Lymphocytic infiltration in endometrial adenocarcinoma. Correlation with histologic differentiation, clinical staging and prognosis. Lymphocytic infiltration in endometrial adenocarcinoma was evaluated in curettings from 50 patients and correlated with histologic differentiation, clinical staging and prognosis. In spite of the absence of any significant correlation between lymphocytic infiltration and the various parameters investigated, the presence of a marked infiltrate in 46% of the cases suggests that in endometrial adenocarcinoma, as in other neoplasms, a cell-mediated immune response does indeed exist, but warrants further investigation by more refined methods.


A quantified approach to the analysis of complications in combined external and intracavitary radiotherapy of uterine cervical neoplasms. A method is presented of calculating biological doses in combined external and intracavitary radiotherapy with long-lived radioisotopes. The method utilizes the well-known "nominal standard dose" concept combined with an original correction, based on a radiobiological model, to account for the nonhomogeneity of the external field therapy. Of practical interest are the points of maximal radiation dose sustained by the rectum and bladder in the treatment of carcinoma of the uterine cervix. Forty patients with malignant tumors of the uterine cervix who were treated at the Beilinson Hospital are described. A high degree of correlation was found between the biological dose and long-term regional complications. There appears to be a practical threshold dose, of about 4,000 rets, below which severe gastrointestinal and genitourinary complications are rare. A strategy of optimal treatment leading to limited complications is presented.


Tissue antigens in the saliva of rats. Demonstration of salivary gland specific and nonspecific antigens. Antisera were raised in rabbits against the half-saturated ammonium sulfate soluble fraction of rat saliva. With these antisera, at least four antigens were demonstrated by immunodiffusion in the saliva and in a saline extract of the parotid gland; three of these antigens were also found in a saline extract of the submaxillary gland. Two of the four antigens were salivary gland specific, the other two being shared by the liver,pancreas and testis. The immunofluorescent pattern indicates that these antigens originate from the acinar epithelium of the salivary glands and probably constitute products of secretion and/or cellular turnover.


Rubella antibodies in women of childbearing age during an epidemic and the two years thereafter. An epidemic of rubella started in the south of Israel late in 1971, and reached the north of the country in the spring of 1972. Between April 1972 and April 1975, 14,149 women of childbearing age from Haifa and the north of Israel were examined for rubella antibodies by hemagglutination inhibition (HI) and complement fixation (CF) tests. During the year of the epidemic, CF antibody titers of greater than or equal 16 were found in 9.3% of cases with HI antibody titers of 64, in 37.7% of cases with HI titers of 128, and in 71.7% of cases with HI titers of greater than or equal 256. Two years later there were no women with CF antibody titers of greater than or equal 16 among those with HI titers of 64, and the percentage with CF antibody titers of greater than or equal 16 had decreased to 4.3% among women with HI titers of 128 and to 38.7% among those with HI antibody titers of greater than or equal 256. These results show that the determination of CF antibody titers may be useful in the serodiagnosis of recent rubella infection, especially diring an epidemic occurring several years after a previous one. The epidemic hardly altered the percentage of women susceptible to rubella. Although vaccination of 12-year-old girs was introduced in Israel in 1972, careful surveillance of women of childbearing age still remains necessary for detection and vaccination of susceptible nonpregnant women.


Variation in the sex ratio in cardiovascular mortality. The favorable mortality rate of adult women compared with that of men is well known in western countries. The sex difference varies in different population groups. In Israel there is a low male:female ratio, as compared with Greece, Holland, and England and Wales. This is due to the relatively high female mortality rates in Israel. Within Israel, the Jewish population of North African origin has a relatively low male:female ratio. In this group, the marked increase in mortality due to ischemic heart disease has occurred in both women and men, in contrast to various western countries in which the male:female ratio increased during the 20th century with the rise in the death rate from ischemic heart disease, especially in middle-aged men. Social disequilibrium, especially family disorganization with its selective stressful effects on women, is suggested as an hypothesis on which to base further study in communities with a low male:female ratio associated with high mortality rates in women.


Continuous measurement of arterial oxygen tension during one-lung anaesthesia. A new type of polarograhic oxygen electrode. A new type of intra-arterial oxygen electrode was evaluated. In 14 patients, undergoing one-lung ventilation for thoracic surgery, the electrode was inserted into the radial artery and changes in PaO2 were recorded continuously. Methods of improving PaO2 during one-lung ventilation were demonstrated.


Delayed lumen obstruction in endotracheal tubes. Delayed onset of obstruction in the Oxford endotracheal tube during anaesthesia is described in five patients. The effects of intra-cuff voluem and pressure on the wall of the tube were investigated and discussed with special refernce to the ffects of body heat and repeated use on the consistency of the tube. It is concluded that inward collapse of the tube wall is caused by a combination of factors, namely: frequent use, softening of the tube wall by body heat, the gradual increasing of intra-cuff volume and pressure by diffusion of nitrous oxide into the cuff, replacing a damaged cuff by a new one and heat sterilization. Deflation and re-inflation of the cuff to minimal occlusive volume at hourly intervals is suggested as a precautionary measure in the prevention of inward collapse of the tube wall.


Cardiovascular effects of ketamine in the pithed rat, rabbit and cat. The effect of i.v. ketamine on arterial pressure and heart rate were examined in pithed rats, rabbits and cats. In all three species ketamine caused a brief decrease in arterial pressure and heart rate. In the rat, but not in the other two species, this initial decrease in arterial pressure was followed by a pressor response which was resistant to alpha-adrenoceptor blockade, depletion of tissue noradrenaline stores and adrenalectomy. It is concluded that the peripherally mediated pressor response found in the pithed rat is specific to this species, is not a result of liberation of peripheral catecholamines and does not explain the pressor effect of ketamine found in man and several animal species.


Effects of ketamine on canine cardiovascular function. Left ventricular contractility was assessed before and during the peak effect of ketamine in eight canine right heart bypass preparations. Myocardial contractility was defined in terms of maximum left ventricular dp/dt, the ejection fraction, and left ventricular end-diastolic and left atrial pressures at constant heart rate and cardiac inflow. Ketamine produced significant decreases in contractility and there were some indications of a dose-response pattern. The action of ketamine was dependent partly on changes in arterial pressure, as the drug caused a mild but sustained vasodilatation which was not dose-dependent. These data offer further evidence of a small and transient negative inotropic effect of ketamine.


Effect of arterial hypotension on anaesthetic requirement in dogs. In the dog a reduction of mean arterial pressure to 40-50 mm Hg decreased MAC by 20+/--EM 4% in the 1st hour. No further change resulted during the ensuing 3 h of hypotension (40-50 mm Hg). Arterial and cerebrospinal fluid lactate and pyruvate concentrations were not affected by hypotension, nor did cerebrospinal fluid pH decrease. In a separate group of dogs, we reduced arterial pressure to 40-50, 30-40 and 20-30 mm Hg successively. MAC decreased concomitantly, being 58+/-11% of control value at the lowest pressure. MAC increased following the restoration of normal pressure, but hysteresis was evident. In a third group of dogs, arterial pressure was reduced rapidly to 10-30 mm Hg. MAC decreased to 29+/-5% of control. This decrease was significantly greater than that associated with the three-stage reduction.


Analysis of chloroform in blood.

Analysis of chloroform in blood. A gas chromatographic method for the analysis of chloroform in blood has been developed in which the anaesthetic agent is extracted into carbon disulphide and trichlorethylene is used as an internal standard. Injection of the extract into a chromatograph resulted in a chloroform peak eluted after 165 s and a trichloroethylene peak at 310 s, both of which are much later than the solvent peak which is completely eluted in 100 s. The mean percentage recovery of chloroform from blood in the range of 1.4--38.12 mg/100 ml was 98.8+/-1.32. This is an improvement on all previous methods described for the estimation of chloroform.


Metabolism of labetalol by animals and man. 1 The disposition and metabolism of labetalol and either 14C- or 3H-labetalol has been studied in mouse, rat, rabbit, dog and man. 2 Radiolabelled labetalol was administered orally at doses of 100 mg/kg to the mouse, up to 50 mg/kg to the rat and rabbit, 20 mg/kg to the dog and 200 mg to man. From measurements of the total plasma radioactivity it was shown that labetalol was well absorbed by all the species. When the measurements of plasma radioactivity and labetalol concentrations were compared, it was found labetaol had been extensively metabolized by the first-pass effect in rat, rabbit and man. Metabolism by this route occurred to a lesser extent in the dog. 3  Radiochemical analysis of the tissues from rats, rabbits and dogs showed that the highest concentrations of radioactivity were found in the lung, liver and kidney. Very little radioactivity was present in the brain. Over 99% of the radioactivity was cleared from the tissues by 7 d. When doses of up to 200 mg 14C-labetalol/kg were given to pregnant rats and 50 mg 14C-labetalol/kg to pregnant rabbits, autoradiographic and radiochemical analysis of the full-term foetuses showed that only small amounts of radioactivity were present in the foetus. 4 The mouse excreted 72%, the rate 48%, the rabbit 61%, the dog 66% and man 60% of the oral dose of radioactivity in the urine. Analysis of themouse and rat faeces showed that the remainder of the dose of radioactivity was excreted in the faeces. 5 Radiochemical analysis of the urine and faeces collected from rats and dogs after an intravenous dose of 1 mg 14C- and 3H-labetalol/kg showed that excretion of radioactivity occurred via both the kidney and bile. 6 An intravenous dose of 1 mg 3H-labetalol/kg to dog and 1 mg 'cold' labetalol/kg to man was not as extensively metabolized as a similar oral dose. 7 The percentage of the dose excreted in the urine as unchanged drug was 2% in the rabbit, 11% in the rat, 19% in the dog and up to 5% in man. The O-phenyl glucuronide was the major metabolite present in the mouse, rat, and rabbit urine. Dog and man also formed this metabolite, but to a lesser extent. A second glucuronide was the major metabolite present in dog urine. This was probably formed through conjugation of glucuronic acid was the secondary alcohol group of labetalol. The major metabolite present in human urine was an unidentified conjugate of labetalol. Minor metabolites of labetalol present in rat, rabbit and dog urine were hydroxylabetalol and its glucuronyl conjugate.


