Periodic acid-Schiff (PAS) staining for glycogen in clinically normal psoriatic and non-psoriatic skin. PAS staining was used to assess the glycogen content of clinically normal forearm epidermis in 32 patients with active psoriasis, 30 patients with inactive psoriasis and 40 non-psoriatic subjects. Glycogen accumulation was greater in patients with active than in those with inactive psoriasis. The inactive psoriatics showed significantly less glycogen accumulation than the controls.


Bacterial flora in psoriasis.

Bacterial flora in psoriasis. The aerobic bacterial flora of psoriatic plaques, uninvolved skin and the anterior nares of forty psoriatic patients was studied. The incidence od Staphylococcus aureus was 30% in the anterior nares, 20% on the plaques and 13% on the uninvolved skin. S. aureus counts were 3 x 10(2)/cm2 on the plaques and 1-5 x 10/cm2 on the normal skin. The total bacterial counts were also higher on plaques (7-9 x 10(3)/cm2) than on normal skin (3-0 x 10(3)/cm2). The incidence of lipophilic diphtheroids was significantly lower on the plaques (4%) than the normal skin (30%). Eighty percent of the strains of S. aureus isolated from psoriatic patients were resistant to 10 units of penicillin. Because of increased desquamation, psoriatic skin is a public health hazard.


Effects of occlusion upon population dynamics of skin bacteria. Quantitative and qualitative changes of the cutaneous aerobic bacterial flora upon 20 sites on the backs of each three healthy subjects were examined before and after one site was occluded, using skin flora maps as a tool. Major local alterations were found to affect the carriage of micro-organisms in distant surrounding areas. Staphylococcus epidermidis was the most successful competitor. Furthermmore, some sites appeared to act as retricted reservoirs for specific types of micro-organisms whereas other areas were less limited in their support of flora.


Changes in dry weight and projected area of human epidermal cells undergoing keratinization as determined by scanning interference microscopy. Cells, obtained from human leg by successive treatments with trypsin, were air dried on microscope slides before mounting in glycerol. Dry weights and projected areas of individual cells were measured using a Vickers M 86 scanning microinterferometer. The dry weights of cells varied from 100 pg for basal cells to 700 pg for large squames. Corresponding projected areas varied from 100 to 1500 mum2.


Histohogical and direct immunofluorescence study of cutaneous hyperreactivity in Behçet's disease. The mechanism of skin hyperreactivity (pathergy) in Behçet's disease is unknown. It has been suggested that the response is due to an Arthus-like reaction. In 18 patients with active Behcet's disease, pathergy was induced and the skin biopsied after 24 h. The histological reaction was that of mold to intense round cell infiltration. perivascular in type. Direct immunofluorescence failed in all 18 patients to demonstrate the local presence of immunoglobulins and complement at the site of lesion. A marked increase in the number of mast cells was observed at the site of reaction and scattered throughout the dermis, the role of which is to be further investigated. The study failed to demonstrate a humoral mechanism in the production of the cutaneous hyperactivity.


Familial Behçet's syndrome.

Familial Behçet's syndrome. Behçet's syndrome is reported in a family of which four generations have so far been affected. The index patient also exhibited an unusual schizo-affective disorder. The histocompatibility antigen haplotype 1-17 was common to four with the disease. Genetic transmission may be responsible for the familial nature of the syndrome and the associated schizophrenia.


Epidermal naevus syndrome.

Epidermal naevus syndrome. A case of the epidermal naevus syndrome is presented. Huge enlargement of both lower limbs was the presenting symptoms together with an extensive veavus unius lateris on the trunk.


Atrophie blanche lesions closely resembling malignant atrophic papulosis (Degos' disease) in systemic lupus erythematosus. Two patients with systemic lupus erythematosus are described who in the course of their disease developed small atrophie blanche lesions that closely resembled those found in malignant atrophic papulosis. Preliminary investigation of these two cases indicates that considerable similarities probably exist in the pathogenesis of malignant atrophic papulosis and the atropie blanche lesions of systemic lupus erythematosus. It is concluded that a diagnosis of malignant atrophic papulosis should only be made after systemic lupus erythematosus has been excluded by full investigation.


Adverse skin reaction from vitamin K1.

Adverse skin reaction from vitamin K1. Two patients with erythematous, tender, indurated plaques at the site of intramuscular injections of vitamin K1 are described. In one of these, condition was reproduced by the injection of a test dose. In both cases, there was associated liver disease. The red, indurated plaques cleared in 4-14 days. There have been two previous reports in the literature of similar lesions. Altough some of the patients showed sclerodermatous changes at the same site within 2 years of the injections. These unusual reactions have only been seen in patients with liver disease and when a variety of drugs have been administered. It may be that these factors are important in the initiation of the cutaneous response.


Congenital triangular alopecia.

Congenital triangular alopecia. Three new cases of congenital triangular alopecia are reported and the differential diagnosis of this rare developmental defect is discussed.


Eosinophilic pustular folliculitis. Report of a European case. A case of eosinophilic pustular folliculitis (Ofuji) is presented. This long-lasting disease of unknown aetiology has hitherto been reported only from Japan. It is characterized by tinea-like slowly growing rings and by blood eosinophilia, The histological changes are epidermal infiltrations and abscesses of predominantly eosinophilic leukocytes, often localized to the follicles.


Intercellular contacts between erythroid precursors in the bone marrow in dyserythropoiesis. The ultrastructure of the cellular interfaces between erythroid precursors has been studied in both congenital and acquired dyserythropoiesis. The types of cell-to-cell contacts found included highly convoluated interdigitating septate-like junctions as well as apparent confluence of the cellular membranes of adjacent erythroid cells. Such findings are not characteristic of the intercellular relationships found in the normal erythroblastic islands. The erythroid plasma membrane modifications are possibly related to the changes in the plasma membranes of erythroid cells known to occur in dyserythropoiesis and their presence may well contribute to the ineffective erythropoiesis with intramedullary cell destruction which characterizes many dyserythropoietic states.


Chelation studies with 2,3-dihydroxybenzoic acid in patients with beta-thalassaemia major. 2-3-Dihydroxybenzoic acid was evaluated as a potentially useful, orally effective iron-chelating drug by performing iron balance studies in patients with beta-thalassaemia major. The administration of this substance at 25 mg/kg/d to five patients for 8 d caused an average increase in iron excretion of 4.5 mg/d. When the drug was administered at 25 mg/kg q.i.d. to eight patients for 21 d, iron excretion increased to 6.5 mg/d. Chelation was highly specific for iron with changes in magnesium and calcium excretion being insignificant. The drug was well tolerated with side effects limited to gastrointestinal complaints which ameliorated when the drug was taken with food. These studies provide a rationale for further evaluation of 2,3-dihydroxybenzoic acid in patients with iron overload.


The use of chang cells cultured in vitro to evaluate potential iron chelating drugs. A number of iron chelating agents, consisting largely of hydroxamic acid and benzoic acid derivatives, have been studied in an in vitro Chang cell culture system to determine their effect on cellular iron uptake, ferritin synthesis and the incorporation of iron into ferritin. The results have been compared with those of a previous study in which iron balance was determined in hypertransfused rats. Both techniques appear to be of value in screening new iron chelating agents for potential therapeutic use in patients with iron overload.


The uptake of ferric iron by rat liver ferritin in vivo and in vitro. The incorporation of ferric iron labelled with 59Fe into rat liver ferritin has been studied in whole animals, into liver homogenate and into purified protein. Uptake of Fe3+ into purified rat liver ferritin followed a pattern similar to that with horse spleen ferritin given Fe2+ and an oxidant. The distributions of iron incorporated as a function of molecular iron content obtained in vivo resembled the in vitro patterns for iron contents above 500 Fe atoms/molecule and times after injection of up to 12 h. At larger intervals a maximum label moved to molecules of highest iron content as the molecules accumulated more iron. The apparently reduced uptake of injected 59Fe into molecules of low iron content might be due either to the chase of cold iron through an existing iron pool or the presence of functionally different ferritins at more than one anatomic site within the cell.


Iron mobilization in the pregnant rat.

Iron mobilization in the pregnant rat. Iron exchange in the pregnant rat was quantitated by repeated determinations of plasma iron turnover (PIT), transferrin iron distribution and measurements of storage iron and food iron utilization employing selective radio-iron probes. Despite a sixfold increase in PIT, intestinal absorption of iron accounted for 40% of the PIT throughout pregnancy, with variations not exceeding +/- 5%. Increased fetal requirements were efficiently compensated for by the mobilization of iron from maternal tissue stores and by increased absorption, and there was no subsequent reduction in iron supply to maternal tissues. Enhancement of iron absorption occurred in the absence of a reduction in serum iron levels or the size of iron stores. In view of the close correlation between PIT and rates of absorption it is postulated that iron absorption in the pregnant rat is regulated by PIT which in turn is determined by the rate of plasma iron clearance by the placenta and the maternal erythroid marrow.


Effect of iron deficiency and desferrioxamine on DNA synthesis in human cells. Desferrioxamine (10(-3) M) caused a fall in the deoxyadenosine triphosphate level after 4 h incubation in normal phytohaemagglutinin-stimulated lymphocytes. There was a rise in the concentrations of the other three deoxyribonucleoside triphosphates (deoxythymidine-,deoxycytidine-and deoxyguanosine-triphosphate). The changes are similar to those caused by hydroxyurea, a known inhibitor of ribonucleotide reductase. Desferrioxamine (10(-3 M) was found to inhibit human lymphocyte ribonucleotide reductase to a mean of 11% of control activity after 45 min incubation. Both drugs, desferrioxamine and hydroxyurea, inhibited incorporation of [3H]thymidine DNA into lymphocytes in the presence or absence of deoxyuridine, and inhibited production of lymphocytic thymidine kinase, having opposite effects to methotrexate on both [3H]thymidine incorporation and thymidine kinase activity. Phytohaemagglutinin-stimulated lymphocytes from patients with chronic iron deficiency showed lower levels of all our deoxyribonucleoside triphosphates than normal lymphocytes. It is suggested that this may be due to reduced ribnucleotide reductase activity of the iron-deficient cells.


