Metabolism of sodium nitroprusside and cyanide in the dog.

Blood cyanide (HCN) and thiocyanate (SCN) concentrations were measured at intervals in anaesthetized dogs given bolus doses of sodium nitroprusside (SNP) 1 mg kg-1 or potassium cyanide 1.07 mg kg-1 and in animals infused with SNP 1.5 mg kg-1 for 1 h. Cyanide appeared rapidly in the red cells to give peak concentrations which accounted for more than 90% of the total blood HCN. A delay between the peak plasma and red cell HCN concentrations confirmed that some of the SNP was degraded in the plasma. Comparison of HCN and SCN concentrations with those measured previously in patients receiving an infusion of SNP suggests that the degradation of SNP and detoxication of HCN may be more rapid in the dog. The various pathways of HCN detoxication are discussed in relation to the reduced formation of SCN in dogs receiving SNP compared with those receiving KCN.


Preservation of the hypoxic pulmonary vasoconstrictor mechanism during methoxyflurane anaesthesia in the dog.

The effect of methoxyflurane on the hypoxic pulmonary vasoconstrictor response in the dog lung was assessed by measuring the redistribution of pulmonary blood flow in response to two levels of unilateral alveolar hypoxia. Methoxyflurane 0.5% had no significant effect on the redistribution of blood flow resulting from the unilateral administration of oxygen 7% or nitrogen and there were no significant differences in PaO2 between the measurements made during unilateral hypoxia with methoxyflurane and those made during unilateral hypoxia in the control periods.


Environmental factors affecting antipyrine metabolism in London factory and office workers.

1 Measurements of antipyrine clearance in saliva have been made in 128 London factory and office workers. 2 Mean antipyrine clearance in 56 Asian immigrants was 40% slower than in 72 White subjects. 3 Although dietary differences existed between the two groups, analysis of their effect independently of race was impossible since all but one of the vegetarians were Asian and the non-vegetarians were nearly all White. 4 In the White subjects, use of the oral contraceptive reduced clearance by 38% in women, while cigarette smoking increase clearance by 38% in men.


Pindolol pharmacokinetics in relation to time course of inhibition of exercise tachycardia.

1 Pharmacokinetics of pindolol were studied in normal subjects given 5, 10 and 20 mg orally and 3 mg i.v. Plasma half time was 2.9 +/- 0.3 (s.e. mean) h for both routes; peak drug levels occurred 1--2 h after ingestion and bioavailability was 53%. Plasma protein binding was 38% and was independent of plasma concentration; the drug was not concentrated in the red cell. 2 Work-heart rate regression lines were calculated from resting heart rate and three grades of 'steady-state' exercise standardized for the maximum work capacity (Wmax) of each subject. The equation was characterized by slope and HR50 (calculated heart rate at 0.5 Wmax). 3 After giving 5 mg i.v. pindolol to produce maximum cardiac beta-adrenoceptor blockade there were differences in inhibition of resting heart rate, slope, HR50 and maximum heart rate suggesting differences in sympathetic components. However, estimates of the degree of inhibition were closely similar for each variable when determined before and after atropinization indicating that the accuracy of estimation was independent of the level of vagal activity. 4 After oral pindolol peak inhibition of resting heart rate, slope and HR50 coincided with peak plasma concentration. Peak reduction of resting heart rate was greatest at the lowest dose, but inhibition of slope and HR50 were similar at all doses. 5 The different heart rate parameters recovered at different rates. After 24 h slope had returned to control, and the residual inhibition of HR50 reflected residual beta-adrenoceptor blockade of resting heart rate, as demonstrated by a shift in isoprenaline-heart rate relationship. 6 Inhibition of HR50 and other exercise parameters were 20% less in the concentration range 5--20 ng/ml than peak inhibition obtained in the range 21--160 ng/ml. The higher potency of pindolol compared with propranolol can be accounted for by the difference in protein binding.


Uricosuric properties of diflunisal in man.

1 The uricosuric effect of diflunisal was studied in eleven normal subjects. 2 Urine and serum uric acid levels were measured and used to assess the effect. 3 Diflunisal caused statistically significant decreases in serum uric acid levels and increases in uric acid clearance at 250 and 375 mg twice daily. 4 Diflunisal taken on an empty stomach caused epigastric discomfort in the high dose group which was obviated when the drug was taken with meals. 5 Diflunisal caused no abnormalities in the measured haematological, urological or biochemical parameters.


Effect of a single oral dose of aspirin on the platelet aggregation response to arachidonic acid.

1 The platelet aggregation response to arachidonic acid ex vivo was measured in six volunteers daily before and for 10 days after a single oral dose of 600 mg aspirin. 2 Arachidonic acid induced aggregation of platelets from all subjects before aspirin and aggregation occurred after an interval which varied inversely with the concentration of arachidonic acid. No aggregation occurred for 4 days after aspirin; a reduced response, compared with pre-aspirin values, was obtained on the 5th, 6th and 8th day. The values on days 7, 9 and 10 were not consistently different from the pre-aspirin values. In 6 subjects 24 h after aspirin ingestion the addition of 10--25% v/v normal platelets restored the aggregation response. 3 It is concluded that aspirin has an effect on the platelet precursors in the marrow in addition to its effect on circulating platelets.


Quinidine dosage, with special reference to an oral loading dose schedule.

1 Plasma concentrations of quinidine were analyzed in 52 cardiac patients after different oral loading doses and during different maintenance dosage intervals using both conventional and sustained release preparations of quinidine. 2 The majority of patients reached therapeutic plasma concentrations in the order of 2--4 microgram/ml within 3 h after the loading dose of 600--800 mg of rapidly absorbed ordinary quinidine sulphate tablets and institution of maintenance therapy with sustained release tablets 1 h after the loading dose. 3 The dosage interval of 12 h during maintenance therapy with sustained release tablets gave as acceptable plasma drug fluctuations as an 8 h interval. 4 A large individual variation in plasma concentrations was noticed. Thus monitoring of therapy by plasma concentration analyses is desirable.


Bioavailability of conventional and slow-release oxprenolol in fasted and nonfasted individuals.