Affinity of labetalol for ocular melanin. 1 The toxicity of labetalol has been studied in mice, rats, rabbits, and dogs, and reproductive studies have been carried out in rats and rabbits. Nothing was observed in any of these species to suggest that patients taking labetalol might be exposed to any toxic hazard. 2 During the reproductive studies 14C-labetalol was used to study placental transfer. Radioactivity was present in the uveal tract of the foetal eye. 3 Radioactive labetalol but not its metabolites was bound to the melanin pigment of the eye. This binding was reversible. It was not possible to saturate the melanin of the cat and dog eye even with prolonged dosing with labetalol. 4 Chloroquine, given orally at doses of 1.5-6 mg/kg/d for 4-7 weeks, produced changes in the cat retina. When oral doses of 20 mg labetalol/d were given to cats for 7 months, no oculotoxic effects were observed. 5 Detailed ophthalmological and histological examinations were carried out on the rats, rabbits, cats and dogs used in these studies. No changes indicative of oculotoxicity were observed. In the reproductive studies no effects were observed in the developing rat or rabbit eye, which could be consequent on the placental transfer of labetalol or its metabolites.


Labetalol in resistant hypertension.

Labetalol in resistant hypertension. 1 The efficacy of labetalol in lowering blood pressure has been assessed in a group of 16 patients with severe hypertension whose blood pressure was inadequately controlled (supine diastolic blood pressure greater than 110 mmHg on two consecutive occasions) on existing therapy or in whom severe side-effects necessitated a change in therapy. 2 All patients had an original pretreatment supine diastolic blood pressure of greater than 120 mmHg and most had evidence of target organ damage. Patients were hospitalized for the purposes of changeover of therapy, labetalol being added to the existing regime which was reduced stepwise and eventually withdrawn. 3 Treatment with labetalol resulted in satisfactory blood pressure control in 10 of 16 patients in this study but high doses were required, the mean daily dose being 3,091 mg (range 1,200-8,000). 4 Treatment with discontinued in 6 patients on account of postural hypotension (3), failure to control supine diastolic blood pressure (2) and retention of urine (1). Long-term treatment was relatively free from side-effects. 5 From preliminary studies in animals it is suggested that an action within the central nervous system may contribute to the hypotensive effect of labetalol.


A study of labetalol in patients of European, West Indian and West African origin. 1 Hypertension in  West Indians and Africans is common and has an unacceptably high mortality in the younger patients. 2 Fifty-three patients received labetalol (a combined alpha- and beta-adrenoreceptor antagonist) as part of an open evaluation of its anti-hypertensive effect. Ten non-caucasian patients were included. 3 Significant reductions in systolic and diastolic pressures were obtained in the caucasian patients, the African and West Indian patients remaining refractory to therapy.


Acute haemodynamic effects of labetalol and its subsequent use of an oral hypotensive agent. 1 Fourteen patients whose lying diastolic blood pressure was persistently 110 mmHg or greater were given labetalol 0.5--1 mg/kg intravenously. 2 The maximum hypotensive effect developed between 20 and 40 min, and on average lasted 3 h. The lying systolic mean blood pressure fell by 30 mmHg and the lying diastolic blood pressure by 17 mmHg (P less than 0.001). 3 This acute hypotensive effect was associated with a significant reduction in the peripheral resistance (P less than 0.02). The hypotension was not associated with significant secondary changes in the stroke volume or pulse rate. 4 The above 14 patients plus 1 additional subject received labetalol orally at a daily dose ranging from 150-2400 mg. The mean lying systolic blood pressure fell by 22 mmHg (P less than0.01) and the mean lying diastolic blood pressure by 26 mmHg (P less than 0.001). The standing values were similar and postural hypotension at this dose did not develop. There was no significant change in the pulse rate. 5 Renal function was monitored by estimates of plasma creatinine and creatinine clearance. Some patients were followed for 2 yr and others for a few months. With the long-term patients, there was no significant reduction in either measurement although in a few patients a slight reduction in creatinine clearance was observed.


Acute effect of intravenous labetalol in the treatment of systemic arterial hypertension. 1 The acute anti-hypertensive effect of intravenously administered labetalol, a combined alpha- and beta-adrenoreceptor antagonist, has been assessed in a pilot study involving 17 hypertensive patients. 2 In 11 patients with mild to moderately severe hypertension, there was a decrease in mean arterial pressure (24 mmHg) after a single intravenous dose of 50 mg labetalol. The peak response occurred approximately 4 min after administration of the drug. 3 In six patients with severe hypertension, there was a reduction in mean arterial pressure of 19 mmHg, but this was achieved after a further 50-mg dose administered 30 min after the initial injection. 4 In neither group was there any clinically significant decrease in heart rate, and drug-related side-effects were insignificant. 5 Labetalol reduced the blood pressure when administered intravenously and thus merits further investigation as a treatment for acute hypertensive crises.


Treatment of phaeochromocytoma and of clonidine withdrawal hypertension with labetalol. 1 Labetalol has been used as medical treatment in five patients with phaeochromocytoma. 2 In four of the five patients, blood pressure was satisfactorily controlled, and symptoms and signs were relieved. 3 Labetalol also provided satisfactory cover across surgery for the removal of tumours in two cases. 4 In one instance, it was not possible to give sufficient labetalol orally to suppress the attacks of phaeochromocytoma. This patient was not effectively controlled at operation with a combination of propranolol and phenoxybenzamine. 5 Intravenous labetalol rapidly lowered blood pressure and relieved symptoms in a patient experienced hypertensive crisis after clonidine withdrawal. 6 Labetalol is a useful addition to the medical treatment of phaeochromocytoma.


Cardiovascular effects of labetalol during halothane anaesthesia. 1 The circulatory effects of labetalol 25 mg intravenously in six patients during 1% halothane anaesthesia were studied. 2Labetalol caused a marked decrease in arterial pressure and a consistent but modest (20%) decrease in cardiac output. Heart rate was slowed and stroke volume did not change significantly. Central venous pressure increased and peripheral resistance decreased. 3 Increasing the halothane concentration of 3% led to marked myocardial depression as evidenced by reduced cardiac output and increased central venous pressure with increasing arterial hypotension. 4 Labetalol may be a suitable drug for controlling induced hypotension under general anaesthesia, although high concentrations of halothane should be used with care.


Exercise tests before and after heart valve replacement. A simple progressive exercise test was performed before and after operation on five subjects undergoing mitral valve replacement and on five subjects undergoing aortic valve replacement. The responses of heart rate and ventilation were related to work rate )kilopond metres/min). The patients were also assessed clinically by the New York Heart Association grading and radiologically before each exercise test. The clinical grading was shown to be a poor guide to observed exercise tolerance, as the improvement noted in symptoms was not matched by the objective measurement of working capacity. Only two patients had normal exercise tolerance after surgery, although six of the ten patients claimed that they had no exertional dyspnoea after operation. The changes in simple ventilatory function tests before and after operation were generally small. We suggest that measurements of exercise tolerance before and after operation should be an essential part of heart valve replacement surgery.


Chest pain in the presentation of sarcoidosis. The cases of six patients are reported illustrating that chest pain may be a prominent feature of sarcoidosis. It may be severe and be the chief presenting symptom. In the cases described it was mostly retrosternal and had few consistent aggravating factors. In each case there was bilateral hilar lymph node enlargement and it is suggested that htis was chiefly responsible for this relatively uncommon symptom of sarcoidosis. It is also suggested that undue persisting pain may respond to corticosteroid administration.


Sex differences in chronic cor pulmonale in delhi. Chronic cor pulmonale is more prevalent in northern India than in the south. It is equally common in men and in women and accounts for 20% of all admissions for heart disorder in Delhi. In a study of 766 patients (239 men and 527 women) carried out over a 15-year period there were some striking sex differences. Some 75% of men and 10% of women smoked. The women came from the poorest class and all of them cooked from an early age over smoky and primitive fireplaces in ill-ventilated huts, while only 7% of the men cooked their own food. Chronic bronchitis and bronchiectasis were the commonest associated lung disorders in both sexes. The women developed heart failure 10-15 years earlier and showed more severe congestive failure with larger hearts and greater derangement of pulmonary function. It is concluded that the cause of chronic cor pulmonale in women in Delhi was damage to the lungs from exposure to smoky cooking fuels from girlhood onwards, followed by repeated chest infections.


Lung cancer model for studying multi-step metastasis. The sequence of the spread of cancer has been a problem for over a century. A simple model based on lung cancer is proposed. It utilizes three well known facts: that lung cancer may selectively attack the adrenals, that such adrenal deposits may or may not invade the outflow venous tract and that contralateral lung deposits are uncommon in this cancer. Therefore any sequence of spread from the ipsilateral lung to the adrenal glands and thence to the contralateral lung merits attention. Its study might throw light on the laws governing multi-step metastasis in man.


Giant 'cryptococcoma' of the lung.

Giant 'cryptococcoma' of the lung. A case is described of cryptococcosis presenting as a large round intrathoracic lesion with superior vena caval obstruction and clubbing of the fingers. The patient was satisfactorily treated by lobectomy under cover of amphotericin B therapy.


Hypersensitivity to bacteria in eczema. IV. Cytotoxic effect of antibacterial antibody on skin cells acquiring bacterial antigens. The sera of persons with generalized eczema (Whitfield-type) or with disseminated nummular eczema were examined for complement-activating antibacterial antibodies to test the hypothesis that some eczematous change results from an antibody-mediated cytotoxic reaction. Bacteria dying in the stratum corneum release soluble antigens, some of which diffuse into the stratum Malpighii and become firmly adsorbed to the epidermal cells. Antibacterial antibody and complement diffusing into the epidermis react with the antigens acquired by the cells and may induce vacuolation or lysis. Phenol-extracted and freeze-press-extracted antigens (both containing teichoic acids) from Staphylococcus aureus and a micrococcus (Baird-Parker types SI and MI respectively) are adsorbed by monolayers of human skin, embryo or amnion. Cells acquiring the antigen(s) are severely damaged when treated with sera containing the appropriate antibacterial antibodies and complement. IgM complement-fixing antibody appears to be much more cytotoxic in this test than IgG. The cytotoxic activity of a serum is specific for the acquired bacterial antigen and appears to depend on a sufficient concentration of the effective antibody, and not on the presence of antibodies with special properties. Explants of full thickness skin treated with bacterial antigen extracts were unharmed by the antibodies that were cytotoxic for monolayers of skin cells treated with the same antigens. The in vitro cytotoxic test should represent a potential in vivo cytotoxic phenomenon, because skin cell monolayers from two patients adsorbed bacterial antigen prepared from cultures obtained from the same patients, and were damaged by autologous serum containing anti-staphylococcal antibody and complement. It seems probable that this may be an aggravating but not necessarily an initiating factor in many cases of eczema.


