactory control over the years in a reasonably high proportion of eyes and with relatively few postoperative complications. There is also a small group of combined trabeculectomy with cataract extraction.


Penetrating wound of the orbit: a 31-year follow-up.

The case is described of a man who at the age of 17 suffered accidental penetration of his right orbit by a rod and spring from a machine gun. Operation was successful, and follow-up 31 years later showed normal vision at 20/20 in both eyes.


Tear calcium levels and contact lens wear.

Hydrophilic contact lens spoliation can be associated with the deposition of calcium salts. The relationship between the concentration of calcium in tear fluid and contact lens use has been studied in healthy volunteers and in patients using various forms of hard and soft contact lenses, tear samples being collected with Schirmer strips and with lenses in situ. Methods are suggested for correcting data on calcium per strip to concentration when this cannot be directly determined. Further data were obtained by direct extraction of freshly removed soft contact lenses. The collected results indicate some variation in tear calcium concentrations, but values are unrelated to use of contact lens and type of lens fitted, and to the rates of tear flow with our procedures for collection. Local calcium concentrations are unlikely therefore to be a significant primary factor in soft contact lens spoliation, but the enlargement of the tear pool associated with the use of a soft contact lens does greatly increase the amount of calcium present, and this may be a factor in secondary deposition.


Autolok band.

A braided polyester band to encircle the globe for retinal detachment has been tested. It has some advantages over other types of band.


Effect of exercise during rehabilitation on swimming performance, metabolism and function of muscle in rats.

1. It has been observed previously that gastrocnemius muscles from rats rehabilitated after early nutritional stress have low endurance and oxidative capacity (Raju, 1974). 2. Exercise during rehabilitation reversed the effects of early malnutrition on muscle function and metabolism. The swimming performance of rehabilitated rats was poor and exercise during rehabilitation improved it. 3. The specific activity of gastrocnemius muscle myosin ATP phosphohydrolase (EC 3.6.1.3) was not altered by either early malnutrition or exercise.


Digestibilities of nitrogen compounds in rumen bacteria and in other components of digesta in the small intestine of the young steer.

1. Mixed bacteria were obtained from the rumen contents of steers and were taken at different times after the animals had been given different feeds. The feeds contained [15N]urea and in some experiments Na2 35SO4 so that the bacteria were labelled with these isotopes. 2. Samples of labelled bacteria were subjected to a simulated abomasal digestion with pepsin and the digests were infused with a non-absorbed marker, polyethylene glycol, into the duodenums of test steers equipped with re-entrant ileal cannulas. Except for samples taken after a 24 h fast, which sometimes gave somewhat lower values, mean values for digestibilities of 15N and 35S in the small intestine were 0-79 and 0-85 respectively. 3. The corresponding value for the digestibility of 15N in similarly-treated wheat-leaf protein concentrate was 0-86. 4. Net digestibilities of total N in the small intestine of the test steers when they were given either a mainly protein-free diet of straw, tapioca and urea or a diet of flaked maize and hay were approximately 0-62. 5. From these and other values it was calculated that approximately 5-2 g intestinally-secreted endogenous N/d flowed through the ileum of a 100 kg steer.


The effect of acute illness on leucocyte and plasma ascorbic acid levels.

1. Serial estimations of plasma (PAA) and leucocyte (LAA) ascorbic acid concentrations were made in geriatric patients in a stable phase of their illness, and compared with a similar ('unstable') group suffering from acute illness. 2. In the 'unstable' group, though PAA levels were unchanged, LAA levels were reduced. 3. The 'follow-up' period of 7-8 d there was much greater variation in both PAA and LAA levels in the 'unstable' group. 4. Acute illness was often accompanied by an increase and then a decrease in LAA levels. Recovery was associated with an increase in LAA levels.


On the role of higher plant and microbial lipases in the ruminal hydrolysis of grass lipids.

1. The galactolipids of heat-treated, 14C-labelled rye grass S24 administered intraruminally to a sheep fed on an autoclaved diet were rapidly catabolized. 2. When grass was homogenized with rumen contents devoid of higher plant lipases the grass galactolipids were rapidly metabolized, but were not metabolized when the rumen contents were boiled to destroy microbial galactolipases. 3. 14C-labelled monogalactosyldiglyceride, digalactosyldiglyceride and triolein were metabolized, with the release of 14C-labelled fatty acids when incubated with a homogenate (100 g/l) of grass or clover in rumen fluid from a starved sheep, but not when the rumen fluid was heat-treated to destroy microbial enzymes. 4. It is concluded that in the sheep the lipases of rumen micro-organisms play a major part in the ruminal degradation of ingested complex lipids of pasture.


The effect of in utero protein malnutrition and subsequent renutrition on rat saliva and some salivary enzymes.

1. Rat pups developed chronic protein insufficiency resulting from malnutrition diet in utero while their dams received 40 g protein/kg diet. 2. Before weaning they were found to have decreased salivary amylase (EC 3.2.1.1) and aminopeptidase activities. 3. A complete diet (250 g protein/kg) rapidly restored salivary amylase activity before weight normalization. However, renourished pups (malnourished pups given 250 g protein/kg for 14 d) still had increased numbers of Streptococcus multans in their plaque.


The effect of dietary protein deprivation on protein synthesis in the isolated liver parenchymal cell.

1. Parenchymal cells were isolated from the liver of rats that had been deprived of dietary protein for 3 weeks. 2. The cells were two-thirds the diameter of those derived from livers of normal animals and consumed oxygen at a rate of 16-2 +/- 2-7 mu1/h per 10(6) viable cells, half the normal value. 3. Albumin and transferrin were synthesized at rates of 0-94 +/- 0-12 and 0-60 +/- 0-07 microgram/h per 10(6) viable cells respectively and urea at a rate of 0-77 +/- 0-12 microgram/h per 10(6) cells. This represents a 25-50% decrease in the rates of synthesis measured in cells isolated from normal livers. 4. The results are discussed in relation to the long-term effects of malnutrition on liver cell function.


A note on the effect of food restriction on tissue ascorbic acid in guinea-pigs.

1. Male, adult guinea-pigs received a scorbutogenic diet and a daily supplement of 1-0 mg ascorbic acid/100 g body-weight. 2. Restriction of food intake for a period of 17 d resulted in a 25% loss in body-weight and a significant reduction in the retention of ascorbic acid by the spleen, liver and adrenal glands.


Characterization of two major neutral glyceroglucolipids of the human gastric content.

Two major neutral glyceroglucolipids (A and B) have been isolated from lipid extract of human gastric content by the procedure involving column fractionation on DEAE-Sephadex, silicic acid, and thin-layer chromatography. Both glycolipids contained glucose, glyceryl ethers, and fatty acids. The structures of these glycolipids were identified by mild alkaline methanolysis, oxidation with periodate and chromium trioxide, and permethylation studies. Based on the obtained data, we propose that glycolipid A is a monoalkylmonoacylglyceryl hexaglucoside and glycolipid B is a monoalkylmonoacylglyceryl octaglucoside. The diglyceride portion of these glycolipids consists mostly of 1-O-alkyl-2-O-acylglycerol.


Hydroperoxidase activities of ferrihemes: heme analogues of peroxidase enzyme intermediates.