Thymidine kinase in megaloblastic anaemia. Thymidine kinase has been measured in phytohaemagglutinin (PHA)-stimulated lymphocytes from 13 normal subjects and eight patients with megaloblastic anaemia. The levels in normal subjects ranged from 0.20 to 2.10 units/mg protein (mean 0.903 units/mg protein) and in megaloblastic anaemia from 2.99 to 9.97 units/mg protein). All the patients showed raised levels of the enzyme which were partly but not completely reduced to normal by addition of folic acid in vitro. Vitamin B12 in vitro had a lowering effect in the five vitamin-B12-deficient patients and two patients with combined deficiencies but not in one 'pure' folate-deficient patient. Thymidine kinase activity was highest in the cells of the least anaemic patients, suggesting that the degree of anaemia in megaloblastic anaemia may be determined in part by the ability of the cells to utilize thymidine by the 'salvage' pathway when the de novo pathway of thymidylate synthesis is failing. The rise in thymidine kinase activity in megaloblastic anaemia is presumably due to induction of the enzyme. Addition of methotrexate or 5-fluorouracil, drugs known to inhibit de novo thymidylate synthesis, caused an increase in thymidine kinase activity in normal PHA-stimulated lymphocytes after 24 h (but not after 1 h) which could be completely blocked by addition of puromycin. Thymidine mono- and di-phosphate kinases were also measured in normal PHA-stimulated lymphocytes. The activities were substantially higher than that of thymidine kinase and their activities were unaffected by methotrexate addition.


HL-A groups in pernicious anaemia.

HL-A groups in pernicious anaemia. There is a significant association with HL-A3 type and pernicious anaemia. In addition there is a negative association with HL-A2. There is no association between HL-A type and the presence or absence of immunity to intrinsic factor.


Factor V in an industrial population.

Factor V in an industrial population. Factor-V levels have been measured in a random sample of 626 men and 307 women working in a variety of occupations in North West London. The method is an automated one-stage assay using the same batch of freeze-dried thromboplastin, all results being expressed in terms of the same freeze-dried standard plasma; it has been shown that only one dilution of test plasma is necessary. Factor-V levels are significantly higher when venepuncture is difficult than when it is satisfactory, the mean levels being about 130% and 117% respectively. Factor-V levels are approximately normally distributed; they are similar in men and women and in blacks and whites, and increase significantly with age at the rate of about 0.6% per annum. Factor-V levels are not affected by oral contraceptives or the menopause, and there are no differences according to blood group or secretor status.


Bleeding from self-administration of phenindione: a detailed case study. A young woman presented with a 2 year history of a severe bleeding disorder and marked deficiencies in all four vitamin-K-dependent factors. Metabolic studies with tracer doses of tritium-labelled vitamin K1 suggested that the patient might be taking an oral anticoagulant; and subsequently her plasma was found to contain a substance identical to phenindione in its spectrophotometric and chromatographic properties. The half-disappearance times of factors II, IX, X were measured after the administration of a concentrate of these factors and were found to conform with published figures. The concentrate controlled the patient's excessive bruising and prolonged skin and gingival bleeding. It would therefore seem that factor VII may not be essential in reversal of the bleeding disorder induced by anticoagulant overdose.


Banked blood microfiltration. I. Microfilter composed of five polyurethane foam layers with graded pore size. Aggregates of amorphous material which develop with storage of banked blood may be a source of pulmonary microembolism in patients having massive transfusions. In order to remove such debris, blood microfilters have been developed and are in routine clinical use. This paper describes the evaluation of one such filter, the Bentley PF 127 model B, which is composed of five layers of polyurethane foam. The time of filtration as a function of the pressure applied, the number of particles removed, the observed changes in blood cells, and the results of scanning electron microscopy of the filter after filtration of human banked blood, are reported.


The establishment of a programme of artificial insemination by donor semen within the National Health Service. A human semen bank has been established in Nottingham for artificial insemination using donor semen (AID). Frozen semen has been used for all inseminations. The introduction and organization of such a programme within the National Health Service is described. Six pregnancies have so far been confirmed and two normal infants have been delivered.


Evaluation of different approaches to obstetric care: Part 1. The obstetric management and results obtained by two obstetric teams working in the Cardiff Maternity Hospital over a five-year period are compared. One team had a more active approach to induction of labour and antepartum monitoring with urinary oestrogen assay and serial ultrasound cephalometry than the other. After controlling for differences in the attributes of the two groups of patients treated, it was not possible to show any striking advantage or disadvantage of the more active approach.


Evaluation of different approaches to obstetric care: Part II. The obstetric management and outcome of normal patients presenting to two obstetric teams working in the Cardiff Maternity Hospital are compared over a five-year period. One team induced labour with amniotomy and oxytocin infusion three times more frequently than the other. No advantage or disadvantage of this practice was demonstrated using various measures of perinatal morbidity and mortality. The implications of these findings are discussed together with those of two previously reported observational studies. The need for experimental research in perinatal medicine is stressed.


The measurement of uterine activity in induced labour. A simple electronic system for quantifying uterine activity in labour has been developed. The basic construction of the system is described. It has been used to confirm the hypothesis of Steer et al (1975) that oxytocin-induced labour has a stable phase during which uterine activity is largely independent of oxytocin infusion rate.


The outcome of pregnancy after gonadotrophin therapy. One hundred and ten women conceived 143 times following induction of ovulation by gonadotrophins. The abortion rate was 21 per cent. Some bleeding occurred in 38-7 per cent of pregnancies and 54 per cent of them ended in abortion. For the 112 pregnancies reaching 20 weeks, the multiple pregnancy rate was 26-8 per cent (21 twins, 5 triplets, 3 quadruplets and 1 sextuplet). Hypertension was present in 8-9 per cent of patients and in 3-3 per cent of those with multiple pregnancy. The length of gestation was related to the number of fetuses at birth and postmaturity did not occur. The Caesarean section rate was 32-1 per cent. The birth weight of the infants was normal and the male to female sex ratio was 0-64 for singleton births and 0-78 for twins. The fetal loss was 15-9 per cent (7-1 per cent for pregnancies of over 28 weeks). Growth and development of the children were apparently normal. The incidence of all congenital malformations was 7 per cent.


Blood coagulation, fibrinolysis and plasma proteins in women with normal and with excessive menstrual blood loss. A group of women with excessive menstrual blood loss (menorrhagia) without any known local or general underlying disease was studied and compared to a group of women with normal menstrual blood loss. Blood coagulation, platelet function and fibrinolysis as well as a variety of plasma proteins were analyzed on six occasions during one menstrual cycle. Women with menorrhagia were found to have a slightly higher concentration of fibrinogen--fibrin degradation products, a higher factor V and VIII activity, higher antithrombin III, alpha 1-antitrypsin  concentrations and higher antifibrinolytic activity than the normal women. The concentration of albumin, beta IE-globulin, orosomucoid, ceruloplasmin and IgG were lower in women with memorrhagia than in normals. The capillary fragility did not differ significantly between the groups. The observed differences in clotting factors between the groups could not have caused the differences in blood losses.


A randomized comparative trial of the performance of the Ayre and the Armovical cervical spatulae. The Ayre and the Armovical cervical spatulae were compared in a randomized trial involving 982 women of childbearing age. It was found that the Armovical spatula was slightly less effective than the Ayre spatula in obtaining adequate numbers of squamous cells. The Armovical spatula was, however, far better at providing a sample of endocervical or metaplastic cells and, as a result, yielded a substantially higher proportion of 'satisfactory' smears (54 per cent as against 39 per cent). Red blood cells were more often present in smears taken with the Armovical spatula than with the Ayre spatula. Although the data in the present study were too few to prove that the Armovical spatula is better than the Ayre spatula at detecting cellular abnormalities, it seems likely that this is so.


Congenital duplication of the lens.

Congenital duplication of the lens. A case of reduplication of the lens with uveal coloboma is described. This is a rare condition and, unlike the two previously reported cases, the other ocular structures and adnexae appeared normal.


Effect of pilocarpine on visual acuity and on the dimensions of the cornea and anterior chamber. The effect of pilocarpine on visual acuity and on the dimensions of the cornea, anterior chamber, and lens were studied in two groups of subjects. Significant changes in ocular tension, corneal curvature, anterior chamber depth, and lens anterior radius were found in a group of 55 glaucomatous eyes as a result of pilocarpine treatment, but there was no change in corneal thickness. Out of 102 glaucomatous eyes 78 became relatively myopic, and this appears to be due to changes in the dimensions of the lens of the eye similar to those occurring in accommodation, as a result of the effect of the drug on the ciliary muscle. The effect of pilocarpine on anterior chamber depth, area, and volume was studied in 125 eyes using a photographic method, and a significant reduction in the dimensions of the anterior chamber was observed as a result of the administration of pilocarpine. A significant correlation between depth and volume was also noted and the implications of this are discussed.


Rapid pneumatic and Mackey-Marg applanation tonometry to evaluate the postural effect on intraocular pressure. A postural study was conducted in three separate groups of subjects. The first group comprised 20 women volunteers with an average age of 20-75 years. In this group, the study was conducted by the pneumatonograph only. Mean pressure recorded was 15-65 +/- 0-25 mmHg and there was an average rise of 1-4 mmHg in supine posture. Groups 2 and 3 comprised 151 non-glaucomatous and 108 glaucomatous eyes respectively in the age range of 30 to 85 years. In these two groups, the study was conducted using the PTG and the Mackay-Marg tonometer. Clinical evaluation of the Mackay-Marg with the PTG gave significant correlation, with mean Mackay-Marg readings being 1-13 mmHg higher. The intraocular pressure when changing from seated to the supine position increased on average by 2-71 and 4-04 mmHg, respectively in Groups 2 and 3 and by 2-51 and 3-72 mmHg by Vackay-Marg, suggesting a higher change in glaucomatous subjects. Pressure on resumption of sitting was found to be lower than the initial pressure. Postural change also showed some direct relationship with age in non-glaucomatous subjects.


Chronic retinal vein occlusion in glaucoma. Asymptomatic chronic retinal vein occlusion that occurs in chronic simple glaucoma is described. The condition is characterized by marked elevation of retinal vein pressure with collateral vessels and vein loops at the optic disc in cases of central vein occlusion, or retinal veno-venous anastomoses along a horizontal line temporal and nasal to the disc in hemisphere vein occlusion. No patient had visible arterial changes, capillary closure, fluorescein leakage, or haemorrhages. The vein occlusion was not limited to "end stage" glaucoma. The role of increased intraocular pressure and glaucomatous enlargement of the optic cup with retinal vein distortion in the pathogenesis of the condition was stressed. Follow-up of these patients revealed persistence of the retinal vein occlusion shown by elevated retinal vein pressures. This would reduce effective perfusion of the inner retina and optic disc and may affect the long-term visual prognosis.


De novo lesions in presumed ocular histoplasmosis-like syndrome. Two patients with multifocal choroiditis similar or identical to POHS are presented. Colour photographs and fluorescein angiography document the occurrence of de novo lesions in the originally involved eye. The cases also demonstrate the development of new choroidal lesions within the originally involved eye, the early evolution of the "basic choroidal lesion", and the need for fluorescein angiography for visualizing the underlying choroidal lesion.