1 The influence of food and plasma levels of oxprenolol was examined in healthy female volunteers using both conventional and slow-release formulations. 2 There were no significant trends in plasma drug profiles due to food with either formulation. Elimination half-lives were longer after food with conventional tablets, presumably due to continued absorption. The opposite effect was seen with slow-release tablets. 3 Slow-release oxprenolol is, under the conditions of this study, a reliable and convenient dosage form, providing similar plasma-drug profiles in fasted and nonfasted individuals.


Eosinophilic cellulitis.

Eight cases of eosinophilic cellulitis are described with acute cutaneous swellings followed by indolent infiltration. The histology is distinctive with focal phagocytosis of eosinophilic material in dermis. Occasionally these histological features are observed in other inflammatory dermatoses when eosinophils have infiltrated the connective tissue.


Acrodermatitis enteropathica.

The first publication identifying acrodermatitis enteropathica as a definite disease (Danbolt & Closs, 1942) is reviewed. Later studies are briefly surveyed, resulting in the recognition of the disease as a zinc deficiency which can be effectively corrected by administration of small oral doses of zinc.


The ribonucleic acid (RNA) skin test in systemic lupus erythematosus and other connective tissue diseases.

A series of 65 patients with different autoimmune diseases was examined using different RNA-solutions for intradermal skin tests. Clinically positive results were obtained most often in patients with mixed connective tissue disease but quite often also in patients with systemic lupus erythematosus and progressive systemic sclerosis or with some symptoms of an automimmune nature. The histological examination of the biopsies from the test sites revealed that there was no correlation between the clinically positive tests and the histological criteria usually used as a sign of a positive test.


Discoid lupus erythematosus in the Nigerians.

Results of a detailed clinical and laboratory study of 37 Nigerian patients with chronic discoid lupus erythematosus are presented. Patients with chronic discoid lupus erythematosus constituted 0.46% of all out-patients seen in the skin clinic between May 1974 and December 1977. A preponderance of females was noticed (female/male ratio of 5:1), while the age distribution of African patients corresponded to values characteristic for the condition seen in other geographical regions. Several morphological types of the condition have been seen. The vitiligoid variant of chronic discoid lupus erythematosus seems to be common in West Africans. Sixteen out of 37 patients presented laboratory abnormalities considered as markers of the association between chronic and systemic lupus erythematosus. Their significance, however is, uncertain as it has been demonstrated on several occasions that in a tropical milieu heavy parasitic infections produce marked immunological disturbances. The problem of the relationship between chronic and systemic lupus erythematosus is discussed and the literature on the incidence of chronic discoid lupus erythematosus in various African countries is reviewed.


Concomitant erythrasma and dermatophytosis of the groin.

Four patients are reported with concomitant erythrasma and trichophyton rubrum dermatophytosis of the groin. The recognition and diagnosis of these associated infections is emphasized.


The change in properties of the stratum corneum as a function of depth.

The constancy of stratum corneum thickness suggests an ordered decrease in intracorneal cohesion binding forces. In this study, intracorneal cohesion was measured in the vertical dimension by cohesography and the number of cells released after applying a standardized stimulus was determined before and after repeated stripping of the same sites with adhesive tape. In addition, surface replicas and the corneocytes from different levels were examined by scanning and transmission electron microscopy and differential phase interference microscopy. The results indicate a gradual loss of cohesion within the stratum corneum towards the surface and that anatomical changes take place in the corneocytes as they progress towards the surface.


Comparison of PUVA and beta-carotene in the treatment of polymorphous light eruption.

Therapy with oral psoralen photochemotherapy (PUVA) and oral beta-carotene was compared in 29 patients with polymorphous light eruption. Complete remission occurred in 90% (9/10) of those treated with PUVA and in 32% (6/19) treated with beta-carotene. In the six patients who were treated in consecutive years with both modalities all had remissions with PUVA while only two had remissions with beta-carotene.


Hereditary angio-oedema: treatment with danazol. Report of a case.

An 8-year-old boy with hereditary angio-oedema was treated with danazol under close endocrinological supervision. The boy's C1 esterase inhibitor (C1inh) and C4 levels increased rapidly to near normal values under a daily dose of 400 mg and were maintained at about 50% of the normal by maintenance doses of 200 mg danazol every other day. Throughout the entire treatment period (11 months) the boy has been maintained free from attacks of angio-oedema. No hormonal imbalance was detected in the follow-up period. Our results indicate that danazol should be suitable for the treatment of HAE not only in adults but also in prepubescent children.


Twenty-nail dystrophy of childhood. Case report and histopathological findings.

A case of twenty-nail dystrophy of childhood is reported. The histopathological findings were distinctive and incompatible with the notion that this condition might be a variant of lichen planus.


Biochemical and histological analysis of bone marrow collagen in myelofibrosis.

Total protein, collagen (hydroxyproline) and glycoproteins (hexosamine) content of control and myelofibrosis (MF) bone marrow samples was determined using a sequential extraction procedure. MF marrow extracts contained higher amounts of collagen than control extracts. The collagen content appeared to increase with the duration of the disease. In more recent MF cases (less than 2 years) at least 60% of the total collagen was extracted in 0.5 M NaCl. this proportion decreases to 33% in older cases (greater than 4 years), indicating a progressive insolubilization (crosslinking) of collagen. The hexosamine content of the extracts decreased in MF as compared to controls reflecting a decrease in glycosaminoglycans (and possibly of structural glycoproteins). The reticulin content of the same bone marrows was estimated by a quantitative morphometric procedure. There was a positive correlation between the morphometrically estimated reticulin surface and the total hydroxyproline content of the marrow samples. The slopes of the least square lines correlating increase of reticulin surface to hydroxyproline content were, however, significantly different in control and MF marrows, indicating a 44% higher increase in histochemically detectable reticulin per unit increase in hydroxyproline content than in the control marrows. This result may indicate a more efficient fibrogenetic process in MF marrow than in normal bone marrow. The above results confirm the collagenous nature of the fibrous reticulin like material deposited in MR marrows and suggests a correlation between the progression of the disease and the rate of synthesis and deposition of collagen fibres.


Alpha thalassaemia in American blacks: a study of a family with five cases of haemoglobin H disease.