By reaction of deuteroferriheme (DFH) with peroxo acids, spectroscopically distinct and peroxidatically active deuteroferriheme-peroxide compounds (DPC) are formed. These species closely resemble, and are probably identical with, the species formed by reaction of DFH with H2O2, which has previously been considered to be an analogue of peroxidase compound I. Stopped-flow spectrophotometric titration studies imply: (a) that DPC is formed by reaction of 1.9 +/- 0.2 mol of DFH with 1 mol of peroxo acid; (b) that the spectral change accompanying formation of DPC is independent of the peroxo acid oxidant used. Titration of the oxidizing power of DPC formed with H2O2 implies that submaximal yields of DPC are obtained, a result that could implicate DPC species as analogues of catalase compound I in the catalase action of ferrihemes. Preliminary results suggest that DPC may involve both monomeric and dimeric heme components.


Rabbit muscle aldolase catalyzed proton exchange of hydroxyacetone phosphate with solvent.

Rabbit muscle aldolase catalyzes the exchange with solvent of all three methyl hydrogens of hydroxyacetone phosphate. Under saturating conditions, rates of the following processes have been measured: deuteration of hydroxyacetone phosphate in 2H2O (by an NMR method), tritiation of hydroxyacetone phosphate in H2O and 2H2O, and detritiation of tritiated hydroxyacetone phosphate in H2O and 2H2O. It is clear from these measurements (1) that there is no primary kinetic isotope effect and hence that hydrogen abstraction is not rate determining to the exchange and (2) that only one (as the closest integer) methyl hydrogen exchanges per turnover. The argument is made that these observations are mutually exclusive in terms of the accepted aldolase mechanism in the absence of further restrictions imposed by the enzyme. Possible restrictions are discussed.


Mouse sperm chromatin proteins: quantitative isolation and partial characterization.

Conditions are described that permit the quantitative extraction of chromatin proteins from the epididymal sperm of the mouse. These proteins have been isolated free of contaminating tail proteins following removal of the tails with cetyltrimethylammonium bromide (CTAB). Without this treatment, numerous acid-soluble tail proteins coextract with the nuclear proteins isolated from partially purified heads. The proteins isolated in this manner do not require prior modification with iodoacetamide and show no evidence of proteolytic degradation. In acid-urea polyacrylamide gels, 99% of the sperm protein migrates as one electrophoretic band. Evidence is presented that suggests that this single band contains two protamine-like proteins.


Characterization of mechanisms for transfer of cholesterol between human erythrocytes and plasma.

The removal from human erythrocytes of cholesterol (mass) and of [3H]cholesterol which had been introduced into the erythrocyte by exchange was studied. Removal was accomplished by incubating erythrocytes in plasma, the free cholesterol content of which had been lowered by the action of lecithin:cholesterol acyltransferase. It was shown that the exchange of cholesterol between erythrocytes and plasma and the net movement of cholesterol out of the membrane into plasma are characterized by the same rate constant and are driven by cholesterol to phospholipid ratios in cells and plasma. The apparent limitation on cholesterol depletion of erythrocytes observed in experiments of this type is explicable as the result of equilibrium between cholesterol in the membrane and in the plasma, an equilibrium reached when there is still cholesterol left in the cells. It is concluded from this study that all the exchangeable cholesterol in human erythrocytes is available for removal from the membrane.


Phospholipid head-group conformations; intermolecular interactions and cholesterol effects.

The predominant orientation of the phosphorylcholine polar head group in phosphatidylcholine and sphingomyelin bilayers and cholesterol perturbations of that orientation have been identified by exploiting the 31P (1H) nuclear Overhauser effect (NOE) in the 31P NMR spectra of phospholipid bilayers. In pure egg phosphatidylcholine bilayers, a NOE of 40% is observed. The magnitude of the NOE has been measured as a function of continuous-wave proton-decoupler frequency in order to identify the proton source of the NOE. In pure egg phosphatidylcholine bilayers, the maximum NOE occurs at the N-methyl proton resonance position of the choline moiety. In a modified phosphatidylcholine in which all the N-methyl protons were replaced by deuterium, the NOE arose from methylene protons next to the phosphate. In mixed systems of phosphatidylcholine and phosphatidylethanolamine, and phosphatidylcholine and diphosphatidylglycerol, both phospholipid resonances attained maximum NOE at the position of the N-methyl proton resonance of phosphatidylcholine. An analogous result was obtained with pure sphingomyelin. These results are explained by orienting the phosphorylcholine portion of the molecule parallel to the surface of the bilayer so that the positively charged N-methyl moiety is located close to the negatively charged phosphate on a neighboring phospholipid in an intermolecular interaction. Addition of cholesterol is shown to disrupt the intermolecular interaction in phosphatidylcholine bilayers.


A theory of phase transitions and phase diagrams for one- and two-component phospholipid bilayers.

A statistical mechanical partition function for phospholipid bilayers is constructed to obtain a theoretical description of the chain melting phase transition in lipid bilayer membranes and of the phase diagrams for two-component bilayers. In addition to providing an accurate representation of the transition temperatures and enthalpies of one-component bilayers composed of 1,2-diacylphosphatidylcholines, the theory can also account for the shapes of the phase diagrams observed for bilayers which are binary mixtures of these compounds with two different hydrocarbon chain lenghts.


Molecular asymmetry in an abortive ternary complex of lobster glyceraldehyde-3-phosphate dehydrogenase.

An abortive ternary complex of lobster glyceraldehyde-3-phosphate dehydrogenase was produced by the covalent attachment of 3,3,3-trifluoroacetone to Cys- 149 in each subunit. X-ray diffraction analysis of the glyceraldehyde-3-phosphate dehydrogenase-trifluoroacetone-nicotinamide adenine dinucleotide complex showed asymmetry with respect to the active-site conformations of the trifluoroacetone substrate analogue and some catalytic groups. These results are consistent with 19F nuclear magnetic resonance observations of this complex (Bode, J., Blumenstein, M., and Raftery, M. A. (1975), Biochemistry 14, 1153--1160). Different substrate conformations were found on opposite sides of the molecular diad relating subunits whose active centers are in close proximity (the R axis).


Distribution of alkali light chains in myosin: isolation of isoenzymes.

Antibodies have been isolated which are specific for the "difference peptide" unique to the alkali 1 light chain (mol wt 20 700) of chicken breast muscle myosin. When coupled to Sepharose as an immunoadsorbent, they are capable of resolving subfragment 1, heavy meromyosin, and myosin into two fractions, one rich in alkali 1 and the other rich in alkali 2. This fractionation provides direct evidence for the existence of two isoenzymic populations in vertebrate skeletal myosin. The ability of antibodies to the difference peptide to distinguish between alkali 1 and 2 provides a marker which will allow the distribution of alkali light chains in muscle fibers and filaments to be investigated.


Optical activity of disulfide bonds in proteins: studies on human choriomammotropin and bovine pituitary somatotropin.

The contributions of the homologous carboxyl-terminal disulfide bonds in human choriomammotropin (human chorionic somatomammotropin, human placental lactogen) and bovine somatotropin (pituitary growth hormone), to the near ultraviolet circular dichroism spectra of these two proteins have been evaluated. The disulfide bond in the human placental protein displays a broad, negative band centered near 260 nm ([theta]M, 260nm = -2100 +/- 160 deg cm2 dmol-1) which is equivalent, within experimental error, to the band previously assigned to the identical disulfide in plasmin modified human somatotropin. The homologous disulfide in the bovine hormone also exhibits a negative band, very similar in intensity ([theta]M, 254nm = -2200 +/- 210 deg cm2 dmol-1), with an estimated band center blue-shifted relative to the human proteins to 252-255 nm. Reoxidation of either partially reduced protein results in complete repair of the circular dichroism spectrum to that of the native protein. No definite contributions could be assigned below 240 nm to the optical activity of these disulfide bonds. Circular dichroism measurements have also been used to approximate the rates of reduction of the two proteins.