Pterygium and its relationship to the dry eye in the Bantu. A comparative study was performed on two groups of Bantus in Johannesburg to see if there was any relationship between the "dry eye" and pterygia, but no correlation was found.


Dacryolith formation around an eyelash retained in the lacrimal sac. A dacryolith was discovered in the lacrimal sac during a dacryocystorhinostomy for chronic dacryocystitis in which there was mucocele formation. Morphological examination confirmed the presence of an eyelash at the centre of the stone and electron microscopy demonstrated the presence of fungi (Candida sp.) in a matrix which was of markedly vairable morphology. The mechanism by which a hair enters the punctum and passes along the canaliculus may be attributed to the step-like pattern of ridges on the surface of a hair. The directional nature of these ridges dictates preferential movement towards the root end of the hair and prevents movements in the opposite direction.


Prognosis of perforating eye injury.

Prognosis of perforating eye injury. The assessment of visual function in a series of 130 consecutive patients of perforating eye injuries, revealed that visual acuity of 6/12 or better was regained in 63 per cent, between 6/60 and 6/18 in 9-2 per cent, less than 6/60 in 15-3 per cent, and enucleation was necessary in 9-2 per cent. In 3 per cent, the eyes were retained as blind, symptomfree, and cosmetically satisfactory organs. Two eyes were found to develop complete traumatic aniridia. None in the series was found to have sympathetic ophthalmitis.


Ocular injuries from liquid golf ball cores. Tissue removed from nine new cases from 18 hours to 20 weeks after injury by a golf ball contained crystalline and other foreign material to which there was a mild inflammatory reaction followed by macrophagic activity and fibrosis. Optical and electron probe analysis showed that the crystalline material was crushed barytes containing small quantities of muscovite as is typical in natural deposits. The centres of several golf balls were shown to contain essentially identical material. By contrast with previous reports, no zinc sulphide was found. The form and frequent location of the deposits in the conjunctiva as compared with cornea and eyelid is related to the structure of these tissues.


Vitrectomy in 125 eyes with diabetic vitreous haemorrhage. A total of 125 consecutive eyes, all registered blind with diabetic vitreous haemorrhage, underwent pars plana vitrectomy with the vitrophage. Sixty-six per cent experienced some improvement in their visual acuity; 24 per cent were unchanged and 10 per cent were worse postoperatively. The major surgical complication was controllable haemorrhage (23 per cent). No retinal dialysis occurred. Significant postoperative complications were transient (71 per cent) and persistent (11 per cent) corneal oedema, early (8 per cent) and late (13 per cent) vitreous haemorrhage, transient (30 per cent) and persistent (6 per cent) rise in intraocular pressure, and rubeosis iridis (5 per cent).


Management of persistent hyperplastic primary vitreous by pars plana vitrectomy. Two children with persistent hyperplastic primary vitreous (PHPV) underwent vitrectomy and lensectomy via the pars plana to remove the fibrovascular stalk. Postoperatively the eyes were quiet, only a slight vitreous haze obscured the fundus view in the immediate postoperative period, and the stumps of the stalks retracted. Early surgical treatment of PHPV may prevent later serious complications.


Lens extraction with ultrasound. Experiments in rabbits. The extraction of the rabbit lens is described using a 25 G irrigating needle and a 22 G aspirating needle; at the latter's bevelled tip lens fragmentation occurs due to the longitudinal ultrasonic vibrations generated there--an 'acoustic horn' causes the tip to vibrate with large amplitudes. The use of small needles allows considerable manoeuvrability in the anterior chamber and usually eliminates the need for corneal suturing. Push-pull coupled syringes equate the volume of irrigation with that of aspiration. This procedure makes possible lens extraction through an aperture in the anterior capsule of the rabbit's lens and a similar machine is being constructed for trial on human cataract.


Perifoveal vascular leakage and macular oedema after intracapsular cataract extraction. Perifoveal capillary leakage of fluorescein was demonstrated in 60 per cent of 50 eyes when angiography was performed two weeks after cataract extraction. Repeat angiography six weeks postoperatively in 17 eyes demonstrated persistence of already established leakage in 11 of 12 eyes and no new leakage in five eyes previously negative. Cystoid macular oedema with visual acuity of less than 20/40 six weeks postoperatively occurred in five eyes (10 per cent). Eyes of patients with vascular disease and those patients of 60 years or older were found to have altered vascular permeability significantly more frequently. Inflammation was no more severe or prevalent in those patients who demonstrated leakage and no inflammation was clinically apparent in 10 of 11 eyes demonstrating dye leakage six weeks postoperatively. We conclude that the constitutional factors of age and vascular disease are of prime importance in causing altered vascular permeability in the early postoperative period after cataract extraction; factors causing sustained leakage with reduction of visual acuity were not demonstrated.


Copper metabolism in retinitis pigmentosa. Clinically and electrophysiologically confirmed cases of primary retinitis pigmentosa have been investigated regarding their copper metabolic state. It is observed that these patients show a normal or near normal serum copper concentration, very low plasma caeruloplasmin concentration, and a very high copper urinary excretion. A similarity between this condition and hepatolenticular degeneration is drawn and it is suggested that retinitis pigmentosa may also be a condition caused by an inborn error of copper metabolism.


The optic disc in glaucoma. I: Classification. Five different descriptive types of glaucomatous optic discs are described, based on the examination of X2 magnification stereophotographs of 252 patients from the files of the Glaucoma Service at Wills Eye Hospital. The method of analysis is described in detail. These types include: overpass cupping, cupping without pallor of the neuroretinal rim, cupping with pallor of the neuroretinal rim, focal notching of the neuroretinal rim, and bean-pot cupping. These morphological types may be caused by variations in factors contributing to the pathogenesis of glaucomatous eyes. Recognition of these differing types may help in determining the factors in each case.


Plasma cortisol suppression response in the South African black population with glaucoma. Plasma cortisol suppression was measured in 25 Black glaucomatous patients and in 19 Black patients of similar age and sex, but without glaucoma, who acted as controls. Initial serum cortisol levels were found to be slightly higher in the glaucomatous group. The response to systemically-administered cortisone was statistically more marked in the glaucomatous patients compared with the control group.


A prospective study of xenon arc photocoagulation for central retinal vein occlusion. Twenty patients with central retinal vein occlusion were randomly divided into two groups in a prospective study to evaluate the effects of xenon are photocoagulation in central retinal vein occlusion. The patients in one group were treated with 360 degrees scatter xenon photocoagulation and the others received no treatment. The average follow-up was 18 months. There were no cases of rubeosis or neovascular glaucoma in the treated group. Two patients in the untreated group developed rubeosis with subsequent neovascular glaucoma. There was no significant difference in the visual prognosis or in fundus neovascularization between the groups.


Laser induced phagocytosis in the pigment epithelium of the Hunter dystrophic rat. The retinae of 14-day-old Hunter dystrophic rats have been subjected to low-energy irradiation by a pulsed ruby laser. Fifteen days after exposure, pigment epithelial cells had proliferated and repopulated the irradiated areas. In all such areas the subretinal photoreceptor debris had been reduced or lost.


Radioactive phosphorus uptake testing of choroidal lesions. A report of two false-negative tests. Two false-negative results from 32P testing for histologically verified malignant melanomas of the choroid are reported. In the first case, a haemorrhagic choroidal detachment caused an increase in probe; additionally, the tumour was necrotic. Both factors are likely to have contributed to the false-negative result. A satisfactory explanation for the false-negative result in the second case was not determined, although it may have accurately reflected a period of minimal tumour activity, inasmuch as repeat 32P testing was strongly positive eight months later, when unequivocal evidence of tumour growth was present. An alternative explanation is that the orally administered 32P was incompletely absorbed. Since 32P testing is frequently accompanied by significant manipulation both in the manoeuvre associated with tumour localization and in that associated with the actual radioactive counting, it would seem desirable to perform indicated enucleation immediately after completion of the 32P testing. While the properly performed 32P test remains a valuable diagnostic test for helping to establish the presence or absence of malignancies of the posterior globe, it is important to guard against the tendency to underestimate careful clinical evaluation.


The utilization of dietary protein by Nigerian men. 1. The excretion of urinary and faecal nitrogen by young Nigerian men of a low income group was found to vary significantly between subjects, but was not significantly different in the same subject, when they were given a mixed diet composed of rice, vegetables and fish on two separate occasions. At the levels of energy and protein supplied by this diet the men gained or lost small amounts of weight, mean values indicating N equilibrium, the variation around the mean being high. 2. The correlation between the urinary and faecal N of these men was significantly negative at and below the level of N equilibrium, becoming positive 2-3 weeks after they had consumed the above diet supplemented by 400 g of lean beef per day, and had already established considerable rates of gain in body-weight and N retention. The diets were made isoenergetic by adding the beef at the expense of cassava flour and red palm oil. 3. The physiological processes involved in the adaptation, by young Nigerian men of low income, to a highly efficient use of low-protein diets are discussed in relation to the biological value and true digestibility of those diets, and to the 'safe level of protein intake' and protein-energy ratios proposed by FAO/WHO (1973, 1975). 4. It is concluded that the protein requirements of apparently healthy men can only be established in the context of their ecological, socio-economic and nutritional backgrounds.


Determination of nitrogen requirement for microbial growth from the effect of urea supplementation of a low N diet on abomasal N flow and N recycling in wethers and lambs. 1. Plasma urea entry rate, urinary area excretion and, by difference, urea recycling in the body, together with the flow of non-ammonia N through the abomasum and digestion of dry matter (DM) before the abomasum were determined in both wethers and lambs receiving cereal-starch diets supplemented with urea to give 60-120 g crude protein (N X 6-25)/kg DM. 2. Lambs excreted less urea in urine than wethers given the same diet. 3. Relationships between plasma urea entry rate or urine urea excretion rate and plasma urea concentration were different for lambs compared to wethers suggesting greater conservation of body N by renal control in lambs. 4. Recycling of urea was not related to plasma urea concentration in wethers but was related exponentially in lambs, suggesting recycling is controlled rather than the result of simple diffusion from the blood to the gastro-intestinal tract. 5. Abomasal non-ammonia-N flow was similar for wethers and lambs and increased linearly with urea supplementation. 6. DM digestion prior to the abomasum was not significantly altered, although there was a tendency for decreased digestion of the basal diet given to lambs. 7. Maximum microbial N flow to the abomasum was estimated as 30 g N/kg organic matter (OM) fermented in the rumen. 8. This work and the literature reviewed suggested maximum net microbial production can be obtained when the diet supplies an amount of fermentable N equal to the microbial N output. It is calculated the diet should supply approximately 26 g fermentable N/kg digestible OM or 1-8 g fermentable N/MJ metabolizable energy. This corresponds to a fermentable crude protein supply varying from 65 to 130 g/kg DM as digestible OM content increases from 400 to 800 g/kg DM.