Five cases of HbH disease were discovered in a large family of American Blacks. Anaemia was mild with PCV ranging from 0.275 to 0.405. The amount of HbH was 2--6%. Studies of haemoglobin synthesis in peripheral blood reticulocytes demonstrated marked deficits in alpha globin production with an average alpha/beta ratio of 0.31 (range 0.22--0.36). Eighteen additional family members had evidence of thalassaemia trait and were provisionally classified as either alpha-thal-1 (average MCV 65.2 fl; range 59--70) or alpha-thal-2 (average MCV 79.6 fl; range 74--88). A subject with altha-thal-1 trait had an alpha/beta ratio of 0.56; the average for five cases of alpha-thal-2 was 0.73. One other family member was thought to be homozygous for alpha-thal-2 trait and exhibited an MCV of 65 fl with an alpha/beta ratio of 0.5. These data reconfirm that in Blacks with alpha thalassaemia the proportion of HbH is lower and the severity of anaemia is less than in certain other racial groups, e.g. Southeast Asians. However, the degree of hypochromia and microcytosis and the imbalance in alpha and beta globin synthesis appear to be similar in Blacks and other races. These results suggest that the milder clinical course of HbH disease in Blacks is not a result of greater alpha globin production in that population of thalassaemics.


Interaction of alpha and beta thalassaemia genes in two Sardinian families.

Our paper describes two Sardinian families with alpha-beta thalassaemia interaction. In the first (family S), the propositus, whose haemoglobin pattern at birth consisted of about 25% Hb Bart's and 75% Hb F, successively developed a clinical and haematological picture typical of Cooleys anaemia. Haematological and globin chain synthesis studies together with these findings suggest that he is homozygous for beta 0 thalassaemia and heterozygous for alpha thalassaemia-1 and alpha thalassaemia-2. This conclusion is further substantiated by the finding of various combination of alpha and beta thalassaemia among his family members. In the P family two twins whose haemoglobin pattern and synthesis data at birth were similar to those of the proband of family S died in the neonatal period. The mother was assumed to be a compound heterozygte for alpha thalassaemia-2 and beta 0 thalassaemia and the father for alpha thalassaemia-1 and beta 0 thalassaemia. The homozygous state for beta 0 thalassaemia in association with the alpha thalassaemia 1 and alpha thalassaemia 2 genes results in a severe clinical picture similar to that of a homozygous beta 0 thalassaemia. The interaction between the heterozygous state for beta 0 thalassaemia and the alpha thalassaemia 1 or alpha thalassaemia 2 genes, or the combination of both, results in a haematological picture similar to that of a beta thalassaemia heterozygote.


The importance of the genetic picture and globin synthesis in determining the clinical and haematological features of thalassaemia intermedia.

Twelve carriers of thalassaemia intermedia were studied. Their clinical and haematological picture was distinctly different from that in both heterozygotes and homozygotes for beta thalassaemia. Several genetic patterns were found responsible for thalassaemia intermedia: beta/delta beta thalassaemia, alpha 2 beta/beta thalassaemia-heterocellular HPFH. In a few subjects the genetic picture indicated that the patients were homozygous for beta thalassaemia, in spite of the mildness of the clinical situation. The lack of genetic uniformity was refelcted in very wide Hb A2 (2.5--8.7%) and Hb F (7.5--96.9%) ranges, as opposed to the noticeable degree of biochemical uniformity indicated by the very similar imbalance of globin chain synthesis: 0.33-0.54 for the non-alpha/alpha chain ratio in the peripheral blood. The mean for this parameter (0.43 +/- 0.05) was significantly different (P less than 0.001) from that observed in heterozygous carriers (0.60 +/- 0.10) and homozygous carriers (0.11 +/- 0.05) for beta thalassaemia. The marrow blood displayed a comparable pattern. It is therefore suggested that the severity of thalassaemia is attributable to the degree of chain synthesis imbalance.


Erythrocyte membrane lipid reorganization during the sickling process.

In order to study possible alterations in membrane lipids during sickling, we have measured the difference in susceptibility to lipid peroxidation, binding of trinitrobenzenesulfonic acid (TNBS) to aminophospholipids, and fatty acid uptake in cells containing sickle haemoglobin under aerobic and anaerobic conditions. We have also examined TNBS binding in irreversibly sickled cells in an attempt to evaluate the permanent effects of any such alterations. We found that when erythrocytes were sickled by deoxygenation, the susceptibility to lipid peroxidation and binding of TNBS to aminophospholipids was markedly increased, while normal control cells showed no change. These effects appeared to be specific for the sickled state rather than a nonspecific consequence of cell age or the concentration of sickle haemoglobin within the cell. In contrast, fatty acid incorporation into membrane phospholipids, representing potential lipid renewal, was decreased in the sickled state. Cell fractions enriched in irreversibly sickled cells showed increased TNBS labelling in air and only modest rises with anoxia. Taken together, these data imply a rearrangement of membrane lipids during the sickling process and suggest a permanent reorganization of membrane lipids in the irreversibly sickled cell.


Protection by ascorbate against acetylphenylhydrazine-induced Heinz body formation in glucose-6-phosphate dehydrogenase deficient erythrocytes.

Ascorbate was found to inhibit oxidation of oxyhaemoglobin and Heinz body formation in glucose-6-phosphate dehydrogenase deficient red cells incubated with acetylphenylhydrazine. It is proposed that ascorbate can substitute for the glutathione which is depleted and act as a scavenger for drug free radicals generated during the reaction. Ascorbate was protective at concentrations only a little higher than that in normal blood, and the possibility that administration of ascorbate could protect GSH deficient cells against the action of oxidative drugs such as APH is considered. A simple method of quantitatively assessing Heinz body formation by measuring the turbidity of the lysed cells is described.


Absence of tight junctions in endothelium of marrow sinuses: possible significance for marrow cell egress.