Isolation and amino-terminal sequence analysis of a new pancreatic trypsinogen of the African lungfish Protopterus aethiopicus.

The purification and characterization of three pancreatic trypsinogens A1, A2, and A3, from the African lungfish, Protopterus aethiopicus, is reported. These zymogens are activated by trypsin, by enterokinase, by an acid protease from Aspergillus oryzae, and by autoactivation. The three trypsinogens contain the same amino-terminal amino acid sequence, beginning with the activation peptide Leu-Pro-Leu-Glu-Asp-Asp-Lys-. Like the activation peptide of the previously characterized trypsinogen B [Reeck, G. R., & Neurath, H. (1972) Biochemistry 11, 503] of the same organism, it lacks the tetraaspartyl sequence characteristic of other vertebrate trypsinogens. Two of the corresponding lungfish trypsins were found to have identical amino-terminal sequences for at least 27 residues. These data suggest that the three enzymes are allelic variants. In contrast, the amino acid sequences differ sufficiently from that of trypsinogen B of the same organism to indicate that trypsinogens A and B are the products of different gene loci.


A Raman spectroscopic investigation of the disulfide conformation in oxytocin and lysine vasopressin.

The conformation of the CCSSCC moiety in oxytocin and lysine vasopressin is investigated using laser Raman spectroscopy. The Raman spectra of solutions of these hormones in water and in dimethyl sulfoxide show an intense band at 508 cm-1 which is assigned to the S-S stretching mode. The presence of shoulders on this band between 490 and 525 cm-1 shows that there is an equilibrium among several conformations for the disulfide unit of these hormones in solution. Most of the CS-SC dihedral angles are within 30 degrees of +/-90 degrees, but some of the molecules have CS-SC dihedral angles strained away from this value by more than 30 degrees. The previously published circular dichroism spectra of these hormones are reinterpreted, and it is shown that the circular dichroism spectra indicate the presence of more than one conformation for the disulfide unit, in agreement with the Raman results.


Incorporation of deoxynucleotides into DNA by diethylaminoethyldextran-treated lymphocytes.

In the presence of diethylaminoethyldextran cultured human lymphocytes will utilize deoxynucleotides for the synthesis of DNA, whereas in the absence of diethylaminoethyldextran no incorporation of deoxynucleotides is detected. Labeled deoxynucleoside mono-, di-, and triphosphates are incorporated into DNA at approximately the same rate. Deoxynucleotide incorporation is essentially linear for 10 min but continues at a gradually diminishing rate for an additional 20 to 50 min. The initial rate of DNA synthesis is at least 20 to 40% of the in vivo rate, and in those cells that are in S phase 0.7-1.5% of the DNA is synthesized. By the three properties examined (restriction to S phase, semiconservative mode, and initial product in short chains), DNA synthesis in diethylaminoethyldextran-treated cells resembles DNA synthesis in vivo.


Intermediates in excision repair by human cells: use of S1 nuclease and benzoylated naphthoylated cellulose to reveal single-strand breaks.

Treatment of human lymphoid cells with methyl methanesulfonate (MMS) results in discontinuities in cellular DNA. Discontinuities can be detected by sedimentation through neutral sucrose gradients after digestion with S1 nuclease on top of the gradient and by benzoylated naphthoylated diethylaminoethylcellulose (BND-cellulose) chromatography. DNA with discontinuities is sensitive to shear which produces single-stranded regions and causes the DNA to adhere to BND-cellulose. The shear-sensitive and S1-sensitive sites produced by MMS treatment are probably identical. Treatment of cells with acetoxyacetylaminofluorene (AAAF) does not result in detectable discontinuities either at doses which produce the same number of adducts as observed after MMS treatment or at doses which inhibit DNA excision repair. Addition of aromatic adducts to DNA can also result in adherence to BND-cellulose but such DNA is not S1 sensitive. We conclude that MMS- and AAAF-induced damage are repaired by different pathways in human cells and that DNA with discontinuities is a readily detectable intermediate only in the repair of MMS damage by the apurinic pathway.


Isolation and characterization of the four major proteins in the virion of bacteriophage phiX174.

A preparative method is described for the isolation of the major protein species from the virion of bacteriophage phiX174. Two proteins, the cistron G and H products, are located in the virion spikes. After removal of the spikes, the capsid contains the cistron F product as well as a small protein which is the product of cistron J and the majority of the DNA. During the removal of the spikes, a precipitate containing the F and G proteins is formed. The proteins from the spike, capsid, or precipitate can be isolated on the basis of size by gel-filtration chromatography. The cistron G protein has an aminoterminal methionine, while the small J protein has an amino-terminal serine. Amino acid compositions as well as peptide maps indicate each species is unique and that, in sum, they account for over half the coding capacity of the viral genome.


Amino acid sequence of the small core protein from bacteriophage phiX174.

The amino acid sequence of small core protein of bacteriophage phiX174 has been determined by a combination of automated Edman degradation of the intact polypeptide and by analysis of tryptic and thermolytic peptides. The six lysyl and six arginyl residues of this 37-residue polypeptide are concentrated in two structurally homologous 12-residue segments of the sequence. The hydrophobic residues of valine, tryptophan, tyrosine, and phenylalanine are contained in the carboxyl-terminal nine residues of the protein, together with one of the two leucyl residues and two of the three glutaminyl residues. The single free carboxyl group in the protein is the alpha-COOH of the C-terminal phenylalanyl residue. The overall sequence of this small core protein suggests that it may function as a DNA-condensing protein. The protein sequence presented here corresponds exactly to the DNA base sequence of the cistron J region of the phiX174 genome determined in another laboratory.


Solanesyl pyrophosphate synthetase from Micrococcus lysodeikticus.

Solanesyl pyrophosphate synthetase from extracts of Micrococcus lysodeikticus was purified by DEAE-Sephadex, hydroxylapatite, and Sephadex G-100 chromatography. This enzyme was found to catalyze the trans condensation of isopentenyl pyrophosphate with geranyl pyrophosphate to afford all-trans-octaprenyl (C40) and alltrans-nonaprenyl (C45) pyrophosphate without accumulation of prenyl pyrophosphate with chain length shorter than C40. all-trans-Farnesyl and all-trans-geranylgeranyl pyrophosphate also were active as cosubstrates, though they were less effective than geranyl pyrophosphate. However, neither dimethylallyl nor cis,trans,trans-geranylgeranyl pyrophosphate was active. The molecular weight of this enzyme was estimated to be 78 000 by Sephadex G-100 filtration. An enzyme preparation from young shoots of potato was found to hydrolyze the polyprenyl pyrophosphates effectively to give the corresponding prenols.


Effects of ultrasound on nucleic acid bases.

To understand the effects of ultrasound in biological systems at the molecular level, sonolysis of nucleic acid bases at sonic intensities less than or equal to 5 W/cm2 was studied. These sonoreactions were followed by UV-spectral decrease and by sonoproduct analysis. The order of reactivity was found to be thymine greater than uracil greater than cytosine greater than guanine greater than adenine. The extent of sonoreactions depends on the exposure time and the rate of the intensity. Aeration was necessary for maintenance of a reasonable reaction rate. "Threshold" intensities for uracil and thymine were observed at approximately 0.5 W/cm2. The effectiveness of the dissolved gases in producing sonoreactions was Ar greater than 02 greater than air greater than N2 greater than He greater than N2O, suggestive of free radicals mediating these reactions since N2O is an effective radical scavenger. Studies of the effects of substituents have shown that electronic rather than steric effects may have a greater influence. Preliminary identification indicates that cis- and trans-uracil glycols are the major products of uracil. Thus, a stepwise mechanism of pyrimidine sonolysis is proposed. Sonolysis of a dilute aqueous solution of uracil yielded pseudo-first-order kinetics in terms of [Ura] with a rate constant of 3.9 X 10(-2) min-1, implying that the rate-limiting step is the reaction of HO- with the base.