The ultrastructure of the human epidermis following ultracentrifugation. Ultracentrifugation of the human epidermis was undertaken to uncover the forces responsible for the establishment and maintenance of the supranuclear melanin cap in basal cells. The wide spacing and elasticity of the tonofibrils caused a selective trapping of melanosomes in this region. At the same time the experiments produced pictures resembling dedifferentiation of the plasma membrane as in the fetal state, and differentiation of the tonofibrillar mass resembling the granular and horny layers. Once the tonofibrils had retracted and massed together, the order of intracellular sedimentation was: melanosome, chromatin, mitochondrion, tonofibril and ribosome.


The epidermis in thyroid disease.

The epidermis in thyroid disease. Epidermal dimensions, replication and anabolic activity have been studied in thirteen patients with thyrotoxicosis and seven patients with hypothyroidism. Epidermal thickness and rete pattern were significantly reduced in hypothyroidism. The rates of epidermal cell division and anabolic activity in the epidermis were estimated by measuring the rates of incorporation of tritiated precursor compounds and were found to be increased in thyrotoxicosis. The mean autoradiographic labelling indices after the intracutaneous injection of tritiated thymidine were 9-1% in thyrotoxic patients and 4-3% in the hypothyroid group. None of the changes observed could be correlated accurately with the degree of thyroid gland activity, but increased thyroid activity was reflected in the epidermal changes better than decreased thyroid activity.


Physical training and connective tissues in young mice. Biochemistry of skin. The effect of physical training on some connective tissue components of the skin was studied in male mice of NMRI-strain. The mice to be trained and their controls were about 2-weeks-old at the beginning of the training, which took place on a 5 degree inclined treadmill 5 days a week for 3-22 weeks progressively. The final exercise bouts were 50 and 80 min for moderate programmes and 180 min for the intensive programme at a speed of 30 cm/s. At the end of each experiment skin samples from trained and control animals were taken for the analyses. We found increased concentrations of nitrogen, hexosamines and hydroxyproline in the skin of the trained mice compared to the controls. The unchanged DNA concentration suggests that the activation of synthesis occurred in a constant number of cells.


Caustic ulcers caused by cement.

Caustic ulcers caused by cement. Seven similar cases with deep ulceration probably due to wet cement are presented. Within 12 hours after making a concrete floor in a kneeling position, curved ulcers developed on both sides of both patellae. The exposure time varied from 2 to 6 h. One patient got wet concrete into her boots and, consequently, ulcers developed on her feet. The high alkalinity of calcium hydroxide in wet cement was the only apparent causative agent in every case.


Metastatic Crohn's disease.

Metastatic Crohn's disease. Metastatic Crohn's disease is a term used to describe a granulomatous reaction occurring in flexures, and separated from the affected areas of the gastro-intestinal tract by normal skin. Previous reports refer to such lesions affecting the groins, the male genitalia, a flexure on the anterior abdominal wall of an obese patient, and submammary regions. A further case, in which the retro-auricular areas are involved, is presented.


Paroxysmal itching in multiple sclerosis. In three women with multiple sclerosis, paroxysmal attacks of itching occurred. There were several similarities between these attacks and other types of paroxysmal phenomena previously described in multiple sclerosis. The attacks were brief, but usually lasted several minutes, they started and ended abruptly, and recurred several times a day. The were controlled effectively by carbamazepine. It is suggested that paroxysmal itching is caused by transversely spreading ephaptic activation of axons within a partially demyelinated lesion in pain-conducting fibre tracts in the central nervous system.


An epidemiological study of the haemostatic and other effects of oral contraceptives. Factors V, VII and VIII (each determined by biological assay), fibrinogen, platelet count and adhesiveness, and fibrinolytic activity were measured in 234 white pre-menopausal women, of whom 57 (24%) were on oral contraceptives and 177 (76%) were not. Cholesterol, triglyceride and blood pressure levels were also recorded. In 20 of the women on oral contraceptives, and in an age-matched group of 20 who were not, prothrombin, factor X, antithrombin III and alpha 2-macroglobulin levels were determined, and factors VII and VIII were also measured immunologically. For the majority of the variables studied, the differences between those using and not using oral contraceptives were greater in younger than older women; in the case of factor VII (biological assay) and fibrinogen, the differences between the regression slopes on age were statistically significant, and mean values were substantially higher in those on oral contraceptives. There was also a significant difference between regression slopes on age for cholesterol. Mean levels of prothrombin, factors VII (immunological assay) and X, triglycerides and blood pressure were significantly higher, and mean levels of antithrombin III significantly lower, in those on oral contraceptives compared with those not. Overall, fibrinolytic activity was significantly higher in the women on oral contraceptives; this difference was, however, almost entirely due to the greatly increased fibrinolytic activity of the non-smokers on oral contraceptives, activity in the smokers on oral contraceptives being similar to that of the women not on these preparations. There were no significant differences in mean platelet count or adhesiveness, or in haemoglobin, packed cell volume, uric acid and blood sugar levels. Among the women on oral contraceptives, there was a significant negative correlation between factor VIII and fibrinolytic activity; this was largely due to five women all of blood groups A and B, in whom, besides high factor-VIII levels and poor fibrinolytic activity, other variables (e.g. fibrinogen) were raised in a direction that might be expected to favour thrombogenesis. It is possible that it is those women whose fibrinolytic activity does not increase in order to compensate for the effects of oral contraceptives on clotting factors, lipids and blood pressure, who are at special risk of thromboembolic episodes. The differential effects of oral contraceptives by age must be borne in mind in evaluating the effects of these preparations on the haemostatic and lipid systems.


Platelet density and size: the interpretation of heterogeneity. Platelets have been separated according to buoyant density using a colloidal silica-polyvinylpyrrolidone system and subjected to electronic sizing. All density populations were found to be heterogeneous in size, the most dense platelets ranging from less than 3 fl to greater than 21 fl in both man and rat. Light platelet fractions contained no platelets greater than 13 fl in either species. Cohort labeling with [75Se]selenomethionine showed no indication of significant change in platelet buoyant density with ageing; greater specific activity found in young, dense platelets appears to be related to increased protein synthetic activity shown in vitro and likely to occur also in their precursor megakaryocytes. It is postulated that dense, intermediate and light platelets are released synchronously by the three different ploidy classes of megakaryocyte, that varying density indicates differing structural characteristics and presumably differences in function. The present findings do not deny the possibility that platelets decrease in size with ageing but if such occurs, it is not associated with a significant change in platelet buoyant density.


Clinical experience with transfusion of cryopreserved platelets. Multiple units of platelet concentrate obtained by plateletpheresis of normal, 'random' or HL-A matched donors were pooled and frozen in polyolefin bags using 5% dimethysulphoxide (DMSO) as a cryoprotective agent and a controlled freezing rate of I degrees C/min. The platelets were stored at approximately-I20 degrees C for as long as 20I days, thawed rapidly at 37 degrees C, washed once and resuspended in ACD plasma prior to transfusion. Two different final concentrations of platelets (approximately 2.7 and 9.0 X 10(12)/1.) were studied. Twenty-three thrombocytopenic patients have received a total of 40 frozen platelet transfusions. The mean freeze-thaw loss was 2I% and was similar for both platelet concentrations. All transfusions were well tolerated and there were no side effects attributable to the small amounts of DMSO infused. Increments in platelet counts I h after transfusion ranged from 0 to 102 X 10(9)/1. with an overall mean corrected increase in evaluable patients of 12 800 (increase x surface area (m2)/number of platelets transfused x 10(11)). Corrected increases tended to be greater with the low concentration of platelets. Overall, the increase in count for the frozen platelet transfusions was 65% of the increments obtained with fresh platelet transfusions administered within 1 week of the frozen platelets. Bleeding times were partially corrected after four out of six transfusions with post-transfusion counts greater than 50 X 10(9)/1., and active haemorrhage was controlled in some patients by frozen platelet transfusions. These results indicate that pooled platelets can be frozen, thawed and transfused with reasonable efficiency. The frozen platelets can circulate and function haemostatically and may eventually play an important role in supportive care.


Utilization of a new strength citrate anticoagulant during centrifugal plateletpheresis. I. Assessment of donor effects. A reduction of donor effects during centrifugal plateletpheresis with the Haemonetics Blood Processor was achieved by reducing the concentration of the citrate anticoagulant. Serum citrate and ionized calcium levels, immediately and 1 h post-pheresis, were affected to a lesser extent by using 5.0 g total ionized citrate (TIC) THAN WITH EITHER 8.0 G OR 11.0 G. Total calcium, bicarbonate, prothrombin time, partial thromboplastin time, ECG, and platelet counts were affected to a similar degree by all three TIC formulations. The total number of platelets collected per litre of blood processed was not significantly different among the three TIC formulations. In vitro studies employing the screen filtration pressure (SFP) technique showed no evidence of platelet aggregates in whole blood collected into either 0.01 M or 0.005 M citrate and agitated or left stationary at room temperature for 5 h. The use of different citrate concentrations in plateletpheresis is discussed.


Preparation and storage of platelet concentrates. I. Factors influencing the harvest of viable platelets from whole blood. Factors affecting the yield and viability of concentrated platelets have been investigated in a blood component programme. It was found that 86+/-1% of the platelets from a unit of whole blood can be concentrated without loss of viability by processing ACD or CPD anticoagulated blood at room temperature. The steps are initial centrifugation at 1000 g for 9 min to harvest platelet-rich plasma; centrifugation of the platelet-rich plasma at 3000 g for 20 min to pack the platelets; and resuspension of the platelet pellet in residual plasma after 1 1/2 h.


Evidence for a splenic release factor of platelets in chronic blood loss plasma of rabbits. Rabbits subjected to chronic blood loss with iron supplementation raised their platelet count and megathrombocyte number 1.7 x and 2.1 x above baseline, respectively. In similarly treated splenectomized animals, no significant response was obtained. When chronic blood loss plasma was injected into recipient animals there was a rise in platelet count and megathrombocyte number of 1.8 x and 3.4 x respectively compared to animals injected with control plasma. In similarly treated splenectomized animals no such rise was obtained; neither was there a rise in platelet count or megathrombocyte number in intact recipient animals when chronic blood loss plasma was obtained from splenectomized animals. It is concluded that the thrombocytosis of chronic blood loss requires the presence of an intact spleen. The data suggest tha presence of a release factor which requires the spleen for its elaboration as well as expression.