The nature of contact between endothelial cells in rat marrow sinuses was studied. Colloidal lanthanum permeated freely into the interendothelial space. Moreover, using freeze-fracture technique, we failed to show junctional structures where these endothelial cells came into contact. With thin sectioning electron microscopy, we observed occasional submembranous densities but the interendothelial distance measured 20 nm and no membrane fusion was seen. Absence of tight junctions between endothelial cells of marrow sinuses may serve a function in marrow cell egress by permitting these cells to slide over each other, thereby changing the luminal size. Within the fixed volume of marrow, an increase in luminal size results in displacement of haemopoietic cells into the lumen.


The lymphocyte production pathway in bone marrow: possible significance of the size spectrum of lymphocytes and their precursors.

It is now generally accepted that transitional ('lymphoid') cells are the precursors of small lymphocytes. Such cells have a heterogeneous size spectrum and show high proliferative capacity. To facilitate the study of the kinetics of lymphocyte production, a detailed investigation of cell sizes of the transitional cell-lymphocyte compartment was carried out using a Coulter counter modified to permit a very rapid and accurate examination of cells in suspension. Enriched populations of 'lymphoid' cells, obtained after 10 d rebound from hypoxia at half an atmosphere, were enriched further by bovine albumin and Ficoll gradients to give density fractions containing two types of cells. Differential counts of stained smears of these fractions enabled a comparison to be made between the size distribution and the specific cell types. Four distinct cell types were characterized in terms of volume and density: small and intermediate-sized lymphocytes (volume 53-59 fl, albumin fractions 19-23%), small transitional cells (154-160 fl, 21-23%), medium transitional cells (206-218 fl, 17-19%) and large transitional cells (350-400 fl, 21-27%). These findings are consistent with the view that there are at least three mitoses in the course of the lymphocyte production pathway in the bone marrow.


Clinical course in 28 unselected patients with aplastic anaemia treated with anabolic steroids.

Twenty-eight unselected patients with histologically proven aplastic anaemia were electively treated with anabolic steroids (75-150 mg orally q.d.) Additional supportive treatment with blood cell components and antibiotics was given if indicated. Response to therapy was defined as favourable if after 3 months of anabolic therapy overt bleeding tendency had disappeared, there was no need for transfusion therapy, a spontaneous increase of haemoglobin had occurred of greater than 3 g/dl above the initial level, and a platelet rise of twofold the initial count (up to at least greater than 30 x 10(9) /L) had occurred. Of 22 patients evaluable for the results of long-term (greater than 3 months) anabolic treatment, six showed a partial response and eleven responded favourably. These 11 are all alive at the end of the study. Five of these patients proved to be anabolic steroid-dependent. The 50% actuarial survival is approximately 4 years after diagnosis, which compares favourably with the best published results from bone marrow transplantation for aplastic anaemia. It is concluded that anabolic therapy in aplastic anaemia should be tried for 2-3 months before the bone marrow transplantation or immunosuppressive therapy is taken into consideration.


Role of serum folate binders in the delivery of folate to tissues and to the fetus.

Specific folate binding protein (FBP) and non-specific binding protein have been prepared from pooled serum from patients in late pregnancy. The two classes of folate binder were labelled with 75Se-selenofolate and injected into non-pregnant and pregnant rabbits. Selenofolate bound to specific FBP was delivered predominantly to the liver, in both pregnant and non-pregnant animals. Fetal tissue took up selenofolate from non-specific FBP only. Specific FBP thus functions to deliver folate to stores and represents a storage pool of folate. The non-specific FBP are responsible for the delivery of folate to the fetus, and probably also to sites of utilization.


Vitamin B6 metabolism in anaemic and alcoholic man.

Physiological and pathological factors affecting intracellular red cell vitamin B6 metabolism in normal, anaemic and alcoholic man were studied using a new assay for pyridoxine kinase (PnK) together with saturated and total aspartate aminotransferase (EGOT) activities as indirect indices of intracellular pyridoxal 5-phosphate (PLP) availability. In studies of anaemic states, subjects with iron deficiency anaemia demonstrated elevated levels of both PnK and saturated EGOT, while seven out of 17 subjects with inflammatory anaemia had subnormal PnK but variable saturated EGOT activities. Despite a high incidence of complicating inflammatory disease, alcoholic subjects with or without ring sideroblastic anaemia had elevated levels of both PnK and saturated EGOT. As judged from the saturated EGOT and the ratio unsaturated EGOT/saturated EGOT, intracellular PLP availability was always appropriate to the higher levels of PnK activity.


Liposome entrapped desferrioxamine and iron transporting ionophores: a new approach to iron chelation therapy.

Liposome-entrapped desferrioxamine was administered to iron-over-loaded 59Fe lavelled mice. When given orally or intraperitoneally entrapment did not enhance the effect of the chelator, but given intravenously liposomal desferrioxamine doubled the 59Fe excretion for a given dose of the drug, and excretion after a single dose continued for up to 3 d. In addition, liposomes containing ionophore A23187 administered concurrently with DTPA caused an excretion of 59Fe, whereas DTPA alone had no effect.


Work capacity, heart rate and blood lactate responses to iron treatment.

Changes in haemoglobin (Hb), work performance, heart rate and postexercise blood lactate were studied in iron deficient, anaemic subjects for 16 d following total dosage infusion of iron dextran, i.v. (30-50 ml). Six adult men and 14 women were subjects with initial Hb levels of 6.6 +/- 0.6 g/dl (mean +/- SEM) for the iron treatment group (n=10) and 8.0 +/- 0.7 for the placebo group (saline infusion, n=10). Serum levels were 0.51 +/- 0.15 and 0.67 +/- 0.12 mg/l for the two groups, respectively. Haemoglobin and maximal work time increased significantly within 4 d after iron treatment and continued to increase up to 16 d. No changes were found in the placebo subjects. Heart rates at a given exercise intensity were lower in the iron treatment group than in control subjects who had the same Hb levels but had not been treated with iron. Post-exercise venous blood lactate was similar on succeeding days after iron treatment even though the subjects reached higher work loads. These results demonstrate the treatment of iron deficient, anaemic subjects with iron dextran results in improved work capacity within 4 d and a lower heart rate at a given work load after treatment which cannot be accounted for totally by the elevation of Hb concentration.


Relation between low erythrocyte acetylcholinesterase activity and membrane lipids in paroxysmal nocturnal haemoglobinuria.