Properties of the partially purified activated glucocorticoid receptor of rat liver. Binding to chromatin subunits.

The activated glucocorticoid receptor from rat liver has been purified over 3000-fold by repeated adsorption to phosphocellulose. The partially purified receptor-triamcinolone complex is stable for sever weeks at -75 degrees C in the presence of 0.1% bovine serum albumin, and sediments at 2.9 (+/- 0.1) S in sucrose gradients containing 0.15 M Na Cl. The Stokes' radius of the partially purified receptor is 26.5 A, and the frictional ratio is 1.14, indicating that at near physiological salt concentration the activated receptor is slightly elongated and has a molecular weight of 33 500. These preparations of receptor are free of exo- and endonucleases and bind to DNA and chromatin, as well as to the native chromatin subunits, the nucleosomes. Removal of histone H1 and further digestion of the nucleosomal DNA to the core size of about 140 base pairs indicate that the integrity of the nucleosome structure determines the extent of interaction with the glucocorticoid receptor. Although the binding of receptor to unfractionated rat liver chromatin is more efficient than to chicken erythrocyte chromatin, the nucleosomes of both tissues bind equal amounts of receptor. Therefore, the factors responsible for this tissue difference do not reside in the nucleosomes, but rather in a higher order structure of the chromatin or in that part of the genome which is not organized as nucleosomes.


Intramitochondrial intermembranal reversible translocation of aspartate aminotransferase and malate dehydrogenase through the inner mitochondrial membrane.

The translocation of aspartate aminotransferase, malate dehydrogenase, and bulk protein from the rat liver inner mitochondrial membrane and matrix toward the intermembranal space induced by certain organic acids (movement effectors) has been studied. Experiments involving a two-stage dissolution of the mitochondrial membranes by the use of detergents strongly suggest that enzymes like aspartate amino-transferase can cross the inner mitochondrial membrane providing exogenous movement effector was present. Experiments which measured the changes in membranal distribution of malate dehydrogenase induced by the movement effectors also suggested the occurrence of a similar phenomenon for this enzyme in intact mitochondria. Control experiments revealed that under our experimental conditions, the inner mitochondrial membrane remained impermeable to small molecules, e.g., sucrose, and that the release of aspartate aminotransferease, malate dehydrogenase, insocitrate dehydrogenase, and bulk protein into the intermembranal space in the presence of succinate occurred at a much lower concentration of digitonin than that required to disrupt the inner mitochondrial membrane.


Measurement of binding constants for protein-DNA interactions by DNA-cellulose chromatography.

We describe the application of DNA-cellulose chromatography to the determination of binding constants for the nonspecific interaction between proteins and DNA. The method involves loading a small DNA-cellulose column to a low binding density and subsequently eluting the protein with buffer at a constant salt concentration. The elution is conveniently followed by monitoring the protein fluorescence of the eluate. From the shape of the elution profile, the binding constant for the interaction can be calculated. Employing columns containing double-stranded calf thymus DNA-cellulose, we have measured binding constants in the range of 10(4) to 10(6) M-1. Extension of this range is possible by varying the amount of DNA on the column. For lac repressor, agreement between our measurements and those of Revzin, A., and von Hippel, P.H. [(1977) Biochemistry 16 (second of five papers in a series in this issue)], who employ an absolute boundary sedimentation method, is good. The column method should be useful for the rapid screening of the effect of a large number of variables on protein-DNA binding constants.


Nonspecific interaction of lac repressor with DNA: an association reaction driven by counterion release.

We have investigated the nonspecific interaction of lac repressor protein with DNA by a quantitative application of DNA-cellulose chromatography (deHaseth, P.L., et al. (1977), Biochemistry 16 (third of five papers in a series in this issue)). The observed association constant for the interaction, KRD obsd, is a sensitive function of ion concentrations and pH. Application of binding theory to interpret these effects gives the results that 11 +/- 2 monovalent ions are released in the interaction and two groups on repressor must be protonated for repressor to bind to DNA. We argue that much of the ion release results from the displacement of cations from the DNA, and estimate on this basis that 12 +/- 2 phosphates are involved in ionic interactions with the protein. Ion release drives the protonation reaction and the overall repressor-DNA interaction. The major role of low molecular weight ions in the repressor-DNA interaction suggests that ion concentration changes must be considered in discussing mechanisms of control of gene expression.


Interpretation of monovalent and divalent cation effects on the lac repressor-operator interaction.

We have investigated the effects of mixed Na+: Mg2+ ionic solutions on the stability of the nonspecific lac repressor-DNA complex. The effects of Mg2+ are simply interpreted in terms of its role as a competitor (with repressor) for DNA sites. From these studies, the binding constant of the Mg-DNA complex can be determined as a function of the concentration of Na+. We have used this information to interpret the data of Riggs and collaborators (Riggs, A.D., et al. (1970), J. Mol. Biol. 48, 67-83; 53, 401-417) on the ion dependence of the repressor-operator interaction. We find that there are approximately 70% as many ionic interactions in the repressor-operator complex as in the nonspecific complex. Our best estimate is that 8 +/- 1 ion pairs are formed. We calculate that the release of counterions in the formation of the specific complex contributes approximately 40% of the favorable free energy change in the association reaction under in vivo ionic conditions. Implications of these findings for the control of the lac operon and for the molecular relationship between the specific and nonspecific complexes are considered.


Composition and template activity of chromatin fractionated by isoelectric focusing.

HeLa cell interphase chromatin has been sheared and fractionated by isoelectric focusing. Chromatin fractions are obtained with a wide range of isoelectric points. No free DNA is observed. While protein/DNA rations are similar in the various fractions, they appear to contain different nonhistone chromosomal proteins. A minor chromatin fraction with isoelectric point congruent to 7.0 does not contain histone H1. This fraction is considerably more active as template with different RNA polymerases than the other fractions. Kinetic studies, in which RNA polymerase activity is assayed at various concentrations of chromatin, indicate that the greater activity of Escherichia coli RNA polymerase is due to an increased rate of transcription at saturating concentrations of template (Vmax) and is not due to a lower concentration required for half-maximal rate of transciption (Km). In contrast, the increased rate of transcription by calf-thymus RNA polymerases II and III is due to a decrease in chromatin concentration required for half-maximal rate of transcription rather than an increased rate of transcription at saturating concentrations of template. These results suggest that chromatin with isoelectric point congruent to 7 offers a greater frequency of binding sites for mammalian RNA polymerases, as would be expected for a "transcriptionally active" fraction.


Active-site labeling of aspartate aminotransferases by the beta,gamma-unsaturated amino acid vinylglycine.