Amino acid metabolism in the piglet. 3. Influence of lysine level in the diet on energy metabolism and in vivo oxidation. 1. Supplementing a lysine-deficient diet (5 g lysine/kg) with five increments of lysine, each of 2 g/kg, resulted in increased in growth rate of Yorkshire piglets, aged between 3 and 7 weeks, up to the highest level of lysine (15 g/kg). 2. The free lysine concentration of plasma tended to increase as the dietary lysine level increased from 13 to 15 g/kg, and plasma threonine concentration decreased significantly as the lysine content of the diet was increased from 11 to 15 g/kg indicating that threonine was the second limiting amino acid in the diet. 3. Oxygen consumption and carbon dioxide production of the piglets were not influenced by supplementing the diets with lysine. The heat production was 0-313 kJ/min per kg body-weight in the 6 h experimental period. 4. Supplementation of the diet with lysine had no consistent effect on the recovery of 14C as 14CO2 from a single dose of L-[U-14C]lysine. 5. Adjustment of the determined recoveries of the tracer dose of lysine for the differences in the plasma concentrations of free lysine for the pigs receiving the graded levels of dietary lysine simplified the relationship between recovery and dietary lysine level: it was linear for the first four increments in dietary lysine and then increased sharply for the fifth increment. This indicated that a marked change in the rate of lysine catabolism occurred as the level of dietary lysine was increased from 13 to 15 g/kg. 6. The results of this experiment indicate that the piglets' requirement for lysine is between 13 and 15 g lysine/kg in a diet which contained 181 g crude protein (nitrogen X6-25)/kg.


Utilization of dried microbial cells grown on methanol in a semi-purified diet for growing pigs. 1. Twenty-four pigs (mean body-weight 44 kg) were used to determine the digestibilities of energy, nitrogen and amino acids in dried microbial cells (DMC) grown on methanol and in white fish meal (WFM). N utilization and the excretion of various nitrogenous compounds were also studied. 2. Semi-purified diets containing DMC, WFM or no protein source were offered at the rate of 1 kg fresh food/d. Daily N intakes were 26-5, 24-7 and 0-65 g respectively. 3. Mean metabolic faecal N determined with the protein-free diet was 1-11 g/d (1-26 g/kg dry matter (DM) eaten) for the two experiments. The corresponding endogenous urinary N value was 2-91 g/d. 4. Daily urinary N output of pigs given the diet with WFM was significantly greater than that of pigs given the diet with DMC, with the result that N retention, net protein utilization and biological value were higher for pigs given DMC. 5. Apparent and true digestibility of amino acids were higher for the diet with DMC than for the diet with WFM. 6. N excretion in the form of ammonia, urea, uric acid and allantoin accounted for 81, 91 and 99% of the total N excreted by the groups given the protein-free diet, DMC and WFM respectively. Pigs given DMC excreted greater quantities of allantoin-N and less urea-N than pigs given WFM.


Nutritional evaluation of the protein of dried tomato pomace in the rat. 1. Nutritional evaluation of dried tomato pomace (DTP-20) as a source of protein was carried out using weanling rats. Comparisons were made with casein (CS), soya-bean meal (SOM-45) and the hydrocarbon-grown yeast Toprina (BP-T). The growth-promoting effects of the diets were evaluated over a period of 28 d of ad lib. feeding. 2. The unsupplemented DPT-20 had a protein efficiency ratio (PER) of 2-18 +/- 0-13 and a net protein utilization (NPU) of 0-55. The addition of DL-methionine (5 g/kg) resulted in a PER of 1-74 +/- 0-15 (t 2-99, P less than 0-01) and a NPU of 0-40, while the addition of an amino acid mixture resulted in a PER of 2-33 +/- 0-08 (t 1-84, P greater than 0-1) and a NPU of 0-70. 3. The reason for the decrease in growth and the reduced PER and NPU values recorded for the methionine-supplemented group of rats is not clear and it is discussed in connexion with an imbalance or a slight toxicity caused by the added amino acid.


Regulation of food intake during growth in fatty and lean female Zucker rats given diets of different protein content. 1. Food intake, and the rates of protein, lipid and energy deposition during growth were measured for lean and congenitally obese (fatty) female Zucker rats given diets of different protein content ad lib. Six semi-synthetic diets were used, which contained 40, 100, 150, 300, 500 and 700 g casein/kg (diets 40C, 100C, 150C, 300C, 500C and 700C). 2. Dietary treatments began when the rats were 34 or 35 d old. Collections of urine and faeces were analysed for energy content. The total carcasses of all the rats were analysed individually for protein, lipid and energy. 3. In the first experiment, twelve rats of each phenotype were given diets 150C or 300C. Four fatty and four lean rats were killed at 50, 66 and 98 d of age. In the second experiment groups of four fatty and four lean rats were given diets 40C, 100C, 500C and 700C ad lib. until they were killed at 66 d of age. Other groups of fatty rats were pair-fed from 35 to 67 d of age on diets 100C and 500C. Rats were also killed at 24 and 34 d of age to provide initial samples for the comparative slaughter procedure. 4. When given food ad lib., fatty and lean rats had identical rates of protein deposition at all ages and for all diets, but lipid and energy deposition were always much greater in the fatty rats. Food intake was also much greater for the fatty rats (except on diet 40C). Differences in food intake and growth rate attributable to diet were most pronounced for the range of diets 40C-150C. 5. Fatty rats pair-fed to lean rats deposited less protein but about twice as much energy and lipid as lean rats on the same diets. 6. The results are discussed in relation to existing theories of appetite control. It appears that food intake is precisely regulated in the congenitally obese Zucker rat. This regulation is intimately linked with the impetus for protein deposition and the rates of retention of lipid and energy appear to be of no importance in relation to appetite control.


The digestion of yeast cell wall polysaccharides in veal calves. 1. The digestibility of the cell wall polysaccharides of an alkane-grown yeast in different parts of the digestive tract of two veal calves fitted with re-entrant cannulas at the end of the ileum was studied by replacing part of the skim-milk powder of their 'normal', milk-substitute (all-milk-protein) diet by yeast (yeast diet). 2. The lactose and glucose of both the all-milk-protein diet and the yeast diet were almost completely digested before the end of the ileum. During this digestion a small amount of oligosaccharides composed of galactose and glucose was synthesized. These oligosaccharides were digested again in the large intestine. 3. The constituent sugars of the water-soluble fraction of the yeast cell wall carbohydrates were glucose and mannose. The 0-5 M-sulphuric acid-hydrolysate of the water-insoluble fraction contained glucose and mannose and the 12 M-H2SO4-hydrolysate only glucose. 4. Digestibilities measured at the end of the ileum varied considerably between the two animals and averaged only about 0-40. 5. These findings suggest that the cell wall polysaccharides of yeast are digested very little by the normal digestive enzymes of the calf's small intestine, but are used as a substrate by the bacterial flora which are mainly concentrated in the large intestine.


Studies on the nutrition of marine flatfish. The effect of different dietary fatty acids on the growth and fatty acid composition of turbot (Scophthalmus maximus). 1. Five groups of juvenile turbot (Scophthalmus maximus) which had been given a diet free of fat for 12 weeks were given diets in which the lipid component (g/kg) was: oleic acid alone 50, oleic acid 40+linoleic acid 10, oleic acid 40+linolenic acid 10, oleic acid40+arachidonic acid 10 or oleic acid 40+cod-liver oil 10. These five experimental diets were given for 16 weeks. 2. Weight gains were highest in the group given the diet containing cod-liver oil and lowest in the groups given diets containing oleic acid alone or oleic acid+linoleic acid. Weight gains in the groups given oleic acid+arachidonic acid or linolenic acid were markedly inferior to those of the group given oleic acid+cod-liver oil. It is concluded that arachidonic acid is inferior to polyunsaturated fatty acids of the omega3 series in maintaining growth rate in turbot. 3. Fatty acid analyses of neutral lipids and phospholipids of liver and extrahepatic tissues did not suggest any evidence of desaturation of dietary oleic acid, linoleic acid or linolenic acid by the turbot. These experiments confirm previous isotopic evidence that turbot lack the necessary microsomal desaturases to perform this metabolic transformation.


Changes in the alkaline phosphatase (EC 3.1.3.1) and inorganic pyrophosphatase (EC 3.6.1.1) activities of rat tissues during magnesium deficiency. The importance of controlling feeding pattern. 1. The adoption of a meal-eating pattern of feeding by rats altered the alkaline phosphatase (EC 3.1.3.1) activity in serum and liver. It was therefore necessary to regulate the feeding pattern of both magnesium-deficient rats and control animals receiving a Mg-adequate diet in order to study the effect of the deficiency. 2. Mg deficiency decreased the activities of alkaline phosphatase and inorganic pyrophosphatase (EC 3.6.1.1) in serum, kidney and tibia, but increased them in spleen. 3. Addition of a standard concentration of exogenous Mg to tissue extracts usually increased the activity of corresponding enzymes from Mg-deficient and control rats by the same proportion, indicating that the main effect of the deficiency was on the amount of enzyme present rather than on the efficiency of its operation. 4. Certain quantitative differences in the response to exogenous Mg and the activity ratio, alkaline phosphatase:inorganic pyrophosphatase were found between tissues from Mg-deficient and control rats. The significance of these are discussed in relation to the association of the two enzymic activities with the same protein molecule, and the possible occurrence of isoenzymes.