Acetylcholinesterase of intact erythrocytes, their ghost and salt soluble extracts obtained from patients with paroxysmal nocturnal haemoglobinuria (PNH) does not differ from normal with respect to Km values for acetylthiocholine, and Ki values for phenyltrimethylammonium iodide. However, the enzyme from PNH sources has lower V max values than normal, has different thermal stability from normal, has less distinctive transition temperature in the Arrhenius plots, and is less subject to inhibition by stearic acid. These results and that from comparison of activation of deoxycholate-extracted enzyme by lipids from normal erythrocytes suggest that the low acetylcholinesterase activity in PNH erythrocytes is due, at least in part, to alteration in the lipid environment of the enzyme.


An examination of complement proteins on membranes of paroxysmal nocturnal haemoglobinuria (PNH) and PNH-like red cells.

Proteins obtained from membranes of PNH erythrocytes and of red cells treated with 2-aminoethylisothiouronium bromide (AET) following lysis in acidified serum (Ham's test) were compared by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The peptide patterns were identical, providing support that AET-treated red cells afford a suitable experimental substitute for PNH red cells in studies involving complement fixation.


Diagnosis of haematological disease using anti-i. I. Disorders with lymphocytosis.

Using cytotoxicity and antibody-binding tests, the i antigen was measured on the blood lymphocytes of normal subjects and of patients in whom a diagnosis of chronic lymphocytic leukemia (CLL) was considered because of a slight lymphocytosis in the blood and bone marrow. Among 25 patients, 15 had a normal amount of i antigen; in 10 there was a marked reduction in i antigen, such as is found in typical CLL. Similar studies were done on lymphocytes from 15 patients with clinical and morphological findings usually associated with lymphosarcoma cell leukemia (LSL). Nine had a normal amount of i antigen; in six there was a marked reduction of i antigen, suggesting a diagnosis of CLL. A study of the subsequent clinical course and final diagnosis in these patients suggests that the reduction in lymphocyte i antigen characteristic of typical CLL occurs in patients with early CLL who have only slight lymphocytosis, but not in patients with slight lymphocytosis from other causes. Similarly, a reduction in lymphocyte i antigen is found in patients with morphologically atypical CLL but not in patients with LSL.


Further studies on a specific platelet antibody found in Bernard-Soulier syndrome and its effects on normal platelet function.

An IgG antiplatelet antibody found in a multitransfused patient with Bernard-Soulier syndrome (BSS), reacted with a normal platelet surface antigen of 150 000 daltons which was similar to the glycoprotein missing from BSS platelets. The BSS platelet antibody (BSS-Pab) aggregated all control platelets which then released ADP and 5-HT and synthesized thromboxane. When mixed with the antibody, BSS platelets did not aggregate, did not release ADP and 5-HT and failed to synthesize thromboxane. The BSS-Pab was not inactivated by incubation with BSS platelet stroma. While the antibody did not aggregate thrombasthenic platelets, its aggregating activity was lost after incubation with their stroma. The BSS-Pab did not provoke ADP or 5-HT release or thromboxane synthesis in thrombasthenic platelets or in the platelets of a patient with platelet cyclooxygenase deficiency or in normal platelets treated with indomethacin. The aggregating, release and synthetic responses of platelets after binding of BSS-Pab to its membrane antigen (probably glycoprotein I) requires the presence of glycoprotein IIb and/or IIa and the normal metabolism of arachidonic acid.


Specificity in the factor VIII response to vasopressin infusion in man.

Structural requirements of the systemic factor VIII response to intravenous vasopressin in man has been investigated using vasopressin analogues. With the analogues available the receptor specificity of this phenomenon could not be distinguished from those associated with the previously described plasminogen activator release or antidiuretic effects of this hormone. Further studies using 1-desamino-[8-D-arginine]vasopressin showed a dose-related release of both procoagulant and antigenic components of the factor VIII complex. The newly released factor VIII could not be distinguished from circulating factor VIII on the basis of molecular size, electrophoretic mobility or in vitro stability despite apparent differences in the duration of response of the procoagulant and antigenic components in vivo.


Unstressed antepartum cardiotocography in the management of the fetus suspected of growth retardation.

A detailed analysis of the antenatal cardiotocographs (CTGs) in 57 patients with suspected fetal growth retardation is presented. Four traces were normal and 53 were 'non-reactive'. 'Non-reactive' traces can be sub-divided into three categories, which, in order of severity are, 'suspect', 'flat' and 'ominous'. When compared with the 'suspect' group, perinatal mortality was significantly increased and the Apgar scores at one and five minutes significantly decreased in the 'flat' and 'ominous' groups. The presence of fetal distress in labour was significantly higher in the 'flat' group as compared with the 'suspect' group. Intrauterine deaths occurred only in the 'ominous' group. With experience, ten patients later in the series with 'ominous' patterns were delivered within 24 hours of the detection of such and nine infants survived. At follow-up between 6 and 34 months after birth, psychomotor development was normal in 47 of the 49 surviving infants.


Metabolism of diphenylhydantoin (phenytoin) during pregnancy.

Plasma levels of diphenylhydantoin (DPH) and the 24 hour urinary excretion of both DPH and its major metabolite 5-(p-hydroxyphenyl)-phenylhydantoin (HPPH) were measured serially during pregnancy in a group of five unselected epileptic patients. Plasma DPH levels fell and on average were some 40 per cent lower at term than they had been in early pregnancy. With one exception, the patients were well controlled during pregnancy but two patients who regularly had plasma DPH levels below 10 microgram/ml both suffered seizures in the puerperium. There was no convincing change in the excretion of either DPH or HPPH which together accounted for less than half the administered dose. Gestational length exceeded 41 weeks in 4 of the 5 patients studied but all were delivered of normal healthy infants, of average birthweight, with no congenital defects or clotting disorders.


The effect of oxytocin in induced labour on neonatal jaundice.

A prospective study in 180 mothers and babies examined the effects of oxytocin in induced labour on plasma bilirubin levels in cord blood, as well as on the incidence of neonatal jaundice. Raised plasma bilirubin levels in cord blood, probably enhanced by breakdown of fetal red cells, appeared to be a dose dependent effect of oxytocin. Commensurate with this was the finding that a larger proportion of babies in the induced group manifested a greater severity of jaundice.