Inhibitor of stem cell proliferation in normal bone marrow. A saline extract from normal bone marrow cells having a molecular weight in the range 50000-1000000 daltons has been found to protect rapidly proliferating haemopoietic spleen colony forming cells (CFUs) from the lethal effects of large doses of tritiated thymidine. This extract is non-toxic to the cells. It is not found in regenerating marrow where the CFUs population is rapidly proliferating. Its effect appears to be specific for the CFUs since it has no effect on the proliferation of its close descendant, the granulocytic precursor cell (CFUc), and no effect on the average cytoplasmic structuredness of the whole bone marrow cell population. The active material is probably protein since it is degraded by trypsin.


Quantitative studies of splenic erythropoiesis in polycythaemia vera and myelofibrosis. A quantitative scanning method employing cyclotron-produced 52Fe has been developed to assess splenic erythropoiesis in patients with myeloproliferative disorders. In 12 patients with myelofibrosis splenic uptake of 52Fe was from 5.0% to 48% of the injected dose. Although a single patient with classical polycythaemia vera had a minor uptake of 2.8% of six other patients with this diagnosis showed no concentration of isotope in the splenic area. The fraction of 52Fe in the spleen of four patients with 'transitional' myeloproliferative disorders characterized by a high red cell mass, hypercellular bone marrow and a leucoerythroblastic blood film varied from 5% to 41%. No clear relationship was noted between the degree of splenic erythropoiesis as defined by this technique and the level of haemoglobin, the degree of splenomegaly, the effectiveness of erythropoiesis of traditional 59Fe surface counting. If splenectomy is considered in patients with myelofibrosis splenic 52Fe quantitation will provide more precise data on the contribution of splenic erythropoiesis than 59Fe surface counting alone.


Folic acid metabolism in normal, folate deficient and alcoholic man. Folate metabolism was studied in normal, folate-deficient and alcoholic man by tracer measurements of plasma clearance, urinary excretion, tissue storage and release of folate using both [3H]pteroylglutamic acid (3H-PteGlu) and 14C-methyl-H4PteGlu. Alcohol ingestion did not adversely affect tissue uptake of folates. Whether in normal or folate deficient subjects, the relative clearance rates of 3H-PteGlu and 14C-methyl-H4PteGlu were maintained in the face of alcohol ingestion and there was no evidence of increased urinary loss of intact vitamin or labelled breakdown products. As measured by the flushing technique, the rate of storage or tissue binding of 3H-PteGlu was not influenced by folate deficiency, folate store depletion or alcohol ingestion. However, alcohol may retard the release of methyl-H4PteGlu from tissue stores to plasma. A significantly greater recovery of 14C-methyl-H4PteGly with flush was observed in those normal subjects who ingested alcohol for 6 d. A partial block in the rate of release of tissue folate stores would be a possible mechanism behind the rapid depression in serum methyl-H4PteGlu levels and early induction of megaloblastic erythropoiesis which has been observed following acute alcohol ingestion.


Autoimmune haemolytic anaemia in children. Forty-four children with autoimmune haemolytic anaemia (AIHA) are described: 31 had acute, subacute or chronic disease with warm autoantibodies and 13 had acute or chronic anaemia with cold autoantibodies. The commonest forms were the acute and subacute types with warm autoantibodies and these were more frequent in young children, while chronic AIHA occurred mainly among children at puberty. In about 16% the anaemia was accompanied by a chronic disorder but in over 50% the anaemia was preceded by an acute infection or immunization. The former gave rise mainly to chronic anaemia, but the latter was associated with the acute and subacute forms. In general the prognosis was good and death was never caused by anaemia per se. The prognosis was worse in patientw with clinical features of thrombocytopenia and bleeding and with the immunological findings of free autoantibodies in the serum and a positive direct antiglobulin test. In acute and subacute forms, treatment with corticosteroids and sometimes with blood transfusions was effective. In chronic forms of the disease it was often necessary to give additional immunosuppressive drugs or/and to perform a splenectomy.


The Negro variety of hereditary persistence of fetal haemoglobin is a mild form of thalassaemia. Further studies have been carried out on blood of the 15-year-old Negro male from Baltimore who was the first reported case of the homozygous state for hereditary persistence of fetal haemoglobin. His red cells contain only Hb F; Hbs A and A2 have never been detected. Over a 15-year period of follow up the red cells of this individual have shown persistent microcytosis with reduced MCH and MCV values. His whole-blood p50 value is decreased, probably because of lack of interaction between Hb F and 2,3-diphosphoglycerate. However, his haemoglobin level at the age of 15 years is lower than would be predicted from the degree of increased oxygen affinity. Globin-chain synthesis studies suggest that this is because he has a mild thalassaemia disorder with an alpha/gamma-chain production ratio of about 1.5, similar to that found in beta-thalassemia heterozygotes. Thus Negro HPFH appears to be a well-compensated form of delta beta thalassaemia.


Globin synthesis in normal human bone marrow. Globin synthesis has been studied by in vitro labelling with radioactive amino acids in 60 normal human bone-marrow samples. Under the conditions routinely used to fractionate alpha and beta chains by chromatography alpha/beta production ratios ranging from 0.5 to 1.0 were obtained, depending on the method of sample treatment. This variation was due entirely to the presence of non-haem proteins derived from white cells which chromagraphy with globin on CM-cellulose. Purification of globin on Sephadex G100 and fractionation of alpha and beta globin chains by a modified chromatographic system resulted in alpha/beta ratios of unity. The relevance of these findings to the study of marrows in which there is unbalanced globin chain production is discussed.


Ultrastructure of sickling and unsickling in time-lapse studies. The denser subpopulation of erythrocytes from patients with sickle cell anaemia was deoxygenated to a pO2 of 4.7 kPa or reoxygenated to a pO2 of 12 kPa with a continuous-flow apparatus. Samples were collected into modified Karnovsky's fixative at intervals between 0.5 and 15 S. .The earliest event after deoxygenation was aggreagation of haemoglobin followed by the formation of fibres of 160-200 A diameter. The polymers were always randomly distributed in a loose network. A highly ordered, close packing of fibres characteristic of the nematic liquid crystal was not achieved within 15 S. Depolymerization involved a shortening of fibres followed by aggregation similar to that observed early in the polymerization process and prior to the return to the unperturbed state. Irreversibly sickled cells were the first to demonstrate polymers following deoxygenation and that last to lose polymers after reoxygenation. Polymerization of the haemoglobin preceded the appearnce of the sickled deformity of reversibly sickled cells and, following reoxygenation, the return to the discoid shape lagged behind the disappearance of polymers. These studies, carried out under physiologic conditions, have demonstrated intracellular changes during time intervals that correspond to the normal venous and arterial circulation that may contribute to the pathophysiology of sickling disorders.


Serum "uracil+uridine" levels in pernicious anaemia. The serum "uracil+uridine" level, expressed as uracil, has been measured in 21 cases of vitamin B12 deficiency, in which the serum folate was normal, and compared with the level in 97 normal subjects. The level in the vitamin B12 deficient group (11.9 mumol/1). was significantly lower than in the controls (15.7 mumol/1., P less than 0.005). Nine of the former were complicated by stystemic illness but the clinical and haematological features in the remaining 12 were consistent with the diagnosis of pernicious anaemia in relapse. The serum uracil level in this group was even lower (10.21 mumol/1., P less than 0.01). This finding is unexpected in view of the generally accepted indirect role of vitamine B12 in the methylation of deoxyuridine monophosphate to deoxythymidine monophosphate. Reasons are given for not accepting these results as reflecting the main biochemical lesion in vitamin B12 deficiency. Although they do not give direct support to an impairment in the methylation of deoxyuridine monophosphate, they do not exclude it as they test only one possible metabolic pathway and moreover they could represent the result of more than one action of vitamin B12 on uracil metabolism. They do show, however, that some aspect of uracil metabolism other than methylation is affected in vitamin B12 deficiency in man.


Serum "uracil+uridine" levels before and after vitamin B12 therapy in pernicious anaemia. The serum "uracil+uridine" levels, expressed as uracil, have been measured in 10 cases of pernicious anaemia both before and after treatment, and compared with the levels in 97 normal subjects. The mean pre-treamtent value (8.82 mumol/1., range 6.0-12.0 mumol/1.) differed significantly from that of the normal controls (15.7 mumol/1., range 5.7-40.5 mumol/1., t = 8.8, P less than 0.001). This confirms the low serum uracil level previously reported in pernicious anaemia relapse. The level rose progressively after treatment, reaching a maximum on the fourth day (mean 17.85 mumol/1., range 9.3-23.4 mumol/1.). This was not significantly different from the mean of the normal control group. The difference between the pre- and post-treatment levels was significant on days 3,4 and 5 (P less than 0.005, P less than 0.001 and P less than 0.005 respectively) and the rise preceded the reticulocyte response by 24 h. A further case was treated with pysiological doses of vitamin B12 (2 mug daily for 6 d) and a similar rise in the serum uracil level noted. These results are not explained by any of the known functions of vitamin B12. They are, however, similar to the changes in the serum methionine levels previously reported in pernicious anaemia. The latter were readily explained by the known action of vitamin B12 on "de novo" methionine synthesis and it is suggested that the synthesis of uracil, like that of methionine, might be influenced by vitamin B12 in man.


Microcytosis, anisocytosis and the red cell indices in iron deficiency. Red cell volume distribution curves have been used to measure microcytosis and anisocytosis in normal subjects, blood donors and patients with iron deficiency anaemia. These measurements were more sensitive than the conventional red cell indices for detecting blood donors with a low transferrin saturation. Three stages are suggested as iron deficiency progressively interferes with haemopoietic function. Anisocytosis and an increased percentage of microcytic cells are the first haematological abnormalities to occur and at this stage haemoglobin concentration is usually normal and trasferrin saturation less than 32%. At the second stage the MCV and MCH decline, haemoglobin concentration is generally sub-normal, though not below 9 g/dl, and transferrin saturation is usually below 16%. The final stage of iron deficiency is associated with a low MCHC, a haemoglobin concentration below 9 g/dl and a transferrin saturation of less than 16%.