Effect of dietary nitrogen source on carbohydrate metabolism in the rumen of the young steer. 1. Steers, fitted with simple rumen and duodenal cannulas, were given diets of approximately equal parts of flaked maize and hay (A) (containing 16 g nitrogen/kg dry matter (DM) or diets of flaked maize and straw supplemented with decorticated groundnut meal (B), fish meal (C), or heated (D) or unheated (E) soya-bean meal (containing 24 g N/kg DM). Chromic oxide was given as a marker with the feeds, and flows of different combined sugars at the duodenum estimated from the values for sugars:marker at this site. Contributions of bacterial constituents to these flows were estimated from amounts of RNA present. 2. Rumen bacteria from steers receiving diet A contained approximately 110 g alpha-dextran-glucose/kg DM and contributed about 60 g alpha-dextran-glucose/d at the duodenum; bacteria from steers receiving diets B, C, D or E contained 25-40 g alpha-dextran-glucose/kg DM and contributed about 20-30 g alpha-dextran-glucose/d at the duodenum. There were no significant differences between different N supplements. About half the alpha-dextran-glucose, varying amounts of the mannose and galactose, and nearly all the rhamnose and ribose in duodenal digesta were contributed by the bacteria. Almost all the arabinose, xylose and cellulose-glucose was of dietary origin. 3. For steers receiving diet A, mean coefficients of digestibility between mouth and duodenum, corrected where necessary for bacterial contribution, were 0-96, 0-73, 0-58, 0-22 and 0-53 for starch-glucose, galactose, arabinose, xylose and cellulose-glucose respectively. Corresponding mean values when diets B, C, D and E were given, which did not differ significantly amongst themselves, were 0-98, 0-79, 0-81, 0-59 and 0-58. Most values for galactose, arabinose and xylose were significantly higher than the diet A values.


Protective effect of zinc supplementation against copper toxicosis in sheep. 1. A study has been made of the effects of dietary zinc supplementation on the development of copper toxicosis in three groups each of eight 12-week-old lambs. 2. None of the lambs receiving 420 mg Zn/kg diet developed Cu toxicosis in the 24-week experimental period, compared with three in the control group receiving 43 mg Zn/kg and possibly one in the group receiving 220 mg Zn/kg. 3. Liver Cu concentrations were reduced by up to 40% in the Zn-supplemented animals, with concomitant reductions, especially in the early stages of the experiment, in the extent of liver damage, as assessed by measurement of plasma aspartate aminotransferase (EC 2.6.1.1) and arginase (EC 3.5.3.1) activities. 4. Plasma and liver Zn concentrations were increased only slightly in the lambs receiving the Zn-supplemented diets, and the only indication of possible toxic effects of the Zn supplements was the development of a slight anaemia in those animals receiving 420 mg Zn/kg diet. 5. The results suggest that the incidence of Cu toxicosis in sheep may be controlled by increasing their dietary Zn intake.


The biphasic nature of protein metabolism during pregnancy in the rat. 1. Additional protein given to malnourished rats early in pregnancy was found to increase substantially the weight and cellularity of their foetuses and placentas at term. 2. This observation confirms the hypothesis that protein is stored in early pregnancy for use during the period of rapid foetal growth.


The rapid determination of carcass fat by the Foss-Let specific gravity technique. 1. Carcass fat was determined by extraction with tetrachloroethylene and measurement of the solvent's change in density. The results were comparable in precision to those of a reference method; the new method extracted storage lipid but little structural lipid. 2. The technique is simple, rapid and appropriate for many nutritional studies.


Sulphur and cystine incorporation into rumen microbial protein. 1. In three series of experiments, Na235so4 was infused into the rumen of sheep consuming a variety of diets. The specific activity of the sulphur in the rumen H2S pool, rumen bacteria and of the S in bacterial cystine were determined. 2. The results showed that, depending upon the diet, the proportion of total sulphur and cystine sulphur in rumen bacteria originating in the rumen H2S pool could be as low as 0-557 and 0-280 respectively.


Protein turnover in rats treated with trienbolone acetate. 1. The anabolic agent Trienbolone acetate (3-oxo-17-beta-hydroxy-4,9,11-estratriene acetate) given subccutaneously to female rats increased their growth ratndrogenic activity. 3. The fractional synthetic and fractional degradative rates of the mixed muscle proteins were smaller in the treated rats than in the control rats.


Evidence for negative cooperativity in the adsorption of phosphorylase b on hydrophobic agaroses. The concept of cooperativity appears to be the key to the understanding of the complex mechanisms underlying the adsorption of proteins to agaroses substituted with hydrophobic alpha-aminoalkanes. The adsorption of phosphorylase b occurs through the positive cooperative interaction of a critical number of approximately 3-5 butyl and a higher number of methyl residues with ocrresponding sites on the enzyme. The amount of adsorbed phosphorylase b per millileter of packed gel (methyl-, butyl-Sepharose) in the absence and presence of 1.1 M ammonium sulfate at temperatures between 0 and 34 degrees C is a power function of the free solute equilibrium concentration (Freundlich isotherm). In contrast, the adsorption of cyanmyoglobin to phosphocellulose is described by the Langmuir equation. The surface coverage dependent isosteric heats of adsorption for phosphorylase b indicate an endothermic reaction only on the butyl-Sepharose in the presence of high salt concentrations. Scatchard plots of the Freundlich isotherms of phosphorylase b are concave upwards, typical of negative cooperativity. Hill plots of these isotherms (5-70% saturation) yield coefficients between nH = 0.39 and 0.71. At high surface coverages, the Hill coefficients approach unity. Apparent association constants (K0.5) of 4-39 X 10(4) M-1 are calculated for the adsorption of phosphorylase b, as compared to 2-9 X 10(4) M-1 for the adsorption of cyanmyoblobin. In general, negative cooperativity of binding may be explained by changes in the affinity of the ligand for the matrix, due to the sequential multivalent adsorption, and competition of phosphorylase b molecules for the critical number of alkyl residues (nonindependence of binding) on one side and to variations in the configuration of binding and entropy on the other.


Effects of the aliphatic carboxylate series of salts on the conformation of proteins. The effects of the aliphatic acid series of salts, formate, acetate, propionate, butyrate, valarate, and caproate, on the conformation of sperm whale myoglobin, human hemoglobin A, and horse heart cytochrome c were investigated by spectral measurements in the Soret region, optical rotation, and intrinsic viscosity measurements. The effectiveness of the aliphatic acid salts as unfolding reagents for proteins is found to increase with increasing hydrocarbon content of the alkyl chains of the salts, which is analogous in behavior to effects of the urea, amide, and alcohol series of protein denaturants. The denaturation midpoints, Sm, as a function of the unfolding reagent were analyzed using the equations of Peller (Peller, L. (1959), J. Phys, Chem- 63, 1199) and Flory (Flory, P.J. (1957), J. Cell. Comp. Physiol. 49, 175) with binding constants based in part on the Scherage-Nemethy theory of hydrophobic bonding or evaluated from free-energy transfer data of nonpolar amino acid side chains from aqueous to nonaqueous solvents. The summation of the polar KP and hydrophobic KHphi contributions of solvent to protein amino acid side chain interactions were found to give best account of the protein denaturation data. Intrinsic viscosity and optical rotation data obtained on hemoglobin and myoglobin at high salt concentrations, above the unfolding transition regions, indicate that the product of denaturation by the aliphatic acid salts is less unfolded than in 6 M guanidine hydrochloride solutions. Residual elements of the helical regions of the proteins seem to either escape unfolding or are reformed at high concentrations of the denaturing salts.


Light-scattering investigations of the subunit dissociation of human hemoglobin A. Effects of the aliphatic acid salts. The subunit dissociation of human hemoglobin A by the aliphatic acid salts at neutral pH has been investigated by light-scattering molecular-weight measurements at 630 nm. Dissociation of hemoglobin tetramers to alphabeta dimers is observed in essentially all experiments at low to intermediate levels of salt concentrations, below the denaturation transitions, described in the accompanying paper (Ibanez, V.S., and Herskovits, T.T. (1976), Biochemistry 15, preceding paper in this issue). The effectiveness of the salts as subunit dissociating agent, reflected by the slopes, s, of the plots of deltaGDdegrees, the standard free energy of dissociation, vs. [D], the salt concentration, is found to increase with increasing alkyl chain length or hydrocarbon content of the salt. Estimates of the apparent number of amino acid sites at the areas of contact per alphabeta dimer formed, N', based on the slopes of the higher members of the series have been obtained using the equation, deltaGDdegrees = deltaGD,Wdegrees - 2N'RTKB[D]. Independent estimates of the binding constant, KB, required for these calculations were based on free-energy transfer data of hydrophobic amino acid alkyl groups and protein denaturation data. Our estimates of N' denaturation data. Our estimates of N' obtained with the more reliable data of the higher members of salt series are in the ranges of 19 and 27 amino acid groups, shown by the x-ray crystallographic structure of horse and human hemoglobin of Perutz (Perutz, M.F., et al. (1968), Nature (London) 219, 131) and Fermi ((1975) J. Mol. Biol. 97, 237) for the smaller alpha1 beta2 contact areas in the tetrameric structure. The lower estimates than 27 based on our dissociation of human hemoglobin suggest that several of the amino acid residues in the contact areas of the subunits are partially exposed to solvent. The increasing effectiveness of the higher mn imporant source of stabilization of the tetrameric structure of hemoglobin.


Intramolecular 1H nuclear Overhauser effect study of the solution conformation of valinomycin in dimethyl sulfoxide. Determination of the mechanism of intramolecular nuclear Overhauser effects (NOE) in peptides and depsipeptides is essential to the use of this technique in conformational analysis of these and related biomolecules. Towards this end, 1H NMR double-resonance studies were conducted on valinomycin in (CD3)2SO at 90 MGZ (FT mode) and 250 MGZ (correlation mode). The NOE's are positive at the lower frequency and negative at the higher frequency. Consideration of the theoretical dependence of the NOE on the proton-proton internuclear correlation time and on the resonance frequency indicates that these results are explained by a predominantly dipolar relaxation mechanism. It is demonstrated that exchange modulation of scalar coupling does not contribute significantly to the NOE. A formalism for the NOE's of loosely coupled spin systems is presented which takes into account the effects of high magnetic-field strengths and long correlation times. An approximate analysis of the NOE data assuming a single correlation time for the entire molecule and ignoring cross-relaxation effects was used to evaluate various models that have been proposed for the conformation of valinomycin. The III-1 model of Patel and Tonelli (Patel, D.J., and Tonelli, A.E. (1973), Biochemistry 12, 486) fits the NOE and peptide NHCalphaH coupling constant data and is probably a preferred orientation in dimethyl sulfoxide. These experiments illustrate how intramolecular NOE data provide a valuable auxiliary method to other techniques for delineating the preferred solution conformation of peptides, depsipeptides, and other biomolecules.