The pyridoxal form of both cytosolic and mitochondrial aspartate aminotransferase is irreversibly inactivated consequent to its interaction with the beta,gamma-unsaturated substrate analogue vinylglycine. Per catalytic cycle, 90% of the enzyme molecules are inactivated while 10% escape inactivation by transamination to the pyridoxamine form. In the presence of vinylglycine plus 2-oxoglutarate, inactivation is complete because of retransamination of the pyridoxamine form to the susceptible pyridoxal form. Peptide analyses after inactivation with [1-14C]vinylglycine showed that vinylglycine alkylates the active-site lysine residue 258 which forms the internal aldimine with the coenzyme pyridoxal 5'-phosphate. The coenzyme itself is left intact; resolution of the inactivated enzyme by base or trichloroacetic acid yields pyridoxal-5'-P. The absorption spectrum of the inactivated enzyme (lambdamax 335 nm) suggests that the cofactor is bound as a substituted aldimine. The proposed pathway of alkylation of Lys-258 involves abstraction of the alpha proton from vinylglycine, isomerization to the alpha,beta-unsaturated enamine, and subsequent nucleophilic attack of the epsilon-amino group of the lysyl residue at the beta carbon of the inhibitor. The determination of the amino acid sequence around the coenzyme-binding lysyl residue in the mitochondrial isoenzyme from chicken gave Ala-(epsilon-Pxy)Lys-Asn-Met-(Gly,Leu,Tyr) which is identical with the other mitochondrial transaminases examined so far.


Rearrangement of chorismate to prephenate. Use of chorismate mutase inhibitors to define the transition state structure.

The enzymically catalyzed conversion of chorismate to prephenate may proceed through either a chair-like or a boat-like transition state. To distinguish between these alternatives, we have prepared a series of structural analogues of the two possible transition state structures and tested them as inhibitors of chorismate mutase-prephenate dehydrogenase from Escherichia coli K12. The results indicate that the enzymically catalyzed reaction passes through a chair-like intermediate. None of the compounds studied is an ideal transition state analogue; it seems likely that the partial bond structure of the transition state precludes the corresponding orientation of the side chain in stable molecules. Nevertheless, the new inhibitors are stronger than any previously available, and the degree of inhibition is consistent with bacteriostatic activity recently observed in some of the compounds.


Volume changes in the formation of internal complexes of flavinyltryptophan peptides.

The effect of pressure, up to 10 kbar, on the fluorescence yield and lifetime of two flavinyltryptophan peptides was investigated. These peptides differed only in the number of methylene groups, respectively three and five, separating the chromophores. At atmospheric pressure the closed nonfluorescent form predominated in both compounds constitutin 94% of the total in the short-linked peptide and 80% in the long-linked one. The fluorescence of both peptides decreased at high pressure and the volume change upon formation of the nonfluorescent complex in the short peptide (--1.8 mL/mol) was less than half of the change in the long peptide (--4.8 mL/mol) or the value for FAD (--4.3 mL/mol). The much smaller compressibility of the short peptides is attributed to the mechanical constraint to the approach of the interacting rings, imposed by the short link. Mechanical constraints of similar nature may be expected to be operative in proteins. Their importance in pressure denaturation is discussed.


Antiproteolytic aldehydes and ketones: substituent and secondary deuterium isotope effects on equilibrium addition of water and other nucleophiles.

Equilibrium constants for hydration of ketones, in dilute D2O solution at 34 degrees C, observed by proton magnetic resonance under conditions of slow exchange, were acetone 0.002, chloroacetone 0.08, 1,3-dichloroacetone 4.17, bromoacetone 0.07, and 1,3-dibromoacetone (an inhibitor of papain) 1.85. Neither acetamidoacetone nor N,N-diacetylaminoacetone showed evidence of appreciable hydration in dilute aqueous solution, nor was any hydrate detectable in solutions of tosylglycine chloromethyl ketone. Substitution of acetaldehyde with acylamido substituents, as in several potent reversible inhibitors of papain, was found to enhance its equilibrium constant for covalent hydration by an order of magnitude; these inhibitors are about 90% hydrated in dilute aqueous solution, and their affinity for proteases may have been underestimated accordingly. The effects of deuterium substitution at C-1 of acetaldehyde, on equilibrium addition of oxygen and sulfur nucleophiles, are substantial and vary with the nature of the nucleophile. These isotope effects may be useful as a mean of distinguishing between alternative structures of complexes formed between enzymes and aldehydes.


Thiohemiacetal formation by inhibitory aldehydes at the active site of papain.

Papain is strongly inhibited by aldehydes resembling carboxylic acids, released by hydrolysis of specific substrates (Westerik, J. O'C., and Wolfenden, R. (1972), J. Biol. Chem. 247, 8195-8197). Inhibitory complexes might involve binding of the aldehyde intact or as a covalent hydrate, or the aldehyde might undergo covalent addition of an active site sulfhydryl group to form a thiohemiacetal derivative. In an attempt to distinguish between these possibilities, benzamidoacetaldehyde-1-d has been synthesized, and its properties compared with those of the undeuterated inhibitor. After correction for differences in hydration, the observed effect on inhibition is found to be compatible with formation of a thiohemiacetal. In keeping with this conclusion, benzamidoethanol (a partial analogue of the covalent hydrate) and benzamide, N-methylbenzamide and N-ethylbenzamide (somewhat similar to the free aldehyde in size and hydrophobic character) are found to exhibit negligible affinity for the active site.


Effect of estradiol-17 beta on  preprolactin messenger ribonucleic acid activity in the rat pituitary gland.

Rat pituitary RNA was translated in the wheat germ system. Preprolactin messenger RNA activity was estimated by adsorption of cell-free products to solid phase antiprolactin. When male rats were injected for 4 days with estradiol-17beta, pituitary preprolactin mRNA activity was increased 2.5- to 3.0-fold over controls. This increase was evident when either total RNA, poly(adenylic acid) RNA, or polysomal RNA was translated in the cell-free system. In male rats receiving daily injections of estradiol-17beta, preprolactin mRNA activity was increased to an apparent maximum of 300% of controls after 7 days of treatment. Our data also indicate that estradiol increases preprolactin mRNA activity per microgram of RNA as well as the pituitary content of RNA. After estradiol treatment was discontinued, preprolactin mRNA activity declined to 50% of the maximum stimulation after approximately 2 days. In ovariectomized retired breeder female rats, a 5-fold increase in preprolactin activity over ovariectomized controls was obtained. In other studies, a 2-fold increase in preprolactin mRNA activity was obtained in male rats 24 h after a single injection of pimozide, a dopamine blocking drug.


Histone composition of nucleosomes isolated from cultured Chinese hamster cells.

Nuclei isolated from cultured Chinese hamster cells were treated with micrococcal nuclease and lysed, and the resulting chromatin subunit classes (nucleosomes) were purified by sedimentation and resedimentation through isokinetic sucrose gradients. Nucleosomes isolated from [3H]thymidine-labeled cells were analyzed for DNA size using both polyacrylamide gel and electron microscopic techniques. Nucleosomes isolated from [14C]lysine-labeled cells were analyzed for protein content using a sodium dodecyl sulfate-polyacrylamide gel system. The results from monitoring the [14c]lysine in each protein indicate that, in the nucleosome classes (monomer through tetramer), the molar ratios of histones H2A, H2B, H3, and H4 are equivalent. Furthermore, in each population of the nucleosome classes monomer through tetramer, it was possible to demonstrate that this histone unit (H2A + H2B + H3 + H4) is present, on the average, in the amount of two for monomers, four for dimers, six for trimers, and eight for tetramers. This is direct experimental confirmation of the prediction of R.D. Kornberg [(1974) Science 184, 868] concerning the substructure of chromatin.


Isolation of the unliganded adenosine 5'-diphosphate, adenosine 5'-triphosphate carrier-linked binding protein and incorporation into the membranes of liposomes.