Two biochemical markers in lymphocyte subpopulations. Human blood lymphocytes can be separated into two populations according to the presence of surface complement receptors. Lymphocytes containing complement receptors (CR+) were found to have a high rate of RNA synthesis or turnover accompanied by increased protein synthesis. Lymphocytes not containing complement receptors (CR-) while maintaining a low profile in RNA synthesis, had a 10-12-fold greater activity in adenosine deaminase enzyme which is believed to be related to lymphocyte-immune responses and cell-mediated immunity. These two biochemical characteristics can be useful tools for future studies in lymphocyte functions. By using these two biochemical markers, we found that CLL lymphocytes were predominantly of the CR+ type, had high active RNA synthesis, and very low adenosine deaminase level. Lymphocytes from two patients with X-linked agammaglobulinaemia showed a picture opposite to that of CLL.


Megakaryocytes and the heterogeneity of circulating platelets. Megakaryocytes mature to the point of cytoplasmic disintergration in three principal ploidy classes: 8n, 16n and 32n. Cells of each of these ploidy classes have been identified, using both microdensitometry and measurement of cell volume and submitted to morphometric analysis. Mature megakaryocytes of the three ploidy classes have been shown to differ in the concentration of cytoplasmic constituents which would be expected to relate to the buoyant density of their platelet progeny. Density separated platelets have been similarly analysed. Light platelets correspond with the 32n megakarycytes and are more liberally endowed with surface connected canalicular system than the progeny of the common 16n megakaryocytes; it is proposed that they have functional characteristics related to this finding. Dense platelets, which are larger in size, correspond with 8n megakaryocytes and show a greater content of granules and mitochondria than platelets of average density. These platelets most probably show specialized function relating to release of granule constituents. Fragments of cytoplasm released from megakaryocytes represent one form of "megathrombocyte" equated by others with newly formed platelets. The differences in structure between these fragments and circulating platelets are emphasized; each such fragment must undergo further disintergration into a number of platelets. It is suggested that the heterogeneity of circulating platelets with respect to both size and density stems from the origin of platelets of varying density from the different populations of megakaryocyte and their release in the form of large cytoplasmic fragments rather than as platelets.


Relationship of factor VIII-like antigen (VIII AGN) and clot promoting acitivty (VIII AHF) as measured by one- and two-stage assays in patients with liver disease. We recently observed a increase in factor-VIII clot promoting activity as measured by a one-stage assay (VIII AHF) in a haemophiliac with hepatitis. However, VIII AHF as measured by a two-stage assay (VIII AHF) was 0.013 u/ml at a time when VIII AHF measured 0.38 u/ml. We then studied seven non-haemophiliacs with liver disease, and attempted to correlate the lvels of VIII AHF and VIII AHF with factor VIII-like antigen (VIII AGN) as measured by quantitative immunoelectrophoresis. In four of the seven patients, disproportionate elevations of VIII AHF compared to VIII AHF were found. Furthermore, VIII AHF values correlated well with VIII AGN vales . No such discrepancy was apparent in four normal control subjects. These findings emphasize the necessity for performing two-stage assays in haemophiliacs as well as non-haemophiliacs with liver disease to assess factor-VIII levels. In addition, they suggest that confirmation of the diagnosis of haemophilia may not be possible in the haemophiliac with hepatitis unless VIII AHF determinations are performed. The reason for the disparity between VIII ahf and VIII AHF levels is not apparent. However, the correlation of VIII AGN and VIII AHF levels in the non-haemophiliacs with liver disease provides further support for the concept that VIII AGN and VIII AHF are closely related or identical molecular entities.


The effects of therapeutic conditions: positive results revisited. This paper surveys evidence relating the 'therapeutic conditions' of Accurate Empathy, Non-Possessive Warmth and Genuineness to psychotherapeutic outcome. The studies reviewed include comparisons of 'good' therapy with no-treatment control groups, and correlational studies relating levels of 'conditions' to outcome. Even leaving aside the question of the validity of the outcome measures used, it is concluded that present evidence suggests no more than a weak relationship between these 'conditions' and patient improvement. It is suggested that a methodologically adequate, experimental, demonstration of their effectiveness has yet to be attempted.


Training in feminine skills in a male transsexual: a pre-operative procedure. This case study describes an attempt to modify the sex-role behaviour of a male transsexual in accordance with the subject's preferred sexual identity. The patient was treated before sex-change surgery. Treatment consisted of a modified form of social skills training and comprised such techniques as modelling, behaviour rehearsal and videotape feedback and focused on female elements of behaviour. Behavioural observations and psychometric testing were carried out before and after treatment and indicated significant change. The results indicated that: (a) behavioural intervention facilitated adoption of a new sex role; (b) the techniques used were particularly useful in modifying sex role behaviour; (c) the adoption of a new sex role in line with preferred identity was successful in terms of patient satisfaction.


Language and cultural influences in the description of pain. Several lines of argument have been pursued in the attempt to study the meanings of pain descriptions in English. First, the component terms of pain descriptions were heuristically classified into primary, secondary, and tertiary groups. Attention was then given to historical features of the primary pain terms. These were handled as morphological structures possessed of a history which has included their entry into the lexicon and their progressive modifications. Besides constituting morphological structures, the primary pain terms are also semantic categories; the history of this 'meaning' dimension was given attention. The sememes which have been bound together with the various primary pain terms were discussed. Brief attention was the given to the contemporary meanings of the secondary and tertiary pain terms. This emphasis on the meanings of the component terms of pain descriptions paved the way for a synchronic analysis of pain descriptions in contemporary English. In this instance, attention was given to the meanings which descriptions of a pain experience seen to have currently. Two modes of analysis were followed. First, the metaphorical roots of pain were explicated. Analyses of the ways in which native speakers use the various pain terms suggest that a 'model' of pain lies behind these descriptions: this model is of a physical process. What pain is being likened to, and what can be likened to pain, in other words, involves a common semantic matrix which is rooted in historical and cultural factors. Many of the sememes originally linked to the various primary pain terms are still 'alive' and implicated when native English speakers describe pain and 'pain-related' phenomena. The sememes which were originally tied to early uses of the primary pain terms no longer have the specific association which they once had with the term; however, it appears that the sememes do enter into the overall contemporary meanings of English pain. Metaphorical transfers implicit in English pain descriptions realize one important semantic component of pain in this language. The second way of analyzing semantic components of English pain involved a grammatical analysis of paradigmatic sentences which realize pain descriptions. A pain experience space was posited on the basis of this grammatical analysis. Each well-formed English sentence describing pain may be seen as associated with a small portion of this space. The sum of the spaces which a pain term can claim constitutes the overall meaning space of that term. The vaious pain terms were associated with different segments of the posited English pain space. It is important that the various spaces of the primary English terms were not sharply bounded and separated one from the other. Instead, the spaces of many terms seemed to be largely contained in the spaces of others...


Factor analysis of analogue scales measuring subjective feelings before and after sleep. A series of 18 visual analogue scales was used to report the subjective feelings of 38 healthy subjects on 732 occasions during experiments concerned with the quality of sleep. Factor analysis of scores from these scales, both for all subjects combined and within each of four subjects separately, yielded two factors consistently -- 'alertness' and 'tranquillity'. Scores derived from the first factor indicated that, in the laboratory setting levels of immediately after waking in the morning were reduced, in relation to those before falling asleep on the previous night.


Cognitive dissonance in the placebo treatment of insomnia -- a pilot experiment. Insomnia in 11 general hospital in-patients was treated by a placebo. The conditions of administration of the placebo were experimentally varied, and a hypothesis derived from Festinger's cognitive dissonance theory was tested. Consistent with this hypothesis, it was found that patients who were required to make meaningful decisions concerning their treatment derived significantly greater hypnotic (therapeutic) value from it than did patients whose treatment was simply administered to them and who were not required to make such decisions. The power of cognitive dissonance theory to explain the placebo effect, and the therapeutic potential of this approach are discussed.


Carcinoembryonic antigen (CEA) in ovarian cancer: factors influencing its incidence and changes which occur in response to cytotoxic drugs. We report the serum levels of carcinoembryonic antigen (CEA) in 109 patients with ovarian cancer. Histology, degree of differentiation, and clinical stage influenced the incidence of positive CEA. Although CEA was significantly raised in patients with a variety of tumours, the highest incidence (77 per cent) was found in those with serious cystadenocarcinoma. Nearly all (94 per cent) of the poorly differentiated tumours were associated with a positive CEA result. Serial CEA levels provided a useful guide to management during cytotoxic chemotherapy, rapidly falling levels indicating a favourable tumour response which was reflected clinically. However, only two-thirds of tumours were associated with detectable CEA levels in serum, day-to-day variations in individual serum levels occurred, and CEA levels tended to fall paradoxically during terminal illness. The significance of persistently low levels in the apparent absence of disease was uncertain.


Haematological changes during normal pregnancy: iron induced macrocytosis. Haematological indices in 300 pregnant and 50 non-pregnant women were obtained by Coulter Counter analysis. In 47 women who did not receive iron supplements during pregnancy, haemoglobin concentration fell from a mean of 12-23 g/dl in early pregnancy to 11-04 g/dl at 34 weeks gestation, a level which was maintained until term. In the 153 women to whom iron supplements were given during pregnancy, the initial fall in haemoglobin concentration was less, was arrested by 28 weeks gestation and then rose to a level equivalent to the booking level. This different pattern in iron supplemented pregnancies was due to an increase in the red cell count together with a change in mean cell volume. Although the changed mean cell volume usually remained within the normal range in the group who did not take iron, a few women developed microcytosis which could reflect iron deficiency; a macrocytic change was noted in a small number of the women who received routine iron supplements and this could reflect iron "over sufficiency". The results are discussed in relation to the possible effects of giving routine oral iron to all pregnant women.


The response of the fetal heart rate of amniocentesis. Fetal heart rate monitoring during 107 amniocenteses suggested that acceleration of the fetal heart rate indicated fetal well-being. Eight out of the 19 fetuses who did not show this response (as against 2 out of the 88 others) had a low Apgar score at birth.