Influence of 5'-terminal m7G and 2'--O-methylated residues on messenger ribonucleic acid binding to ribosomes. Removal of 80% of the 5'-terminal 7-methyl-guanosine (m7G) from methylated reovirus mRNA by beta elimination results in a concomitant loss of the ability to bind to wheat germ ribosomes. The mRNA molecules that retain the m7G account for most of the residual binding. Removal of the m7G from all molecules in preparations of methylated reovirus and vesicular stomatitis virus mRNA reduces the extent of binding to wheat germ ribosomes from 80% to 5-7%. In the reticulocyte lysate, however, significant binding (17-34%) of the beta-eliminated viral RNAs occurs. This m7G-independent binding appears to be due to recognition by ribosomes of other structural features of the 5'-proximal sequences. Initiation complexes involving beta-eliminated animal virus mRNAs and rabbit reticulocyte ribosomes appear to be more stable than the more heterologous combination of the same viral mRNAs and wheat germ ribosomes. In addition, evidence is presented to show that the 2'-O-methylated nucleoside of the 5'-terminal cap has a positive influence on the ribosome binding of viral mRNA and of capped synthetic ribopolymers. A model involving recognition of multiple structural features of the 5'-terminal region of mRNA by ribosomes during initiation of protein synthesis is presented.


Natural abundance carbon-13 nuclear magnetic resonance studies of bovine white matter and myelin. Whole bovine white matter yields a poorly resolved natural abundance 13C nuclear magnetic resonance (NMR) spectrum. The spectrum principally reflects carbon atoms of the constituent membrane lipids: several resonances could be specifically assigned but no resonances attributable to cholesterol are detectable. Except for the methyl group at the terminus of fatty acyl chains, lipid carbons giving rise to the 13C NMR spectrum have values of spin-lattice relaxation time between 140 and 500 ms, indicating significant restrictions on segmental and rotational mibolities but consistent with a generally fluid structural organization. The 13C NMR spectrum of myelin isolated from bovine white matter is similar to that for the whole white matter itself. In both white matter and isolated meylin, the integrated intensities for several carbon atoms are considerably less than those for the same carbon atoms in total lipid extracts. The data for white matter and myelin are consistent with a model in which observed line broadening is due to restrictions in the amplitude of chain flexing rather than to severe restrictions on chain segmental motion. Failure to detect resonances of cholesterol ring system carbon atoms may reflect marked anisotropy of rotational reorientation.


Utilization of serum lipoprotein lipids by the monoacylglycerol acyltransferase. The plasma membrane of rat liver contains an enzyme which is stimulated by heparin and hydrolyzes liposomes composed of phosphoglycerides as well as mono-and diacylglycerol (Waite, M. and Sisson, P (1973) J.Biol, Chem. 248, 7985-7902). Further, in liposomes this enzyme catalyzes a transacylation in which the acyl group is removed from position 1 of an acyl glyceride donor, and combined with the hydroxy group of a variety of acyl acceptors. To investigate the possible role of this enzyme in lipoprotein metabolism, we have incorporated specific labeled glycerides into lipoproteins. The high density, low and very low lipoproteins were then separated by molecular sieving and characterized by their physical and chemical properties. Using the labeled substrates as model lipoproteins, we found the following: 1)The enzyme is capable of hydrolyzing monoacylglycerol, diacylglycerophosphoethanolamine and diacylglycerophosphocholine; monoacylglycerol however is the preferred substrate in all three lipoprotein fractions. 2)Relative to the activity found on liposomes, the transacylation activity is low. 3)The specific radioactivity of the substrates in fraction B (low density lipoprotein) did not change during the reaction, which indicates that the labeled lipid is not a pool separate from the endogenous lipid of the lipoprotein.


Enzymes of phospholipid synthesis in Bacillus Calmette-Guerin induced rabbit alveolar macrophage. Characterization and localization of cytidine diphosphocholine phosphotransferase and monoacylphospholipid acyltransferase. The rabbit alveolar macrophage is capable of renewing its plasma membrane by at least two metabolic pathways. It contains (1) a monoacylphospholipid acyltransferase, which catalyzes the synthesis of diacylphospholipids by recycling monoacylphospholipids produced by the action of phospholipases and (2) a cytidine diphosphocholine phosphotransferase (CDPcholine phosphotransferase), which catalyzes the last step in the synthesis de novo of diacylglycerophosphocholine. These activities have been characterized in the cell homogenate with respect to time, protein, pH optimum (for CDPcholine phosphotransferase), substrate specificity (for monoacylphospholipid acyltransferase) and cation requirement ( for CDPcholine phosphotransferase). Monoacylphospholipid acyltransferase activity is localized solely in the endoplasmic reticulum. On the other hand, the CDPcholine phosphotransferase activity can be measured in the endoplasmic reticulum and in the plasma membrane, characterized by both differential and gradient sedimentation techniques. In addition to the normal route of phospholipid synthesis in the endoplasmic reticulum, the rabbit alveolar macrophage may thus possess the capacity for in situ synthesis of phospholipids of plasma membrane as a mechanism for membrane renewal following phagocytosis.


Phospholipase A2 activity of the regularly arranged surface protein of Acinetobacter sp.199A. The regularly arranged surface protein, the a-protein, of Acinetobacter 199A has been shown to have phospholipase A2 activity. Since half of the a-protein synthesised by Acinetobacter 199A is secreted into the growth medium, the bacteria are producing extracellular phospholipase A2.


Properties of purified bovine milk lipoprotein lipase. Lipoprotein lipase has been purified from bovine milk by affinity chromatography on Sepharose containing covalently linked heparin. In addition to an enzyme eluted by salt, further activity could be eluted with detergent. Rechromatography experiments suggested that the two activities were due to the same enzyme. This assumption was further verified by several other criteria as follows: (a) both require a serum activator, (b) their apparent molecular weights (55 000), their amino acid compositions and amino sugar contents were similar and (c) they had identical immunological reactivities. Thus, the enzyme appears to be bound to the heparin-Sepharose matrix by both salt-reversed and detergent-reversed interactions. Sodium deoxycholate stimulated the activity eluted by high salt, but had no effect on the detergent-eluted enzyme.


On the binding of bile salt to pancreatic lipase. The binding of taurodeoxycholate to pancreatic lipase and a few other proteins has been studied with equilibrium dialysis and in gel filtration experiments. A three compartment dialysis cell has been used; with this cell, complete equilibration is not necessary for calculation of the binding even at bile salt concentrations above the critical micellar concentration. The results indicate that taurodeoxycholate does not bind to lipase below the critical micellar concentration, that the binding starts in the critical micellar concentration range of the bile salt and reaches around 12 mol taurodeoxycholate per mol of lipase at taurodeoxycholate concentrations well above the critical micellar concentration. Previous results indicating a binding of maximally 1-2 mol taurodeoxycholate/mol lipase were too low, depending on the experimental conditions in which complete equilibration was not obtained. The binding isotherm for taurodeoxycholate to lipase is similar to that for co-lipase; colipase and lipase in mixture bind as much taurodeoxycholate as the sum for the single proteins. Taurodeoxycholate binds to ribonuclease and chymotrypsinogen to a similar extent as to lipase.


Influence of trypsin on lipolysis in human fat cells. Comparison with rat adipocytes. 1. Trypsin-treated human and rat fat cells were obtained by digestion of adipose tissue with collagenase plus trypsin and their lipolytic response to insulin, catecholamines and dibutyryl cyclic AMP were compared with the lipolytic response of human and rat fat cells isolated with collagenase only. 2. In both human and rat fat cells, no significant modification occurred in the intracellular lactate dehydrogenase content and in the basal release of glycerol after trypsination. 3. In rat fat cells, trypsin abolished the antilipolytic effect of insulin but maintained a normal lipolytic response to epinephrine, norepinephrine and isoproterenol. 4. In human fat cells, on the contrary, trypsin failed to modify the antilipolytic effect of insulin, but markedly potentiated the lipolytic response to epinephrine, norepinephrine and isoproterenol. Trypsin also increased the rate of intracellular 3' :5' cyclic AMP accumulation in response to catecholamines. Under these conditions, however, trypsin-treated human fat cells had a normal reponse to the lipolytic agent dibutyryl cyclin AMP. 5. These data suggest that human fat cells differ from the rat ones by the existence in human adipocyte membranes of a trypsin-sensitive component which inhibits the catecholamine induced lipolytic process and which is different from the alpha receptors.


Deposition and mobilization of cholesterol ester in cultured human skin fibroblasts. Human skin fibroblasts in culture served as a model system to study intracellular cholesterol ester deposition in mesenchymal cells. Confluent cultures were exposed to homologous low density lipoprotein alone and together with chloroquine. In the presence of low density lipoprotein alone, even at half circulating serum concentrations, cellular free cholesterol increased no more than 12%, while the increase in cholesterol ester ranged from 13--100% during 48 h of incubation. Addition of chloroquine to the culture medium containing low density lipoprotein resulted in a very marked increase in cholesterol ester and the ratio of cellular esterified cholesterol to free cholesterol rose up to 2.2. In the presence of chloroquine the sum of uptake and degradation of 125I-labeled low density lipoprotein was enhanced and at higher chloroquine concentrations the more pronounced inhibition of degradation resulted in the intracellular retention of undegraded protein. Upon removal of the chloroquine-containing medium, there was a slight fall in the cellular cholesterol after 24 h incubation in a medium containing 10% fetal calf serum. Replacement of the fetal calf serum by lipoprotein-deficient serum and a mixture of high density apolipoprotein and sphingomyelin increased very significantly the loss of total cholesterol from the cells. At the same time the rate of loss of the retained labeled low density lipoprotein was also increased. The present results provide an adequate and reproducible model system for the study of cholesterol accumulation in human mesenchymal cells, which is one of the basic changes in atheromatosis. The availability of cholesterol ester laden cells also provides a good system for the study of agents active in cholesterol removal.


The effect of diether phosphatidylcholine on the enterohepatic circulation of biliary sterols. The effect of diether phosphatidylcholine on the enterohepatic circulation of bile salts and cholesterol was determined using the bile duct cannulated rat. Animals were infused intraduodenally with a solution containing radioactive bile salts and cholesterol. Phospholipid was also present in the infusate. In control animals it was supplied by pig liver phosphatidylcholine (diester). For experiments, equimolar amounts of diether phosphatidylcholine were used. No difference was observed between control and experimental groups for absorption and subsequent secretion of bile salts into bile. Significantly less cholesterol was absorbed, however, by experimental rats. This was correlated with a decreased secretion of radioactive cholesterol into bile. In controls, almost 14% of the administered dose of cholesterol appeared in bile over the period of infusion while the value for experimentals was 5%. It is concluded that diether phosphatidylcholine inhibits the enterohepatic circulation of cholesterol but not of bile salts.