The ADP,ATP carrier-linked binding protein of beef heart mitochondria was isolated free of ligands, using the detergent 3-lauramido-N,N-dimethylpropylamine oxide. Unlike the preparation of the carboxyatractylate (CAT)-protein complex described earlier, this protein enables direct binding studies to be made with the inhibitor ligands. The protein was characterized with respect to its polypeptide composition, stability against degradation, and immunological properties; the identity of the binding protein with the previously isolated CAT-protein complex was thereby shown. As a step toward reconstitution studies, the isolated binding protein was incorporated into liposomes by a simple rapid mixing process. The complete insertion into the vesicular membrane was demonstrated by chromatography on Sepharose 6B and by immunoprecipitation reactions.


Reconstitution of inhibitor binding properties of the isolated adenosine 5'-diphosphate,adenosine 5'-triphosphate carrier-linked binding protein.

We studied the binding of carboxyatractylate (CAT) and bongkrekate (BKA) to the solubilized ATP,ADP carrier-linked binding protein, which had been incorporated into liposomes. After solubilization with 3-lauramido-N,N-dimethylpropylamine oxide the binding protein had largely lost it affinity and binding capacity for both CAT and BKA. On incorporation into phospholipid vesicles, CAT binding was restored to a considerable extent (3.5 mumol of CAT/g of protein), reaching the original affinity as observed in mitochondria (Kd = 10(-8) M). With high amounts of CAT and under the influence of ADP the binding can be increased to 6.8 mumol of CAT/g of protein, indicating a movement of binding sites in the liposomal membrane. The binding of BKA was also reconstituted with high affinity (Kd = 8 X 10(-8) M) and to the same extent (6.4 mumol of BKA/g of protein). As in the case of intact mitochondria, this reconstituted binding depends on the presence of ADP. This dependence on ADP has an apparent Km = 7 muM, similar to the carrier affinity for ADP in intact mitochondria. The reorientation model of Klingenberg for the ADP,ATP carrier implicating an ADP-catalyzed transition between the CAT binding form (c state) and BKA binding form (m state) in the inner mitochondrial membrane has been confirmed in this reconstituted system.


Oxidative titrations of reduced cytochrome aa3: correlation of midpoint potentials and extinction coefficients observed at three major absorption bands.

Anaerobic oxidative titrations of purified cytochrome aa3 were monitored at three wavelengths (444, 604, and 820 nm), in both the absence and the presence of carbon monoxide. Computer simulation of each titration curve was utilized to ascertain the midpoint potentials of the four oxidation-reduction centers of the enzyme. For experiments performed under nitrogen, two components were found to titrate with low potential (heme aL = 220 mV, CuL = 240 mV) and two with high potential (heme ath, cuH = 340 mV), consistent with results obtained previously in reductive titrations. Unequal heme extinction coefficients were observed at 444 nm. Oxidation by either potassium ferricyanide or 1,1'-bis(hydroxymethyl)ferricinium ion showed that the low potential heme component contributed 75% of the absorbance change at 444 nm. At 820 nm, the entire absorbance change could be attributed to a single, low potential copper component. Midpoint potentials calculated for the carbon monoxide complexed enzyme agreed with previously reported values. The copper components retained the values observed under nitrogen, while the titratable heme group gave an apparent midpoint potential of 260 mV. These results enable us to assign absorbance changes at various wavelengths to specific redox components of cytochrome aa3.


Complete primary structure of the major component myoglobin of Pacific common dolphin (Delphinus delphis).

The complete amino acid sequence of the major component myoglobin from Pacific common dolphin, Delphinus delphis, was determined by the automatic Edman degradation of several large peptides obtained by specific cleavages of the protein. More than 80% of the covalent structure was established by the degradation of the apomyoglobin and five peptides from: (1) cyanogen bromide cleavage at the two methionine residues, (2) trypsin cleavage of the acetimidated apomyoglobin at the three arginine residues, and (3) 2-p-nitrophenylsulfenyl-3-methyl-3'-bromoindolenine cleavage at the two tryptophan residues. The rest of the sequence was determined by use of the peptides prepared from further digestion of the central cyanogen bromide peptide with staphylococcal protease and trypsin. The primary structure of this myoglobin proved identical with that from the Atlantic bottlenosed dolphin, Tursiops truncatus, but showed four substitutions with respect to the sequence reported for the Black Sea dolphin which has also been given the designation Delphinus delphis.


Mitochondrial neutral amino acid transport: evidence for a carrier mediated mechanism.

Swelling of rat liver mitochondria induced by various neutral amino acids indicates that the L isomers of serine, alanine, methionine, valine, threonine, and leucine enter the mitochondrial matrix by a stereospecific process. Chemical modification of mitochondria with diazobenzenesulfonate or p-mercuribenzoate inhibited the rapid uptake of these compounds, as well as that of L-proline and glycine, to various extents. Diazobenzenesulfonate did not inhibit the transport of compounds that enter the mitochondrial matrix by simple diffusion, i.e., thiocyanate, nitrate, formate, bicarbonate, and acetate. Inhibition by p-mercuribenzoate was reversed by treatment with dithiothreitol. Inclusion of various neutral amino acids in the p-mercuribenzoate preincubation mixture substantially prevented inactivation of transport. Glycine, D- or L-serine, L-threonine, L-methionine, L-alanine, and L-valine all individually protected against the inactivation of glycine, L-serine, L-threonine, L-methionine, L-alanine, L-valine, L-proline, and beta-alanine transport, while L-proline, beta-alanine,


Isolation and characterization of rat liver free and membrane-bound polysomal messenger ribonucleoprotein particles.

Rat liver mRNA-labeled free and membrane-bound polysomes uncontaminated with nuclear or cytoplasmic ribonucleoprotein particles were dissociated with EDTA and the released messenger ribonucleoprotein particles isolated using an oligo(dT)-cellulose column. Seventy percent of the labeled mRNA applied bound to the column. Binding of mRNP was dependent on the presence of a poly(A) segment in the RNA. The nonbound material contained most of the ribosomal subunits and also messenger-sized poly(A-)RNA molecules associated with protein. The free mRNP fraction bound to the column, washed with 250 mM NaCl, and subsequently eluted contained seven proteins ranging in molecular weight from 52 000 to 138 000 only one of which was found in the fraction not bound to the column. Furthermore, these proteins were shown to have a higher affinity for poly(A+)RNA as compared with rRNA. The membrane-bound mRNP contained five proteins, four of which were identical with those associated with free mRNP. Membrane-bound mRNP were disrupted at a lower salt concentration than the free. The proteins found associated with free and membrane-bound polysomal mRNP appeared to be clustered in the poly(A) region of the molecule. The implications of these findings are discussed.


Some photoreactions of isolated cytochrome b-559.

Cytochrome b-559 was isolated from spinach and the alga Bumilleriopsis filiformis (Xanthophyceae) and characterized by functional properties: (a) It was active as electron acceptor in a diaphorase system using NADPH as donor and ferredoxin and ferredoxin-NADP reductase as redox proteins. (b) It exhibited photooxidation with Photosystem-I particles, when illuminated with 707 nm light. (c) It was photooxidized by Photosystem-II particles and 652 nm light at room temperature. Light greater than 702 nm was ineffective. The data corroborate previous reports on redox reactions of bound cytochrome b-559.


Influence of the energetic state of rat liver mitochondria on the sensitivity of the phosphate carrier towards SH reagents.