Further clinical experience in the treatment of hirsutism with cyproterone acetate.

Further experience in the treatment of female hirsutism with the anti-androgen cyproterone acetate is reported. Two-thirds of treated patients have shown improvement but relapse occurred within two or three months of stopping treatment. Acne also healed in two patients.


Giant cell arteritis of the female genital tract.

In a patient who had a hysterectomy and bilateral salpingo-oophorectomy for endometrial adenocarcinoma, a giant cell arteritis was found in many of the myometrial and tubal vessels and in a few of the small arteries of the cervix and ovaries. On subsequent questioning, a history of treatment for polymyalgia rheumatica during the preceding 12 months was elicited. Of the three previously reported patients with giant cell arteritis of the uterus, two were suffering from polymyalgia rheumatica whilst one possibly had disseminated visceral giant cell arteritis.


Familial carcinoma of the ovary. Case report.

A patient was successfully treated by surgery, progestogens and chlorambucil for a poorly differentiated cystadenocarcinoma of the ovary. Four of her relatives had died of ovarian cancer; it was ascertained that two of them also had poorly differentiated cystadenocarcinoma.


Activity and characterization of a low molecular fraction present in human amniotic fluid with broad spectrum antibacterial activity.

The isolation of a low molecular weight fraction from amniotic fluid with broad spectrum antimicrobial activity is reported. This fraction consists of a family of peptides whose bactericidal activity is zinc dependent and can be inhibited by phosphate. Peptides are present in early gestation in low concentration increasing in amount from about 28 weeks gestation when antimicrobial activity becomes detectable in whole amniotic fluid. A simple bacteriological assay for this material is described. Its relation to the occurrence of perinatal infection is discussed.


The relation between plasma oestrogen, progesterone and prolactin concentrations and the efficacy of vaginal prostaglandin E2 gel in initiating labour.

In an attempt to relate the efficacy of treatment to endogenous hormone levels, plasma oestrogen, progesterone and prolactin levels were analysed in women at term following the vaginal administration of prostaglandin E2 gel to induce labour. Patients who went into labour after treatment had significantly higher oestradiol-17 beta levels before treatment compared with those requiring formal surgical induction the following day. No difference could be demonstrated in levels of progesterone or prolactin in the two groups of patients. The significance of these results is discussed.


Inhibition of prostaglandin-induced uterine activity by nifedipine.

The effects of the calcium antagonist, nifedipine, on uterine activity induced by prostaglandins F2 alpha (PGF2 alpha) and E2 (PGE2) were studied in women undergoing therapeutic midtrimester abortion, and in patients with a missed abortion in the 16th to 27th week of pregnancy. In the five subjects receiving intra-amniotic PGF2 alpha (25 to 40 mg) for midtrimester abortion, nifedipine (30 mg orally) decreased uterine activity from a mean of 372 to 203 Montevideo Units. The effect on the intensity of the contractions was pronounced; frequently and basal tone were little affected. In patients with missed abortion, uterine contractions were induced by extra-amniotic application of PGE2 (0.5 to 1.5 mg) in a viscous gel. The activity was often more irregular than that in the women receiving intra-amniotic PGF2 alpha. However, nifedipine (30 mg orally) had a marked inhibitory effect on the uterine contractions. It is concluded that nifedipine can be used for treatment of uterine hyperactivity induced by prostaglandins. Combined treatment with beta 2-adrenoceptor stimulants might be considered.


The relation between plasma unconjugated oestriol and cystine aminopeptidase concentrations and uterine contractions.

The relation between maternal plasma unconjugated oestriol (E3) concentrations and uterine activity was investigated by very frequent, carefully timed determinations of E3 during synchronously recorded uterine contractions in six normal subjects. There was an initial rise (10 to 50 per cent above the mean) in E3 levels reaching a peak coincident with the contraction peak (range 20 seconds before to 15 seconds after) followed by a fall of similar magnitude below the mean reaching a nadir about 80 seconds (range 60 to 105 seconds) after the contraction peak. Although the pattern of E3 fluctuation was most marked in labour the overall degree of variation was no greater than at other times. Cystine aminopeptidase (CAP) concentrations in the same serum samples showed no significant correlation with uterine contractions. The reasons for, and practical applications of, the E3 results are discussed.


Computer analysis of fetal heart rate variation during normal pregnancy.

Ninety-seven one-hour recordings of the abdominal fetal electrocardiogram (ECG) were made from 59 normal patients between 21 and 41 weeks of gestation. The heart intervals, measured between successive R-waves, were analysed by computer. The signal-to-noise ratio of the fetal ECG limited the precision of the interval measurements to approximately one millisecond. The characteristics of the baseline heart rate changed significantly as gestation advanced, the mean R-R interval, the standard deviation of the intervals and the standard deviation of the interval differences all increasing with gestation (p less than 0.001). In later gestation the baseline heart rate during periods of fetal rest differed significantly from that during periods of fetal activity; during rest the mean R-R interval was greater (p smaller than 0.001) and the standard deviations of the intervals and interval differences were smaller (p smaller than 0.001). Examination of the coefficient of variation of the heart intervals gave a result which contradicted the significance of this measurement as an index of fetal welfare as proposed by Curran and MacGregor (1970).


Haemodynamics of mild hypertension in pregnancy.

One hundred and sixty-three haemodynamic studies were done serially in 23 normotensive and 25 mildly hypertensive women during pregnancy. With all subjects resting in the left lateral position, heart rate, cardiac output, stroke volume, left ventricular work index, total peripheral vascular resistance and haematocrit were all significantly higher while plasma volume per unit body weight was significantly lower in the hypertensive than in the normotensive pregnant women. It is postulated that this hyperkinetic circulatory state is due to hyperactivity of the sympathetic adrenergic component of the autonomic nervous system in hypertensive pregnancy.


The possible role of prostaglandins in the hyperfunction of the renin-angiotensin-aldosterone system in the newborn.