The effects of detergents and phospholipids on a glycolipid galactosyltransferase. 1. Phospholipids activated the enzyme, lactosylceramide: UDP-galactose alpha-galactosyltransferase in hamster cells (NIL 2E clone 8) when assayed in the presence of neutral detergents. 2. Phosphatidylserine and phosphatidylinositol were the most effective phospholipids in activating the enzyme. Other phospholipids were also effective, but sphingomyelin and lysophosphatidylcholine were inhibitory. 3. Considerable enzyme activity was obtained in the absence of any detergent. Most of this activity was due to glycosylation of endogenous acceptors. 4. There was a complex effect of detergents on the enzyme activity. Very low concentrations were sharply inhibitory, but higher concentrations, above the critical micelle concentration for detergent, caused regeneration of activity. 5. The phospholipids, in the absence of a detergent, are required to maintain the lipid substrate, lactosylceramide, in a suitable dispersion where it can be acted upon by the enzyme. In the presence of detergents, it is proposed that the phospholipids also act by affecting the state of the lipid substrate.


The subcellular distribution of carotenoids in Phycomyces blakesleeanus C115 car-42 mad-107 (--). 1. Subcellular fractions of the C115 car-42 mad-107(--) strain of Phycomyces blakesleeanus, prepared by differential centrifugation, were irreversibly associated with extraneous lipids. Such contamination was avoided by the use of density gradient centrifugation. 2. The two beta-carotene-containing fractions, a particulate fraction and lipid globules, also contained sterol, phospholipid and alkaline p-nitrophenylphosphatase activity. Both fractions revealed close similarities on amino acid analysis.


Formation of [17-2H]androsta-5,16-dien-3beta-ol from [17,21,21,21]pregnenolone by the microsomal fraction of boar testis. After incubation of [17,21,21,21-2H4]pregnenolone, which had been synthesized from nonlabeled pregnenolone by base-catalyzed exchange procedure, with the microsomal fraction of boar testis, [17-2H]androsta-5,16-dien-3beta-ol was identified as a metabolite. The identification has been performed by gas chromatography-mass spectrometry of the metabolite and of its hydrogenation product. Conclusive evidence on the location of the 2H atom at C-17 has been obtained by transforming the metabolite to androsta-4,16-dien-3-one and then to androst-4-ene-3,17-dione and by proving that the former compound contained a 2H atom while the latter did not. The result indicates that, in boar testis, androsta-5,16-dien-3beta-ol is synthesized from pregnenolone by a pathway in which 17 alpha-hydrogen atom of pregnenolone is retained throughout the reaction, excluding 17 alpha-hydroxypregnenolone and 3beta-hydroxyandrost-5-en17-one as intermediates.


Conversions of prostaglandin endoperoxides by glutathione-S-transferases and serum albumins. Serum albumins of certain animal species (cow, sheep, pig) accelerate the decomposition of prostaglandin endoperoxides, with formation of large amounts of prostaglandin D. The reaction is inhibited by arachidonic acid, which suggests an interaction of the endoperoxide with the fatty acid binding sites of serum albumin. Glutathione-S-transferases, in the presence of glutathione, convert the endoperoxide into a mixture of prostaglandin F2alpha, E2 and D2. The prostaglandin D/E-ratio depends on the transferase used. The known rat liver transferases give mainly prostaglandin F2alpha and E2, but a new transferase in sheep lung was discovered which gives rise to large quantities of prostaglandin F2alpha and D2. The sheep lung transferase was purified to homogeneity. Two iso-enzymes with identical enzymic activity were obtained. The major component (transferase SL 2, an iso-enzyme of glutathione-S-transferase, EC 2.5.1.18) has a molecular weight of 45 000 and consists of two subunits. Its isoelectric point is 9,8-9.9. These properties, as well as the amino acid composition and the substrate specificity for typical transferase substrates, indicate a close resemblance to transferase B (ligandin), a major binding protein of rat liver. Although purified glutathione peroxidase from erythrocytes is very active in catalysing the reduction of the 15-hydroperoxy group of prostaglandins, it does not have any effect on the decomposition of the endoperoxide group.


Stimulation of the microsomal alkylglycerol monooxygenase by catalase. Liver cytosol contains a heat-sensitive nondialyzable soluble factor necessary for maximal activity of the alkylglycerol monooxygenase present in rat liver microsomes. In this report, we demonstrate that the stimulatory component in rat liver supernatant is catalase. Catalase functions to protect the enzyme from inactivation by H2O2 in addition to its documented ability to retard the noenzymatic oxidation of the pterin cofactor. The protective effect of catalase on alkylglycerol monooxygenase and on the aromatic amino acid hydroxylase systems indicates that H2O2 sensitivity is a general feature of pterin-dependent hydroxylases.


Subcellular distribution of a gonadotropin-induced form of mouse ovarian alkaline phosphatase. The subcellular distributions of alkaline phosphates I (the major activity of prepubertal mouse ovaries) and alkaline phosphatase Ib (a kinetically distinct isoenzyme induced in large amounts by injection of human chorionic gonadotropin or luteinizing hormone) were studied by differential rate centrifugation and discontinuous density gradient centrifugation of ovarian homogenates from control and gonadotropin-treated mice. The distributions of the two alkaline phosphatases were alike and were similar to those of nucleotidase, Mg2+ -dependent ATPase and Co2+ -stimulated naphthylamidase activities, suggesting that they were associated with plasma-membrane vesicles.


The isolation and characterisation of hyaluronic acid in egg shell. Analysis showed that the organic part of the chicken's egg shell consisted of a series of proteins and polysaccharides, probably present as glycoproteins and glycosaminoglycans. A purified preparation of a glycosaminoglycan (minimum mol. wt. 25 000), homogeneous by sedimentation velocity analysis and sedimentation to equilibrium in a density gradient, contained equimolar amounts of N-acetylglucosamine (36.3% s/w) and glucuronic acid 35.6% w/w). Digestion with testicular and streptomyces hyaluronidases and identification of the degradation products showed the glycosaminoglycan to be hyaluronidases and identification of the degradation products showed the glycosaminoglycan to be hyaluronic acid.


Quantitative aspect of the myofibrillar protein turnover in transient state on dietary protein depletion and repletion revealed by urinary excretion of N7-methylhistid;ne. The fractional rates of synthesis and breakdown of myosin and actin in skeletal muscle of young adult male rats were measured during 2 weeks of ad libitum feeding of a protein-free diet, and 8 days of refeeding with an adequate protein diet. Daily urinary excretion of NT-methylhistidine (3-methylhistidine) by the NT-methylhistidine pool of the body gave the fractional breakdown rate of the myosin-actin pool. The fractional synthesis rate of the myosin-actin pool was calculated from the fractional breakdown rate and the size of NT-methylhistidine pool in the body. The feeding of the protein-free diet resulted in a decrease in body weight and a decrease in daily urinary excretion of NT-methylhistidine. Refeeding caused an increase in body weight and a progressive increase in daily urinary excretion of NT-methylhistidine. At the start of the experiment, the fractional breakdown rate of the myosin-actin pool was 4% per day and with prolonged protein depletion, the rate decreased to 1.25% per day. The fractional synthesis rate also decreased more rapidly than the breakdown rate. On refeeding for one day with an adequate protein diet, the fractional synthesis rate increased from 0.75 to 5.75% per day. Accumulation of skeletal muscle protein by refeeding was accompanied by a difference between the faster rate of synthesis and slower rate of breakdown even though the fractional breakdown rate increased during the rehabilitation period.


The selective uptake and transmission of proteins to the circulation from the small intestine of the suckling rat. The relative concentration of various immunoglobulins attain in the sera of 14-day-old rats have been compared 4 h after their oral and intraluminal administration. The circulatory concentration of these proteins attained higher values when they were administered into the isolated proximal small intestina than when they were administered into the distal small intestine. Selective uptake of proteins was shown to predominate in the proximal small intestine.


[The fate of alpha and epsilon lysine amino groups in rat liver in vitro (author's transl)]. The fate of alpha and epsilon lysine amino groups has been explored in rat liver homogenate by means of L-lysine labelled selectively in the two positions. alpha-15NH2 and epsilon-15NH2 are rapidly incorporated into the amino group of glutamic acid and it seems at first that both transaminations occur simultaneously. But the reversible transfer of the amino group between alpha-aminoadipic acid and glutamic acid, determined by means of labelled alpha-aminoadipic acid, proceeds swiftly, and the incorporation of alpha-15NH2 from the corresponding labelled lysine in glutamic acid may be easily explained by epsilon-transamination and saccharopine formation. The direct transamination of the alpha-amino group of L-lysine is most improbable and might be limited to some microorganisms and to epsilon-N-substituted lysine derivatives.


Glycoprotein biosynthesis by organ cultures of hamster trachea. Conditions have been developed for maintaining hamster tracheas in organ culture for at least 10 days. Secreted glycoproteins labelled with [14C]glucosamine and [3H]fucose were isolated from the spent medium and digested with papain, and the digest was fractionated on DEAE-Sephadex by stepwise elution with NaCl. The fractions eluted by 0.1 and 0.2 M NaCl and some of the products eluted with 0.4 M NaCl were shown to be derived from epithelial glycoproteins. Glycosaminoglycans were eluted by 0.4 M and by 1.25 M NaCl. Glycopeptides isolated from the epithelium by homogenization, ethanol precipitation and papain digestion, and defined as "intracellular", gave a very similar profile on DEAE-Sephadex. The 0.1 M glycopeptide peak was the major fraction of epithelial origin from both secreted and "intracellular" material; it labelled extensively with both glucosamine and fucose and had a molecular fraction was purified further by chromatography on Sephadex G-75 and DEAE-Sephadex; its amino acid and carbohydrate compositions were determined.


Glycoprotein synthesis in gastric epithelial cells of the rat. Properties of microsomal glycoprotein glycosyltransferases. 1. Optimal assay conditions were determined for a microsomal glycoprotein galactosyl- and fucosyltransferase derived from gastric epithelial scrapings with both exogenous and endogenous acceptor glycoprotein. 2. Subcellular fractionation of the homogenate yielded microsomal fractions enriched in glycosyltransferases. 3. The effect of feeding on galactosyltransferase activity per cell was examined. 4. Endogenous acceptor molecules were identified as glycoproteins after labeling by means of UDP-[3H]galactose in the cell-free system.


Chemical synthesis of bilirubin glucuronide conjugates. In dimethylformamide, the two carboxyl groups of bilirubin react with the bifunctional coupling agent, carbonyldimidazole, to form bilirubin diimidazole, which was isolated and crystallised. The bilirubin diimidazole, termed "activated bilirubin", was shown to react spontaneously with primary alcohols to form diesters of bilirubin. After addition of the tetrabutyl ammonium salt of glucuronic acid, compounds with chromatographic mobilities similar to those of bilirubin mono- and diglucuronides from bile were formed. Bilirubin diglucuronides were isolated by barium precipitation followed by solvent extraction. The bilirubin diglucuronides were considered to be a mixture of alpha and beta glucuronides esterified at positions 1,2,3, or 4 of glucuronic acid because the compound(s) was resistant to hydrolysis with glucuronidase and gave multiple sponts by chromatography after diazotization with ethyl anthranilate. The model compounds lauryl glucuronides were synthesized similarly; the most polar product by chromatography and had identical chromatographic mobility to synthetic lauryl 1-D-glucuronide prepared by reductive debenzylation of lauryl (benzyl (2,3,4-tri-O-benzyl))-D-glucuronide. It is concluded that bilirubin-1-di-beta-D-glucuronide can be synthesized when suitable protecting groups for the 2, 3, and 4 hydroxyl groups of glucuronic acid become available.