Plasma levels of pregnancy-specific beta1-glycoprotein in normal pregnancy. Pregnancy-specific beta-glycoprotein (PSbetaG) is a major product of the trophoblast which has only recently been identified. Like human placental lactogen, the concentration of PSbetaG in maternal blood rises throughout pregnancy until about 34 weeks, thereafter tending to remain constant until term, with only a small day-to-day variation in individuals. The circulating maternal levels of PSbetaG between 34 weeks and term are about 200 mug/ml, 20 to 30 times greater than the levels of placental lactogen, thus allowing detection and measurement of PSbetaG by relatively simple techniques. The levels of PSbetaG are above the normal range in the majority of twin pregnancies. It is suggested that PSbetaG measurements may be useful in assessing placental function and may help in the detection of multiple pregnancies at an early stage of gestation.


The effect of oxytocics on the human cervix during midtrimester pregnancy. Observations of the effects of oxytocics on the human pregnant cervix have been made in vivo using a double open ended catheter technique. Prostaglandin E, prostaglandin F2alpha and oxytocin had similar but no specific effects upon the intracervical canal pressure; ergometrine caused contractions of the cervix. The significance of these findings is discussed in relation to cervical rupture and cervico-vaginal fistulae that have been reported following second trimester abortion induced with prostaglandins.


The concentrations of prostaglandin E2 and prostaglandin F2alpha in tissues within the fetoplacental unit after spontaneous or induced labour. The concentrations of prostaglandin E2 (PGE2) and prostaglandin F2alpha (PGF2alpha) were measured by radioimmunoassay in portions of umbilical cord, placenta, amnion, chorion and decidua. The samples were obtained during 12 confinements immediately after the delivery of a healthy baby. Labour had either occurred spontaneously (6 subjects) or had been induced approximately two weeks after the estimated date of delivery. The results showed that the concentration of PGE2 was significantly higher (Student's 't' test) in samples of placenta (P less than 0-05), decidua (P less than 0-025), umbilical cord (P less than 0-0125) and amnion (P less than 0-0025), from those patients who had started labour spontaneously. In contrast, the concentration of PGF2alpha was only raised to a significant extent (P less than 0-025) in the amnion. The findings are discussed.


Cellular development of some embryonic organs and the chorion during the first trimester of human pregnancy. The DNA, RNA and protein content of chorionic tissue, heart, liver, kidney, lung and brain was estimated in 27 fetuses obtained at termination of pregnancy between 50 and 105 days of menstrual age. For brain, heart and liver, growth appeared to be most rapid at the earliest period studied (50 to 60 days) whereas kidney and lung development was most rapid later in the first trimester. It is suggested that these periods of particularly rapid growth may be the times at which those tissues are most vulnerable to injury.


Study of the uterine cavity by ultrasound in the early puerperium. The uterine cavity length, maximum width and area were determined by ultrasonic scanning in ten women after their first full term delivery (primiparae) and ten women after their second full term delivery (secundiparase) during the first eight days of the puerperium. A consistent and progressive decrease in all three measurements was found; although faster between days 1 and 3 than between days 3 and 8 the difference was statistically significant only for cavity area. The rates of involution for both parity groups were similar and were not influenced by breast feeding. The cavity length in primiparae and secundiparae was also similar, but the maximum width on days 3, 5 and 8 and the area on days 3 and 5 were significantly greater in secundiparae. A correlation between the infant birth weight and cavity length on day 1 and between infant birth weight and cavity area on days 1, 3 and 5 was found in secundiparae.


Diagnostic ultrasound in the management of patients using intrauterine contraceptive devices. A group of 80 patients was scanned by ultrasound within 24 hours of insertion of an intrauterine contraceptive device (IUCD) and re-examined clinically and/or by ultrasound after the next menstrual period. An attempt was made to identify factors such as uterine size, uterine flexion and the position of the IUCD which might be related to its expulsion or removal when, on initial scanning, the device was not located in the fundal area; removal was necessary in 5 out of 6 patients. In patients with an endometrial cavity of less than 40 mm length and/or acute uterine flexion, the overall figure for expulsion and removal was 54 per cent (13 out of 24). It is hoped that a consideration of these factors will lead to a reduction of the failure rate of the IUCD.


Angiomatous lymphoid hamartoma of the pelvis. A mass removed from the pelvic retroperitoneal tissues of a 24-year-old women was found to be an angiomatous lymphoid hamartoma. The clinical, radiological and histological features are described, and the problems encountered in diagnosis discussed.


The theory, feasibility and accuracy of an ultrasonic method of estimating fetal weight. We present the theory, method, feasibility and accuracy of estimating fetal weight by measuring the fetal volume using compound ultrasonic scanning. The two parameters had a very high coefficient of correlation (0-9794), and the standard error in one series of 20 patients was only +/- 106 g. The correlation coefficient achieved by one of us who was new to ultrasound techniques was 0-82, which compared favourably with correlations between fetal weight and biparietal diameter measurements (r = 0-26). A discussion as to the correction factors required to allow for the unknown value of the velocity of ultrasound in fetal tissues, the clinical use of this method, and the possible means by which the accuracy of estimating fetal weight may be further improved is included.


Relation of plasma oestriol to changes in blood pressure in pregnant patients with hypertension. Serial plasma oestriol levels were determined by radioimmunoassay in 24 patients with severe hypertension in pregnancy. No significant correlation could be found between plasma oestriol and percentage change in diastolic blood pressure. It seems unlikely that hypotensive therapy significantly improves fetoplacental function.


Plasma levels of oestriol-17 beta, oestriol and human placental lactogen during bed rest. Plasma unconjugated oestradiol-17 beta, total oestriol and human placental lactogen levels were measured in twelve healthy volunteers admitted for bed rest in the last trimester of pregnancy. No significant alteration in levels was observed.


Umbilical vein cortisol after spontaneous and induced labour and at elective Caesarean section. Umbilical vein (UV) plasma cortisol levels were assayed at delivery after spontaneous labour, oxytocin-induced labour, prostaglandin E2-induced labour and after delivery by elective Caesarean section. Higher mean UV cortisol levels were observed after vaginal delivery than after delivery by elective Caesarean section. The highest mean UV cortisol level was observed in the PGE2 group which accords with the known ability of prostaglandins to increase steroid production. The significance of these findings is discussed.


Relation of umbilical vein cortisol to neonatal bilirubin concentrations. Umbilical vein cortisol levels were assayed following spontaneous labour in 30 patients. Serum bilirubin was measured in the infants born at three days after delivery. No correlation was demonstrated between third day bilirubin concentration and the cord blood cortisol. The implications of this finding are discussed.


Placental transfer and fetal uptake of 3H-digoxin in humans. Placental transmission and fetal distribution of 3H-digoxin were studied in seven pregnant women undergoing legal termination of pregnancy during the first half of gestation. The radioactivity in fetal and maternal plasma and in fetal tissues was estimated using the oxidation method, and the integrity of the labelled drug by thin layer chromatography. The 3H-digoxin activity was clearly demonstrated in the umbilical cord blood five minutes after injection of the drug into the maternal blood, and the fetal plasma concentrations of 3H-digoxin approximated to the maternal value 30 minutes after drug administration. The distribution of 3H-digoxin in the fetal tissues was relatively even, with the highest 3H concentrations found in the heart and placenta, the lowest in the brain. The results suggest that the capacity of human fetal heart to bind digoxin during the first half of gestation is limited.


Termination of abnormal intrauterine pregnancies with intramuscular administration of dihomo 15 methyl prostaglandin F 2alpha. A synthetic compound, 2a, 2b dihomo 15 (S) 15 methyl prostaglandin F 2 alpha methyl ester given 8-hourly by intramuscular injection was used to terminate pregnancies in 72 patients with a missed abortion, intrauterine fetal death, molar or anencephalic pregnancies. Treatment was successful in all cases. A 4 mg dose of loperamide given orally at 15 to 30 minutes before the injection of prostaglandin analogue prevented diarrhoea as a side effect.


Carcinoma of the cervical stump.

Carcinoma of the cervical stump. A study was made of 56 patients with carcinoma of thecervical stump after subtotal hysterectomy who were seen between 1946 and 1972 at the National Women's Hospital, Auckland, New Zealand. The duration of symptoms before diagnosis, stage distribution and five-year survival rates were examined and compared with those in 1459 patients with carcinoma of the cervix, and no previous subtotal hysterectomy, who presented over the same period.


Adenocarcinoma of the uterine cervix: cytological evidence of a long preclinical evolution. Adenocarcinoma of the cervix occurred in 13 women who had previously had "negative" cervical smears. These smears were reviewed, and abnormalities were found in six cases, the intervals ranging from two to eight years. It is concluded that adenocarcinoma, like squamous carcinoma of the cervix, can have a prolonged evolution during which it is cytologically detectable.


The effect of prolactin on the response of human lymphocytes to phytohaemagglutinin. Lymphocytes were collected from ten normal human males and incubated with or without phytohaemagglutinin (PHA) in the absence or presence of ovine prolactin in concentrations found in normal individuals and in human pregnancy. Prolactin consistently inhibited lymphocyte transformations. The degree of inhibition became progressively less as PHA concentrations were increased.


Cataract after exposure to non-ionizing radiant energy. The case histories of two individuals exposed to thermal radiation emitted from an electric oven and range were presented. In one patient, earlier exposure to medical diathermy appears to have initiated delayed or late-appearing, capsular cataracts. Instead of the anticipated slow progression, the cataractogenesis was accelerated following recent, repeated exposure to the intense, infrared radiation. In the other patient, exposed solely to infrared radiation, a chorioretinal lesion indistinguishable from the type characteristically secondary to repeated, thermal radiation was observed. More recently, the earliest sign of thermal radiation cataractogenesis, capsular opacification, has become evident. The widespread availability of radiant energy sources such as diathermy machines, microwave ovens, and electric ovens and ranges makes it imperative to examine carefully any possible hazards that may result from their use. With this in mind, radiant energy should be considered in the differential diagnosis of cataractogenesis.


Treatment of uniocular aphakia. A comparison of iris clip lenses with hard corneal contact lenses. The visual results, stereopsis, and complications have been assessed on 50 patients in a presenile age group treated for uniocular aphakia. Twenty-five patients received a Binkhorst four-loop lens implant at the time of cataract surgery and 25 were offered a conventional hard contact lens trial postoperatively. The period of follow-up ranged from one to three years.