Plasma renin activity and plasma aldosterone concentration were measured by radioimmunoassay in eight newborn infants born vaginally to mothers on indomethacin treatment. Measurements were made in venous cord blood at birth and on the sixth day of life. The results were compared to those obtained in eight healthy control newborn infants of similar gestational age and birth weight. Maternal treatment with indomethacin was associated with a decrease of plasma renin activity (mean +/- SD) from 16.71 +/- 2.76 ng/ml/hour in the control group to 9.60 +/- 3.84 ng/ml/hour in the treated group. Plasma aldosterone concentration was lower in the cord blood of the treated group and a similar trend was demonstrated in the sixth day samples. It is suggested that the increased endogenous prostaglandin production during labour may be one of the factors responsible for the development of hyperactivity of renin-angiotensin-aldosterone system in the newborn.


The significance of abnormal glucose tolerance (hyperglycaemia and hypoglycaemia) in pregnancy.

Maternal hypoglycaemia (plasma glucose below 5th centile) had a highly significant association with fetal growth retardation, and perinatal mortality was significantly increased in the presence of both hypoglycaemia and hyperglycaemia (plasma glucose above 95th centile) when pregnancy outcome was analyzed in 5000 consecutive patients who had a glucose tolerance test performed during the third trimester of pregnancy. This study confirms the significance of abnormal glucose tolerance as a causative factor of feto-placental dysfunction. The flat glucose tolerance test pattern had no significance beyond the presence of associated hypoglycaemia, but reactive hypoglycaemia, and persistent abnormalities of plasma glucose levels during the test, were associated with higher incidences of complicated outcome. Hypertonic dextrose therapy administered to the patient with persistently subnormal urinary oestriol excretion was less likely to cause a favourable response in oestriol excretion if glucose tolerance was abnormal, perhaps because the adverse influences of abnormal glucose tolerance were not reversible by the third trimester of pregnancy. Hypoglycaemia and hyperglycaemia, additional to diabetes mellitus, are significant factors in the aetiology and diagnosis of abnormal pregnancy, and point to the need to investigate therapeutic measures.


Protease inhibitors in serum and amniotic fluid during pregnancy.

Antitryptic and antichymotryptic activities in amniotic fluid and maternal serum were measured at various stages of pregnancy using the caseinolytic assay method for proteases. The inhibitory activities increased up to 20 weeks in amniotic fluid and remained in the same range up to 30 weeks; a sharp fall was observed at term. In serum, there was a gradual rise of activities with peak values around 30 weeks. The decrease with further advance of pregnancy was not as sharp as in amniotic fluid. The ratios of antitryptic to antichymotryptic activities remained fairly constant (1.54 +/- 0.07) in amniotic fluid throughout pregnancy. In maternal serum the ratio varied over a range of 1.28 to 2.67 and the increase in antichymotryptic activity was relatively greater between 16 and 30 weeks resulting in a lower ratio between the two activities. Amniotic fluid was found to contain a higher proportion of a heat stable inhibitor compared to serum and its contribution to total antitryptic activity varied from 8.7 to 15.6 per cent.


Acute menopausal transition associated with clomiphene therapy: Two case reports.

Two patients presenting with anovulation and secondary infertility were treated with clomiphene citrate. Intermittent blood samples were obtained for the first three months of therapy in each case. One patient failed either to ovulate or menstruate in response to clomiphene and the other patient had only two episodes of cyclical bleeding before she too became amenorrhoeic. Hormonal analyses revealed that both women had undergone a rapid and precocious menopausal transition which has persisted throughout the subsequent three years.


An extraperitoneal operation for vaginal vault suspension to the anterior abdominal wall.

A completely extraperitoneal abdomino-vaginal operation for vaginal inversion, using Mersilene mesh, is described. There were no major postoperative complications and at follow-up of the 13 patients at 10 to 26 months after operation the results were satisfactory.


Antenatal search for fetal beta-thalassaemia in a twin pregnancy.

The antenatal search for fetal beta-thalassaemia in a twin pregnancy is described. Thalassaemia major was correctly excluded in both fetuses, and some of the problems of identifying patients at risk are outlined as well as those of obtaining fetal blood in the second trimester of pregnancy.


Solar keratosis, pterygium, and squamous cell carcinoma of the conjunctiva in Malawi.

The histological features of 234 conjunctival biopsies from Africans in Malawi have been re-examined.  The appearances of solar keratosis, pinguecula, and pterygium are presented as part of a continuous spectrum of the same pathological process and aetiology, which may lead to carcinomatous change.  The results are discussed with regard to the specific geographical distribution of such lesions found by other workers, with particular emphasis on ultraviolet radiation as the main aetiological factor.


Isolation of Chlamydia trachomatis from different areas of conjunctiva in relation to intensity of hyperendemic trachoma in school children in Southern Tunisia.

The simplified one-passage technique of culture in irradiated McCoy cells, in conjunction with certain other developments in technique, was used to isolate Chlamydia trachomatis from specimens collected from 78 children with trachoma in Douz, Southern Tunisia. The results show that C. trachomatis is not confined to the upper tarsal area of the conjunctiva in hyperendemic trachoma. The higher isolation rate and corresponding increase in the number of inclusions obtained from swabbings of the upper fornix and lower lid in addition to the conventional collection from the upper tarsus show the superiority of collecting specimens for culture from the whole conjunctiva. Specimens could be collected from the whole conjunctiva by using 1 swab for each eye and pooled for subsequent inoculation, so that the laboratory incurred no additional work. A close correlation was observed between isolation rate, together with the number of inclusions, obtained in cell culture, and intensity of inflammatory disease in hyperendemic trachoma.  The sensitivity and practicability of this cultural test should provide a valuable laboratory index for use in epidemiological and therapeutic studies of trachoma.


Surgical correction of senile entropion of the lower lid with fascia lata sling.

A method of surgical correction of senile entropion of the lower lid with heterogenous fascia lata sling is described with no tissue loss and minimum of surgical trauma.


The contralateral upper eyelid in ptosis: some observations pertinent to ptosis corrective surgery.