Phosphate transport into rat liver mitochondria was measured by the swelling technique in 0.1 M ammonium phosphate. Energized or non-energized mitochondria were preincubated with different thiol reagents and evidence is given that with a slow-reacting thiol reagent, ethacrynate, the inactivation of the phosphate carrier is obtained when mitochondria are energized, while poor or no inactivation occurs when mitochondria are non-energized or preincubated with Pi. Moreover, the inactivation depends on the presence of Mg2+ and on the nature of the substrate. Some comparative essays were done using N-ethylmaleimide as a thiol reagent, but no energy-linked variation of N-ethylmaleimide inhibition on phosphate transport was obtained. Taking into account the fact that both thiol-reagents incorporation into rat liver mitochondria is sitmulated by the presence of substrate, the different behaviour of these two thiol-reagents towards Pi transport is discussed on the basis of their different reactivity with SH groups.


Studies on the inner mitochondrial membrane localization of proline dehydrogenase.

1. The site of proline dehydrogenase (EC 1.5.99.-) activity in blowfly (Phormia regina) flight muscle mitochondria has been investigated employing the inner membrane-impermeable Fe(CN)63-as electron acceptor. Antimycin had no inhibitory effect on ferricyanide reduction due to proline dehydrogenase activity. Ferricyanide reductase activity due to inside localized dehydrogenase activity was antimycin sensitive. These results indicate that the interaction between proline dehydrogenase and ferricyanide was direct and not dependent on respiratory chain activity. 2. The stimulatory action of the effector, ADP, on proline dehydrogenase activity was insensitive to atractyloside, an indication that the site of dehydrogenase interaction with ADP was external to the atractyloside barrier. 3. Swelling studies revealed that proline does not readily penetrate the matrix space. 4. An outside localization for proline dehydrogenase is discussed in terms of the role of proline in insect flight muscle metabolism.


Excited state annihilation in the photosynthetic unit.

The kinetics of the in vivo fluorescence decays and fluorescence yields, as a function of excitation intensity, have been analysed with a model using excited state annihilation and time-dependent quenching processes. Triplet states, formed in the singlet-singlet annihilation processes, account for additional quenching of singlet states and the persistence of annihilation at longer times than the fluorescence life-time. Together these processes give a satisfactory account of existing experimental data of the intensity dependence of fluorescence in vivo.


The relationship between the activity of chloroplast photosystem II and the midpoint oxidation-reduction potential of cytochrome b-559.

The role of cytochrome b-559 in Photosystem II reactions has been investigated using hydroxylamine treatment of chloroplast membranes. Incubation of chloroplasts with hydroxylamine in darkness resulted in inhibition of water oxidation and a decrease in the amplitude of cytochrome b-559 reducible by hydroquinone. The loss of water oxidizing activity perfectly correlated with the decrease in amplitude of cytochrome b-559 reduction. Potentiometric titration of cytochrome b-559 after hydroxylamine treatment revealed a component with Em7.8 at +240 mV in addition to a lower potential species at +90 mV. This compared to control chloroplasts in which cytochrome b-559 exists in the typical high potential state, Em7.8 = +383 mV, in addition to some of the low potential (Em7.8 = +77 mV) form. Photosystem II activity could be further inhibited by incubation with hydroxylamine in the light. In these chloroplasts only low rates of photooxidation of artificial electron donors were observed compared to 'dark' chloroplasts. In addition, the hydroxylamine light treatment caused a further change in cytochrome b-559 redox properties; a single component, Em7.8 = 90 mV is seen in titration curves. The role of cytochrome b-559 in Photosystem II functioning is discussed on the basis of these observations which suggest a dependence of photooxidizing ability of Photosystem II on the redox properties of this cytochrome.


13 C NMR measurements of unsonicated phosphatidylcholine bilayers of different fatty acid and sterol composition.

(1) 13C NMR linewidths were measured for various 13 C resonances in unsonicated dispersions of synthetic and natural phosphatidylcholines both in the absence and presence of cholesterol at temperatures where the acyl chains are in the liquid-crystalline state. (2) In the absence of cholesterol the linewidths of the various resolved chain resonances were decreased with increasing unsaturation and decreasing chain length. The motion of the delta9-cis olefinic carbon atoms in dioleoylphosphatidylcholine was more restricted than the motion of the delts9-cis olefinic carbon atoms in 1-stearoyl-2-oleoyl-phosphatidylcholine despite the higher overall fluidity of dioleoylphosphatidylcholine. The polar head-group motion was not dependent upon the fatty acid composition. (3) Incorporation of cholesterol broadens all observed chain resonances of all phosphatidylcholines, thus demonstrating a reduction in chain motion by cholesterol. For both the saturated and unsaturated phosphatidylcholines the reduction of the chain motion is decreased with increasing chain length. (4) The chemical shift of the carbonyl resonances of sonicated dipalmitoylphosphatidylcholine vesicle labelled with 13C in both chains in the 1-position was slightly decreased by the incorporation of 50 mol% of cholesterol. In contrast, 50 mol% of epicholesterol, the 3alpha0OH isomer, produced a large downfield shift and a splitting of the carbonyl resonance.


The interaction of spectrin - actin and synthetic phospholipids.

Using differential scanning calorimetry and freeze fracture electron microscopy interactions were studied between lipids and a spectrin - actin complex isolated from human erythrocyte membranes. With dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol and mixtures of these two compounds, which for experimental reasons were chosen as the lipid counterpart, such an interaction could clearly be deduced from changes in the temperature and the enthalpy of the phase transition. Furthermore it was demonstrated that the interaction with this membrane protein protects the bilayer against the action of Ca2+ and Mg2+ and prevents fusion of lipid vesicles which easily occurs in some of the systems when divalent ions were added to the pure lipid vesicles.


Theory of self-assembly of lipid bilayers and vesicles.

A simple theory is developed that explains the formation of bilayers and vesicles and accounts quantitatively for many of their physical properties: Properties including vesicle size distributions and bilayer elasticity emerge from a unified theory that links thermodynamics, interaction free energy, and molecular geometry. The theory may be applied to the analysis of more complicated membrane structures and mechanisms.


Small-angle X-ray scattering from mitochondria.

X-ray (CuKalpha) scattering curves of rat liver mitochondria are characterized by continuously decreasing intensity from 0.5 to 5 mrad and a broad maximum centered near 20 mrad. The condensed-to-orthodox morphological transition of the inner membranes of intact mitochondria causes a dramatic decrease in scattering at very small angle and a marked shift of the 20 mrad maximum to smaller angle. A similar small-angle scattering maximum is observed with inner mitochondrial membrane fractions prepared by digitonin treatment and osmotic shock/step gradient centrifugation procedures. However, the small-angle X-ray scattering curves of mitochondria after acetone treatment and osmoticlysis/sonication are essentially continuous. These characteristics of mitochondrial X-ray scattering are discussed in terms of known structural features of the organelle.


Movement of sodium into human platelets induced by ADP.

1. In normal human platelets the concentrations of Na+ and K+ were 42.1 +/- 4.3 and 98.8 +/- 3.7 mequiv/l of platelet water respectively (mean +/- S.E. of 22 samples). 2. When platelet-rich plasma was incubated with 22Na+ at 37 degrees C for 2-3 h an increase in platelet Na+ concentration was found which was significant after 210 min. Platelet K+ concentration did not change significantly. The platelet 22Na+ radioactivity increased faster than did the total Na+ suggesting a Na+o-Na+ exchange process in unactivated platelets. 3. Addition of ADP to platelet-rich plasma resulted in platelet aggregation and a rapid rise (within seconds) in 22Na+-radioactivity within the platelets and after 300 s this increase diminished toward control levels. 4. Under the same experimental conditions, ADP did not bring about an increase of 36Cl- in the platelets. 5. Ouabain (10-(6) M) added to platelet-rich plasma induced an increase in Na+ concentration and 22Na+ radioactivity in the platelets, as well as a decrease in K+ concentration. ADP produced a further increase in 22Na+, which did not return toward control values, in the presence of ouabain. 6. The association of an increase in 22Na+ but not of 36Cl- accompanying aggregation by ADP suggests a selective mechanism for the movement of Na+ into platelets rather than a movement of NaCl together with water under an osmotic gradient.