[Determination, at equilibrium, of association constants of labelled or unlabelled ligands by a non-graphical method (author's transl)]. The present work deals with the determination of association constants at equilibrium by a non-graphical method in binding systems containing one specific receptor. Equations have have been derived from that originally described by Lea (Biochim. Biophys. Acta, 322, 68--74), the terms of which are obtained from the data of simple displacement curves of a bound radioactive ligand by unlabelled competitors identical or different in nature. By knowing the function relating the variations of the bound ligand (B) to the affinity constant (Ki) and the quantity (Mi) of competitor for a given system, it is possible to calculate any of these parameters when the two others are measured. Thus, it becomes easy to compare the relative affinities of differents receptors for the same ligand or that of one receptor for various labelled or unlabelled ligands. Furthermore, theoretical displacement curves can be drawn and compared to experimental data, when only knowing the affinity constant of a specific binding system in given conditions. These modes of calculation have been tested in a study of interactions between various steroids and a fraction of human serum proteins precipitated by ammonium sulfate (30-45%) and containing the sex hormone-binding globulin. Association constants thus obtained agree well with those reported in the literature and determined by graphical procedures.


A study of the diffusion of compact particles in polymer solutions using quasi-elastic light scattering. This paper describes an investigation, using quasi-elastic light scattering, of the diffusion of polystyrene spheres through solutions of dextran. The diffusion coefficient, D, of the spheres is shown to vary inversely with the volume fraction, phi, of dextran according to D = D0/(1 + vphi + kphi2). Changes in the molecular weight of dextran are shown to reflect changes in the macromolecular shape parameter, v, and the interaction parameter, k. This result differs from previous studies which suggested an exp( Bphi1/2) dependence and no molecular weight dependence [8].


Glucocorticoid receptors in corticosensitive and corticoresistant thymocyte subpopulations. I. characterization of glucocorticoid receptors and isolation of a corticoresistant subpopulation. 1. Separation of mouse thymocytes by centrifugation on a discontinuous bovine serum albumin gradient leads to the isolation of four subpopulations of cells. 2. The study of I13H]uridine incorporation in vitro by these subpopulation in the presence of steroid shows that one of them is corticoresistant. 3. However, the binding capacity of these subpopulations measured by incubation with [3H]dexamethasone is very similar. 4. It is therefore concluded that in mouse thymus, in contrast with lymphoma cells, corticoresistance may not be explained by a defect of cytoplasmic glucocorticoid receptors.


Experiments and calculations concerning a thermal enzyme probe. A simple device capable of measuring almost any reactant in an enzyme-catalyzed reaction is created when an enzyme is immobilized onto one thermal sensor of a differential thermometer. Experiments are described in which two thermistors, one bare and one coated with immobilized enzyme, are immersed in a well-stirred solution. The response of this device to increases in glucose-ATP concentration was observed using hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), and to increases in glucose concentration using glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4). A simple model is presented whose predictions are in reasonable agreement with the experimental results.


Kinetics of human erythrocyte hexokinase. Influence of temperature, ATP4- and magnesium ions. Human erythrocyte hexokinase (EC 2.7.1.1) is inhibited competitively with respect to Mg-ATP2- by uncomplexed Mg2+ (Ki = 16--18 mM) and ATP4- (Ki = 1.6 mM). No real activation by low concentrations of Mg2+ could be detected and no allosteric behaviour was observed under the conditions tested. The temperature dependence of the enzyme was studied in relationship to the presence of Mg2+ or ATP4-. At equal concentrations of Mg2+ and ATP4- a break in the Arrhenius plot was observed at 27.5 degrees C, the higher temperature form of the enzyme having the lower activation energy. This break point in the Arrhenius plot was shifted to 36 degrees C in the presence of 5 mM Mg2+. A straight-line relationship was observed in the presence of 2.5 mM ATP4-. The Km for Mg-ATP2- showed a linear increase at temperatures over about 36 degrees C independent of the presence of Mg2+ or ATP4-. The nature of these phenomena is discussed.


A note on the dual role of glucose in the protection of glucokinase against inactivation. Protection against thermal denaturation, urea denaturation and tryptic inactivation of rat liver glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2979192) is provided by glucose and to a lesser degree sorbitol. 2. Protection by glucose exhibits two distinct stages, one occurring at the physiological substrate levels, while the other occurring well above that necessary for enzyme saturation. Sorbitol protection increases uniformly with increasing sorbitol concentration. 3. Several other enzymes also are protected against heat inactivation by high concentrations of glucose or sorbitol.


Nucleotide specificity of pyruvate kinase and phosphoenolpyruvate carboxykinase. Various analogues of adenosine 5'-diphosphate with modifications in the heterocyclic base residue were tested as substrates of rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC. 2.7.1.40) and guinea pig liver mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32). The significance of different structural elements for the enzyme-substrate interaction is discussed. While pyruvate kinase shows a rather broad specificity for these analogues, phosphoenolpyruvate carboxykinase has a more stringent requirement for nucleotides, the intact keto and NH groups at C6 and N1 of the pyrimidine ring representing essential sites for the phosphoenolpyruvate carboxykinase substrate interaction. The biological significance of the different substrate specificities of pyruvate kinase and phosphoenolpyruvate carboxykinase is discussed as a possible metabolic control factor.


Inactivation of acetylcholinesterase by propanol and sodium dodecyl sulphate. Departure from first-order kinetics was observed for the inactivation of bovine erythrocyte and electric eel acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) by n-propanol. This was attributed to the presence of isoenzymes in the commercial preparations, although inactivation via a two-step process cannot be eliminated. The rate of inactivation of bovine erythrocyte enzyme by propanol or sodium dodecyl sulphate was decreased by the presence of added protein and increased by Triton X-100. The presence of a substrate (acetylthiocholine) decreased the rate of inactivation of bovine erythrocyte acetylcholinesterase by sodium dodecyl sulphate, but increased the rate of inactivation by propanol. Re-interpretation of earlier data on the inactivation and reversible inhibition of acetylcholinesterase by simple aliphatic alcohols indicated that they do not bind to hydrophobic regions of the active site.


Enzymic activity at interfaces. II. Enzymic activity of microsomal nuclease and bovine pancreatic ribonuclease at the air/water interface. The adsorption isotherms and the spreading tendency of microsomal nuclease (nucleate 3'-oligonucleotidohydrolase, EC 3.1.4.7) and bovine pancreatic ribonuclease (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) in the presence of isopropyl alcohol as spreading agent have been determined using enzymes radioactively labelled by acetylation. In parallel, the concentration-surface pressure relations have been established. The enzymic activity of microsomal nuclease spread from isopropyl alcohol containing aqueous solutions was only a few percent of its activity in bulk, while the activity of the adsorbed enzyme was only slightly reduced. Adsorbed monolayers of RNAase were almost inactivated, while the spread monolayers in the presence of isopropyl alcohol became reactivated after exposure to the substrate for several hours. The exposure time for the reactivation decreases with increasing surface concentration.


The sulphatase of ox liver. XX. The preparation of sulphatases B1alpha and B1beta. Sulphatases B1alpha and B1beta (EC 3.1.6.1) have been prepared as apparently homogeneous proteins by chromatography on ConA-Sepharose. Both have a mol. wt. of 56 000, and E1%280nm of 17 and a turnover number of 8600 min-1 with nitrocatechol sulphate as substrate. Their amino acid compositions are identical: like sulphatase A, the sulphatases B are rich in proline and yield glucosamine on hydrolysis. They are not altered by treatment with neuraminidase. Both fractions show strong UDP-N-acetylgalactosamine 4-sulphatase activity, weak iduronate sulphatase activity, but no significant heparan N-sulphatase activity. It is suggested that the physiological activity of sulphatase B is that of the N-acetylgalactosamine 4-sulphatase which is lacking in the Maroteaux-Lamy Syndrome.


Purification and characterisation of polygalacturonases from a commercial Aspergillus niger preparation. The polygalacturonase (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) activity of Pectinol is resolved into two fractions (E1 and E2) of about equal total activity on DEAE-cellulose. These fractions are purified from other pectinolytic enzyme activity by Sephadex G-75 chromatography. Both E1 and E2 reduce the viscosity of polygalacturonate by 50% after 7% of the glycosidic bonds are hydrolysed. Their activities are not affected by iodoacetate (1 mM) or EDTA (10 mM). E1 and E2 have different molecular weights (35 000 and 85 000, respectively) and different electrophoretic mobilities on sodium dodecyl sulphate polyacrylamide gels. Their pH (4.1 and 3.8 respectively) and ionic strength optima and specific activities also differ. Both enzymes are inhibited at similar rates by diethyl pyrocarbonate at pH 6 but only E2 is protected from this inhibition by 2% (w/v) polygalacturonate. The rate of change of protein absorbance at 250 nm accompanying this inhibition, and the residues are essential for the activities of both E1 and E2. About 2 molecules of carbethoxyhistidine per subunit of E2 and 0.6 molecules per subunit of E1 are present in the completely inhibited enzymes.20


Proteinases of human epidermis; a possible mechanism for polymorphonuclear leukocyte chemotaxis. Three neutral proteinases (EC 3.4.--.--) and cathepsin D have been identified in human epidermis utilizing a highly sensitive radioactive method. The proteinases were extracted in 1.0 M KC1 and 0.1% Triton X-100 and separated by Sephadex G-75 chromatography. The neutral proteinase peaks were all inhibited by diisopropyl fluorophosphate and thus were serine proteinases. Incubation of the enzyme fractions with [3H] diisopropyl fluorophosphate followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the two larger molecular weight proteinases were enzyme mixtures. The small molecular weight [3H] diisopropyl fluorophosphate proteinase migrated as a single band. Injection of the small molecular weight neutral proteinase into rabbit skin produced a polymorphonuclear leukocyte infiltration and edema. The reaction was not observed with the diisopropyl fluorophosphate-inhibited enzyme fraction. The release of neutral proteinases may be one of the signal events in the epidermal inflammatory response.