Internuclear ophthalmoplegia. An electro-oculographic study of peak angular saccadic velocities. Peak angular saccadic velocities were measured during 30 degrees saccades in 18 patients with internuclear ophthalmoplegia who had full or nearly full adduction range, and 25 normal subjects. The following observations were made: 1. In the normal group adduction velocities were significantly faster than abduction velocities and centring movements were faster than decentring movements. 2. In patients with internuclear ophthalmoplegia adduction movements were significantly slower than in normal subjects and were significantly slower than abduction velocities. 3. Abduction velocities in patients with internuclear ophthalmoplegia were significantly slower than abduction velocities in normal subjects. It was concluded that measurements of peak angular velocities during saccades may be useful in detecting internuclear ophthalmoplegia or confirming the presence of suspected internuclear ophthalmoplegia. Slow abduction suggests that many patients with internuclear ophthalmoplegia (60 per cent in this series) have lesions affecting the supranuclear pathways subserving horizontal conjugate gaze.


A study of head and eye movement in spasmus nutans. A case of spasmus nutans was studied using objective recordings of head and eye displacement in order to generate a precise description of the ocular oscillations and head nodding and to investigate their interrelationships. The ocularoscillations consisted of 11-Hz sinusoidal convergence movements. The head nodding consisted of a 3-Hz, 3 degrees peak to peak, sinusoidal oscillation in the horizontal plane. The occurrence of the nodding always abolished the ocular oscillation which was replaced by normal compensatory eye movements which assisted visual acuity. The hypothesis is made that the head nodding is not pathological; it is a learned behavioural pattern which permits the patient to nullify the pathological eye movements. In order to test this hypothesis further, objective evidence is required to determine the precise order of appearance of the abnormal head and eye movements in spasmus nutans.


Lactate dehydrogenase isoenzyme pattern in the eye muscles. Deviation in myopia. The lactate dehydrogenase isoenzyme patterns in 46 extraocular eye muscle samples removed at surgery for squint were determined by acrylamide-gel electrophoresis and reduction of NAD coupled with formazan reaction. Muscle type subunits predominated in the isoenzymes of the medial and lateral rectus muscles of emmetropic and hypermetropic eyes, whereas heart type subunits predominated in those of myopic eyes.


Causes of blindness among students in blind school institutions in a developing country. Out of 270 students in 17 blind school institutions in Malawi 73 per cent were blind before the age of three. The most common cause for the blindness was ocular infection (75-2 per cent). Meales, as a single cause, was responsible for 43-7 per cent of the cases and smallpox for 5-2 per cent. Bacterial infections were incriminated in 26-3 per cent of the cases. Most of these had received traditional medicine during the acute phase of the disease. Hereditary factors as causes of blindness were found in 7-8 per cent of the cases. These included congenital cataracts (2-6 per cent), optic atorphy of unknown origin (3-0 per cent), microphthalmos (1-5 per cent), and macular degeneration (0-7 per cent). Careful ophthalmological examination showed that in 37 cases an intervention could be attempted in order to improve the vision. In the 11 most favourable cases this was attempted, with the result that nine cases gained a useful vision of 4/60 to 6/18 in the better eye.


Assembly of DNA with histones and nonhistone chromosomal proteins in vitro. We have examined, by protein binding assays, thermal denaturation, and circular dichroism, the possible effects of histones on nonhistone chromosomal protein (NHCP) interactions with DNA. For these studies, we have fractionated mouse Krebs II chromosomal proteins into three discrete fractions: Mo, 5 M urea-soluble NHCP; M1, 5 M urea-1 M NaCl-soluble NHCP from 5 M urea-extracted chromatin; and M3, 5 M urea-3 M NaCl-soluble chromosomal proteins from 5 M urea-1 M NaCl-extracted chromatin. These fractions contain heterogeneous populations of NHCP, and were found to differentially affect histone binding to DNA by methods of reconstitution, or by direct binding of M0, M1, or M3 to urea-salt reconstituted DNA with histones. M0 was found to exert a unique effect on the thermal denaturation and circular dichroic spectra of DNA-histone complexes. M0 from Krebs II chromatin was also found to complete for DNA sites in the presence of M0 from mouse liver chromatin. In addition, in 5 M urea, pH 8.0, histone binding to DNA reached saturation at 1.85 mg/mg of DNA, higher than the in vivo ratio of 1.00 mg/mg of DNA. Saturation of histone binding to DNA occurred only in the presence of 5 M urea, resulting in a reduction of nonspecific histone-histone interactions on DNA.


Interactions of hydrated metal ions with nucleotides: the crystal structure of barium adenosine 5'-monophosphate heptahydrate. The crystal and molecular structure of barium adenosine 5'-monophosphate heptahydrate was determined from x-ray diffraction data. Crystals of barium adenosine 5'-monophosphate heptahydrate are monoclinic, space group C2, with a = 32.559(3), b = 6.969(3), c = 9.597(1) A, and beta = 100.31(1) degrees. Intensity data were collected with an automated diffractometer. The structure was solved by the heavy-atom method and refined by least-squares to R = 0.034. This structure provides an example of an outer-sphere metal-nucleotide complex, in which a completely hydrated metal ion interacts with the nucleotide only through water bridges. The barium ion is coordinated to eight water molecules, which form a slightly distorted square antiprism. Seven of the eight water molecules from the barium hydration shell are hydrogen bonded to phosphate groups; three of these water molecules are also hydrogen bonded to other suitable acceptor sites on the base and ribose moieties. The conformation about the glycosidic bond is anti, with chiCN = 69 degrees, and, as in most nucleotide structures, the conformation about the C(4')-C(5') bond is gauche-gauche. However, the ribose displays an unusual conformation (best described as C(4')-exo) not previously observed in crystal structures of nucleosides or nucleotides, other than 3',5'-cyclic nucleotides. It is possible that this unusual conformation is a consequence of the metal-water-nucleotide bridging interactions.


Relaxation of chromatin structure by ethidium bromide binding: determined by viscometry and histone dissociation studies. The effects of ethidium bromide intercalation on chromatin structure were monitored by viscometry and analysis of histone dissociation. Investigation of the NaCl concentration dependence of chromatin viscosity showed that the reduced viscosity (etared) was very low up to 0.4 M NaCl and increased gradually when the salt concentration was raised further. In chromatin intercalated by ethidium bromide, etared was not significantly different at low salt concentrations (up to 0.2 M NaCl). However, when the salt concentration was raised further, the viscosity response curve increased sharply to reach viscosities about 4-5 times higher than those for nonintercalated chromatin. The increase in viscosity was proportional to the increase in fluorescence intensity, when the ratio of ethidium bromide to DNA mucleotide was raised. The transition of intercalated chromatin into the relaxed form was reversible, dependent on the nature of the electrolyte and cooperative, as indicated by the small increase in salt concentration required to obtain chromatin relaxation. Investigation of the NaCl concentration dependence of histone dissociation revealed that total histones and each individual histone fraction were released from intercalated chromatin at much reduced NaCl concentrations. The midpoints of the dissociation curves of the individual histones ranged from 0.30 to 0.45 M NaCl and fell within the same range where the drastic viscosity change occurred. These results indicate that intercalation of ethidium bromide labilizes chromatin structure to relaxation by moderately elevated salt concentrations. It is suggested that the labilization is caused by changes in the DNA helix conformation due to dye intercalation decreasing the stability of histone-DNA interactions.


A nuclear membrane-associated DNA complex in cultured mammalian cells capable of synthesizing DNA in vitro. A DNA-nuclear membrane complex has been isolated by two different methods from the nuclei of cultured mouse fibroblast (3T3) cells. One method, utilizing the detergent sarkosyl (sodium lauroyl sarkosinate), yields a DNA-nuclear membrane complex (the M band), which contains virtually all of the DNA in the nuclei. However, treatment of the M band by sonication, vortexing, or freeze-thaw reduces the amount of DNA in the complex by approximately 50-80%, depending upon the phase of the cell cycle from which the complex was extracted. The remaining DNA is tightly bound to the nuclear membrane and resists further shearing procedures. Over 90% of the choline-labeled phospholipid present in nuclei is also found in these sheared M bands. The percentage of DNA associated with the nuclear membrane varies during the cell cycle and correlates well with the onset, continuation, and cessation of DNA synthesis. Thus, although DNA-membrane complexes can be detected throughout the cell cycle, the percentage of DNA bound to membrane increases during late G1 and S and decreases during G2. In addition, there are distinct qualitative differences in the type of DNA present in the membrane fraction, with a more highly d(A-T) rich DNA being present in confluent (G0) cells than in cells during the S phase. This d(A-T) rich DNA may be related to the mouse satellite DNA identified by others. The M band can be separated into two DNA-nuclear membrane subfractions by centrifugation through a continuous sucrose gradient. The relative proportions of these two subfractions depend upon the percentage of sarkosyl present in the M band prior to centrifugation, with complete removal of sarkosyl resulting in a very large increase in the sedimentation velocity of the complex and in the formation of only one fraction. Evidence that this is a complex of DNA with membrane is given by the finding that DNA is dissociated from the complex with Pronase, deoxycholate, or high levels of sarkosyl. Removal of virtually all of the DNA with DNase from this rapidly sedimenting complex does not dissociate any of the phospholipid which still sediments rapidly as a single band. A second method, which yields a DNA-membrane fraction from nuclei, utilizes sedimentation of lysed nuclei to equilibrium in CsCl density gradients. This low-density CsCl fraction contains only 10-15% of the total DNA, but contains most of the nascent DNA, which may be chased into a membrane-free fraction. The DNA-membrane fraction from CsCl gradients possesses properties in common with the M-band fraction and can be converted into an M band. DNA membrane complexes from sucrose gradients, as well as the crude M-band preparation and a non-membrane-associated DNA fraction from nuclei can synthesize DNA in vitro without the addition of an external DNA template or DNA polymerase. In contrast to the activity in the non-membrane-associated DNA fraction, the membrane-associated polymerase activity is strongly stimulated by adenosine triphosphate and is unaffected by ethidium bromide...