Specific interaction of concanavalin A with glycolipid monolayers.

The effect of 131I-labelled concanavalin A on the surface pressure and surface radioactivity of monolayers formed from phospholipids and from natural and synthetic glycolipids has been studied. The lectin binds to and penetrates dipalmitoyl phosphatidylcholine monolayers at a surface pressure of 15 dynes/cm and this interaction is inhibited by the presence of alpha-methyl mannose in the subphase. At surface pressures of 25 dynes/cm or higher, concanavalin A will interact with monoglucosyl diglyceride or diglucosyl diglyceride from Acholeplasma laidlawii and with synthetic glycolipids containing 2 or 3 alpha 1 leads to 4-linked D-glucose residues in the headgroup, but not with phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, or with the ganglioside II3NeuAc-GgOse4-Cer. The binding to the glycolipid sugar group and penetration of the hydrocarbon region seem to occur simultaneously, as the time courses for the development of surface pressure and surface radioactivity coincide.


Physicochemical studies of non-histone protein HMG17 with DNA.

Non-histone protein high mobility group-17 (HMG17) isolated from calf thymus consists of 89 amino acids and the complete sequence is known (Walker, J.M., Hastings, J.R.B. and Johns, E.W. (1977) Eur. J. Biochem. 176, 461-468). We have studied its conformation and interaction with DNA by a variety of technics. The results show that the protein has a random structure. It binds non-cooperatively, non-specifically and reversibly to DNA. It is estimated that each molecule of protein binds to 57 nucleotides of calf thymus DNA. The equilibrium constant for binding is approx. 1 - 10(6) M-1. HMG17 induces conformational changes in DNA similar to some of the histones in particular H1.


Electron microscopic studies of purified eukaryote messenger RNA.

Rat liver mRNA, hen and rabbit globin messenger RNA (mRNA) were investigated by electron microscopy. No secondary structures were visible in the molecules of these mRNAs under conditions where short secondary structures of other types of RNA, i.e. MS2 phage RNA and 28 S rRNA, were clearly demonstrated. The contour lengths of these mRNAs were also determined by electron microscopy and compared with the sizes estimated by other techniques. The contour lengths of rabbit short and long globin mRNA are 0.1498 +/- 0.0019 and 0.1908 +/- 0.0021 micrometer, respectively. The former is assumed to be globin mRNA for alpha chain and the latter for beta chain. Similarly, hen "alpha" and "beta" globin mRNA have mean lengths of 0.1449 +/- 0.0011 and 0.1891 +/- 0.0017 micrometer, respectively. Hen and rabbit reticulocytes contain 1.8-2.0 times as much mRNA for alpha globin chains as for beta.


Induction of thymidine kinases in phytohaemagglutinin-stimulated human lymphocytes.

Thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) activity increased approx. 200-fold during transformation of human lymphocytes with phytohaemagglutinin. Two peaks of thymidine kinase (peak IS and peak IIS) were separated from stimulated lymphocytes on DEAE-Sephadex. The activity in peak IS was 20-fold the activity in peak IIS. Only one peak was obtained from normal lymphocytes (peak IIN). The elution volume of this peak was identical to that of peak IIS. The Km values for ATP were 1 mM for peak IS, 0.2 mM for peak IIS and 0.3 mM for peak IIN. 90 micronM dTTP gave 50% inhibition of the activity in peak IS, while the same inhibition of the activities in peak IIS and peak IIN was obtained with only 15 micronM dTTP. Km for thymidine was about 6 micronM for peak IS. The kinetic relation between thymidine and the activity in peak IIS was complex but very similar to that of peak IIN. It is suggested, that a new form of thymidine kinase appears in the lymphocytes due to phytohaemagglutinin stimulation.


Suppression of phytohemagglutinin-induction of thymidine uptake in guinea pig lymphocytes by methylglyoxal bis(guanylhydrazone) treatment.

Treatment with methylglyoxal bis(guanylhydrazone), a specific inhibitor of S-adenosylmethionine decarboxylase (EC 4.1.1.50), suppressed the phytohemagglutinin-induction of [3H]thymidine uptake by guinea pig lymphocytes. The kinetics of [3H]thymidine uptake revealed that the Km value for thymidine was not changed, but the V value was markedly lowered by the methylglyoxal bis(guanylhydrazone) treatment. The induction of ATP: thymidine 5'-phosphotransferase (EC 2.7.1.75) (thymidine kinase) activity by phytohemagglutinin was suppressed to about the same extent as the induction of thymidine uptake. These suppressions were dependent on the methylglyoxal bis(guanylhydrazone) doses and on duration of the methylglyoxal bis(guanylhydrazone) treatment. Analysis of [3H]thymidine labelled compounds of the acid-soluble fraction showed that conversion of thymidine to thymidine 5'-triphosphate was inhibited by the methylglyoxal bis(guanylhydrazone) treatment. DNA polymerase activity was less inhibited by the methylglyoxal bis(guanylhydrazone) treatment in comparison with the methylglyoxal bis(guanylhydrazone) inhibition of thymidine uptake by whole cells. These results strongly suggested that blocking of polyamine accumulation by the methylglyoxal bis(guanylhydrazone) treatment influenced phytohemagglutinin induction of thymidine phosphorylation, resulting in a decrease of thymidine incorporation into DNA.


The canine erythroid cell system: a new model for study of regulatory mechanism in hemoglobin synthesis.

The total methodology for a new mammalian erythroid cell system which permits direct investigation of the terminal stages of hemoglobin synthesis and assembly is described. The canine system allows quantitative separation of native heme containing alpha and beta chains which recombine to for tetrameric hemoglobin with normal functional properties (n = 2.17). These chains can be utilized for investigation in a cell-free system in which the rates of synthesis of alpha and beta chains are equal (alpha/beta = 0.96 +/- 0.05). Methodology for the quantitative separation of globin allows study of the effects of balanced and unbalanced globin chain synthesis in both the intact cells and the cell-free system.


DNA damage in ultraviolet-irradiated HeLa and CHO-Kl cells examined by alkaline lysis and hydroxyapatite chromatography.

DNA damage and repair activity have been examined in cultured mammalian cells by the technique of alkaline lysis followed by analysis of the single-strand content of DNA by hydroxyapatite chromatography; the rate of unwinding of DNA in alkali is related to the number of single-strand breaks present. When HeLa cells are ultraviolet-irradiated and incubated, on subsequent alkaline lysis the DNA unwinds at a faster rate than the DNA of unirradiated cells. The rate is increased if inhibitors of DNA synthesis (hydroxyurea, deoxyadenosine or 1-beta-D-arabinofuranosyl cytosine) are present during the incubation. Similar effects are seen in CHO-Kl cells synchronised in G1 phase, though mitotic CHO-Kl cells show little effect of post-irradiation incubation or of the presence of inhibitors. It appears that agents known to block replicative DNA synthesis can also inhibit repair DNA synthesis following ultraviolet irradiation, leading to an accumulation of single-strand breaks in DNA produced by repair nuclease activity. This phenomenon is probably responsible for the retarded sedimentation of such DNA on alkaline sucrose gradients.