Membrane microviscosity and human platelet function. An increased sensitivity to epinephrine-induced aggregation has been observed both in platelets obtained from patients with type IIa hyperlipoproteinemia and in normal platelets following incubation with cholesterol-rich lecithin dispersions. We have reported previously that the membrane fraction of platelets is enriched with cholesterol relative to phospholipid under each of these conditions. To further explore the effect of cholesterol on platelet membranes, we have examined the fluidity (microviscosity) of whole platelets and platelet subcellular fractions using a hydrophobic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), under conditions in which the cholesterol-to-phospholipid mole ratio (C/PL) of platelets was varied by incubation with various cholesterol-lecithin sonicated dispersions. The C/PL of platelets directly influenced the rotational diffusion of DPH, as indicated by changes in fluorescence polarization. This was reflected in an increase in microviscosity at 37 degrees C (ETA37) from 2.84 P in normal platelets to 4.06 P in platelets with a 118% increase in C/PL. Conversely, platelets with a 43% decrease in C/PL had a 13% decrease in eta37. A strong correlation (r = 0.94) existed between C/PL and eta37 throughout this entire range. However, C/PL had no effect on the excited-state fluorescence lifetime of DPH. Both C/PL and eta37 were lower in isolated platelet membranes than in the platelet granule fraction. When platelets were incubated for 20 h with cholesterol-rich dispersions, there was an increase in C/PL and eta37 in both the membrane and granule fractions. However, this occurred more rapidly in membranes so that, at 5 h (a time when an increased sensitivity of whole platelets to epinephrine is evident), membrane C/PL had increased 55% and eta37 had increased 42%, whereas granule C/PL and eta37 had changed minimally. Cholesterol-rich platelets and subcellular fractions had a lower fusion (or flow) activation energy for viscosity (deltaE), reflecting a higher degree of order, and the converse was true in cholesterol-poor platelets. Moreover, a strong negative correlation existed between the percent change in deltaE and the percent change in eta37 induced either by cholesterol incorporation or depletion. These data demonstrate that cholesterol influences the fluidity and the degree of order within the hydrophobic core of platelet membranes. Changes induced in these physical properties by an excess of cholesterol relative to phospholipid may underlie the abnormal reception or transmission of the aggregation stimulus in cholesterol-rich platelets.


Phosphorus-31 nuclear magnetic resonance detection of unexpected phosphodiesters in muscle. In the examination of intact muscles by 31P nuclear magnetic resonance spectroscopy, a number of signals have been detected in the phosphodiester region (-0.5 to 0.5 ppm) of the spectrum which could not be correlated with the known common phosphates of muscle tissue. These signals arise from perchloric acid extractable compounds with several common chemical properties, one of which is a ready solubility in nearly anhydrous ethanol solutions. A component contributing to the major resonance has been identified as glycerol-3-phosphorylcholine. This characterization is based on both 31P nuclear magnetic resonance and chromatographic data.


Phosphorus-31 Fourier transform nuclear magnetic resonance study of mononucleotides and dinucleotides. 2. Coupling constants. Stereosensitive 3JPH and 4JPH phosphorus-proton coupling constants have been measured directly from the phosphorus-31 nuclear magnetic resonance (NMR) spectra of a variety of adenine, thymine, and uracil 3'-mononucleotides, 5'-mononucleotides, their cyclic analogues, and the corresponding dinucleotides, under various conditions of pH and temperature. For all 5'-mononucleotides, the identical 3JPH coupling to phosphorus of the two H5' protons is found essentially independent of the nature of the base, the presence of a 2'-OH on the sugar ring, the temperature, and the pH; the "gauche-gauche" rotamers about C5'-O5' and C4'-C5' remain overwhelmingly (85%) preferred. The "gauche" arrangement during C3'-O3' is favored in all cases for 3'-mononucleotides. However, while no sizeable pH effect is noted on 3'-monodeoxyribonucleotides, the pH dependence of 3JPH3 in 3'-monoribonucleotides strongly suggests an interaction between the 3'-phosphate and the 2'-OH. Molecular features affecting the magnitude of 4JPH coupling constants are discussed together with pH and temperature effects. The time-averaged preferential structural features of mononucleotides are found in dinucleotides with a higher probability; hence, dimerization induces an increase in the statistical conformational purity of the phosphodiester-sugar backbone, even at extreme pH. Temperature studies point out that the thermal unwinding of stacked dinucleotides occurs mainly via rotation about P3'-O3' and P5'-O5' bond axes.


Equilibrium binding of thrombin to platelets. Binding of human [125I]thrombin to washed human platelets was studied in order to analyze the nonenzymic aspects of the thrombin stimulation of platelets. Highly purified alpha-thrombin that was iodinated with lactoperoxidase retained full clotting and esterase activities and full activity toward platelets, was not distinguished from native thrombin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel chromatography, and bound to platelets the same as unlabeled thrombin. Bound and free [125I]thrombin were measured after rapid separation of platelets from the suspending medium by centrifugation through oil. Maximum binding was within 15 s, the shortest time measured. At concentrations of thrombin sufficient to cause less than maximal platelet stimulation, 90% of the total thrombin was free in the suspending solutions. Equilibrium binding was established, with both free thrombin and free platelets retaining activity, and with rapid reequilibration after dilution or addition of unlabeled thrombin. The equilibrium was complex, with the apparent number of binding sites and dissociation constants dependent on thrombin concentration. Analysis of bound thrombin as a function of thrombin concentration by double-reciprocal and Scatchard plots indicated 300-400 high affinity sites (Kdiss = 1.8-2 nM); these correlate with thrombin stimulation of Ca2+ secretion, which shows half maximal effect at 1.5 nM thrombin and maximal effect with 500-600 thrombins bound per platelet.


Proteolytic activation of protein C from bovine plasma. Protein C is a vitamin K dependent protein present in bovine plasma (Stenflo, J. (1976), J. Biol. Chem. 251, 355). It is a glycoprotein (mol wt approximately 62 000) composed of a heavy chain (mol wt 41 000) and a light chain (mol wt 21 000). The heavy chain has an amino-terminal sequence of Asp-Thr-Asn-Gln and contains nearly three-fourths of the carbohydrate. The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe. Incubation of protein C with either factor X activator from Russell's viper venom or trypsin resulted in the cleavage of an Arg-Ile bond between residues 14 and 15 of the heavy chain. Concomitant with this cleavage was the formation of a serine enzyme which was inhibited by diisopropyl phosphorofluoridate. Liberation of the tetradecapeptide decreased the molecular weight of the heavy chain from about 41 000 to 39 000 and resulted in the formation of a new amino-terminal sequence of Ile-Val-Asp-Gly in the heavy chain. No change in the molecular weight of the light chain was observed during the activation reaction. These results indicate that protein C, like the four vitamin K dependent coagulation proteins, exists in plasma in a precursor form and is converted to a serine protease by hydrolysis of a specific Arg-Ile peptide bond. The biological substrate for the enzymatic form of protein C and the physiological mechanism whereby protein C is converted to a serine enzyme are not known.


Factor X activating enzyme from Russell's viper venom: isolation and characterization. The protease from Russell's viper venom that activates factor X (Stuart factor), factor IX (Christmas factor), and protein C was purified by gel filtration on Sephadex G-150 and QAE-Sephadex A-50 column chromatography. The purified enzyme migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 79 000. A minimal molecular weight of 78 500 +/- 800 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. Upon reduction with 2-mercaptoethanol, a heavy chain (mol wt 59 000) and a light chain were observed. The light chain migrated as a single band (mol wt 19 000) in 7.5% polyacrylamide-sodium dodecyl sulfate gels but appeared as a doublet (mol wt 18 000 and 20 000) in 10% polyacrylamide-sodium dodecyl sulfate gels. The amino-terminal end of the heavy chain was heterogeneous and contained isoleucine, valine and serine. The amino-terminal sequence of the light chain was Val-Leu-Asp. The factor X activator contained 13% carbohydrate including 6.0% hexose, 1.7% N-acetyleneuraminic acid, and 5.3% galactosamine. Most of the carbohydrate was found to be present in the heavy chain, although some was also observed in both forms of the light chain. The factor X activator had no esterase activity toward benzoyl-Phe-Val-Arg-p-nitroanilide or benzoylarginine ethyl ester and was not inhibited by 0.05 M diisopropyl phosphorofluoridate. These data indicate that factor X activator from Russell's viper venom is a highly specific protease composed of one heavy chain and one light chain, and these chains are held together by a disulfide bond(s).


Isolation, purification, and cross-linking profiles of elastin from lung and aorta. Elastin from anatomically defined regions of young calf lung and dog aorta was isolated and purified by a procedure which sequentially removed lipids, collagen, structural glycoproteins, and the microfibrillar proteins without apparent damage to the cross-linking residues, which have been shown to be sensitive to autoclaving and hot alkali treatment. One of the methods described was effective in obtaining pure elastin from lung parenchyma. Visceral pleura was found to be the richest source (25% dry weight) of elastin in the lung tissues examined. The amino acid compositions of the elastins purified by different methods were compared for purity and for the detection of possible damage to cross-linking compounds. Cross-linking profiles were obtained by column chromatography either after reduction with 3[H]NaBH4 or after reaction with 14[C]NaCN and NH3. The 3[H]NaBH4 method, under carefully controlled conditions, proved not to be quantitatively reproducible. The reaction of elastin with 14[C]NaCN and NH3 appeared preferable due to its reproducibility; this procedure required one type of hydrolysis for the analysis of all the cross-linking compounds. Examination of the cross-linking profiles of the elastins from various tissue regions revealed differences in the type, distribution, and quality of cross-links.


Resonance Raman spectra of iron(III)-, copper(II)-, cobalt(III)-, and manganese(III)-transferrins and of bis(2,4,6-trichlorophenolato)diimidazolecopper(II) monohydrate, a possible model for copper(II) binding to transferrins. Fe(III), Cu(II), Co(III), and Mn(III) complexes of ovo- and human serum transferrins show resonance enhanced Raman bands near 1600, 1500, 1270, and 1170 cm-1 upon excitation with laser frequencies which fall within the visible absorption bands of those metalloproteins. Comparison of the visible absorption and resonance Raman spectra of the Cu(II)-transferrin complexes with those for the Cu(II) model compound, bis(2,4,6-trichlorophenolato)diimidazolecopper(II) monohydrate, indicates that the resonance Raman bands are due to enhancement of phenolic vibrational modes. For the model (Cu(II) compound, a normal coordinate analysis was used to aid our assignment of the observed resonance bands at 1562, 1463, 1311, and 1122 cm-1 to A1 vibrational modes of the 2,4,6-trichlorophenolato moiety. These assignments are consistent with those made for Cu(II)-transferrins. The latter assignments were based upon calculated A1 frequencies for p-methylphenol (Cummings, D.L., and Wood, J.L. (1974), J. Mol. Struct. 20, 1). The wavelength shifts in the resonance bands for the model compound from those for Cu(II)-transferrins are due to the influence of the chloro substituents on the planar vibrations of phenol. These results clearly identify tyrosine as a ligand in copper binding to transferrins.