The validity of Hering's law in corrective surgery for ptosis is demonstrated for the first time. It is also shown how Hering's law has been utilised in corrective surgery for unilateral ptosis to elicit a predominant response from the unoperated contralateral upper eyelid. Other implications of reciprocal innervation of the 2 upper eyelids are discussed and a tentative hypothesis is postulated that in a case of true unilateral levator ptosis the fellow upper eyelid will show compensatory retraction.


Pathology of a unique type of teratoid medulloepithelioma.

A unique teratoid medulloepithelioma showing a conspicuous vascular component with orbital involvement is described.


The retinal-pigment epithelial interface.

The interdependence of the outer retina and pigment epithelium is illustrated by the functional changes and structural alterations which occur in each in response to choroidal ischaemia, retinal detachment, vitamin A deficiency, and other causes. The pluripotential role of the pigment epithelium is stressed, particularly in relation to the phagocytosis of outer segment material and the ability of this layer to undergo metaplasia into a variety of different cell types.  Similarities of all disturbances of the retinal-pigment epithelial interface to retinitis pigmentosa are pointed out, and the significance of the findings in relation to this disease is discussed.


Senile disciform macular degeneration: features indicating suitability for photocoagulation.

During a 1-year period 398 new patients were seen with disciform macular degeneration (530 eyes). The lesions were studied retrospectively, and those in which the neovascular tissue did not underlie the fovea, and therefore were treatable, were identified. The following conclusions were drawn: (1) Treatment is possible in most patients with good acuity and few with poor acuity. (2) Treatment is possible in a large proportion of patients with a short history and few with a long history of visual loss. (3) As many as 50% of all patients with senile disciform macular degeneration may be amenable to treatment if seen early enough in the course of their disease. (4) Over one-third of eyes with lesions that are untreated have a visual acuity of 6/60 or better 3 years after the onset of symptoms. (5) If a controlled trial proves that treatment is beneficial, these results emphasise the need for rapid referral and show that these patients will generate a large additional clinical load.


Cryotherapy of proliferative sickle retinopathy, II: triple freeze-thaw cycle.

Complete closure of retinal neovascularisation due to proliferative sickle cell retinopathy was achieved in 28 'sea fans' (areas of neovascularisation) in 9 eyes of 9 patients by means of a triple freeze-thaw technique.  However, 2 eyes developed subsequent rhegmatogenous retinal detachments, presumably related to vitreous traction on necrotic retina.  We therefore recommend photocoagulation as the best treatment of proliferative sickle cell retinopathy. If opacities in the media prevent photocoagulation, a single freeze-thaw cycle is preferable.


Body growth, puberty and undernutrition in the male guinea-pig.

1. Male guinea-pigs were assigned to four different groups at birth. The heaviest group of animals was severely undernourished from 21 d of age (weight gain: 1.2 g/d v. 10 g/d for normally-fed animals). 2. At 35, 45, 55 and 65 d of age in normally-fed animals, and at 45 and 55 d of age in undernourished animals, blood testosterone levels were determined by radioimmunoassay, epididymidal tissue examined for the presence of spermatozoa and testes sectioned for rating of spermatogenesis using light microscopy. 3. Sexual maturity in terms of potential fertility (spermatozoa in the epididymis) was reached by all normally-fed animals between 45 and 55 d of age. High birth-weight animals had a higher incidence of the most advanced stages of spermatogenesis than low birth-weight animals had a higher incidence of the most advanced stages of spermatogenesis than low birth-weight ones at the various ages studied. In the undernourished animals spermatogenesis was clearly delayed. 4. Plasma testosterone concentrations were already in the adult range at 35 d and independent of age and birth-weight. The undernourished animals, however, had very low levels. Maintenance of spermatogenesis seemed compatible with low plasma levels of testosterone. 5. It is concluded that the timing of testicular development seems to be determined by the time the animals are born and appears to be unaffected by severe undernutrition from 21 d of age.


Trace nutrients. 2. Manganese in British food.

1. The amount of manganese in nationally-representative samples of prepared and cooked groups of foods, and in a wide variety of individual foods, was determined by atomic absorption spectroscopy. 2. The average British diet was calculated to provide 4.6 mg Mn/d of which half was derived from tea and other beverages, 30% from cereals, and 15% from vegetables and fruit. Animal products provided little Mn. 3. Individual foods other than tea which were particularly rich in Mn in Britain were unrefined and partially-refined cereals, and some spices and herbs. Some vegetables and fruit, coffee, wine, chocolate and brown sugar also contained significant amounts of Mn.


Lipid composition and metabolism in liver and brain of vitamin B12-deficient rat sucklings.

1. Rat sucklings (18-d-old) bred from vitamin B12-deprived dams were compared with vitamin B12-supplemented dams' offspring, which were considered normal rat sucklings. 2. The vitamin B12-deficient rat sucklings had lower body-weight, liver weight and brain weight. 3. Vitamin B12 deficiency was also evident from the tenfold lower concentrations of vitamin B12 in liver and cerebellum. 4. The concentration of liver lipid was markedly increased in vitamin B12-deficient rats; triacylglycerol accounted for most of the increase. In brain the lipid concentration was slightly decreased (less than 0.05). 5. The methylation of ethanolamine phosphoglyceride to choline phosphoglyceride was reduced in both liver and brain in vitamin B12-deficient rats, as measured after the administration of [14C]ethanolamine. A slight decrease in choline phosphoglyceride concentration could be a consequence of this finding. The composition of phospholipids was otherwise normal. 6. Odd-chain fatty acids (pentadecanoate and heptadecanoate) accumulated in both liver and brain of the vitamin B12-deficient rat sucklings and constituted approximately 1% of total fatty acid. 7. The biosynthesis of fatty acid and cholesterol from intraperitoneally-injected 3H2O and [14C]propionate was unchanged in vitamin B12 deficiency.


Growth of muscle fibres during recovery from severe malnutrition in Jamaican infants.

1. The growth of muscle fibres was analysed by light microscopy in biopsies from subjects when malnourished, during nutritional rehabilitation, and after clinical recovery. 2. Muscle fibres from malnourished subjects were extremely atrophic (cross-sectional area, 110 micrometers2). The fibres doubled in size during the early period of rehabilitation. Growth of muscle fibres during later periods of rehabilitation occurred at a slower rate. 3. The absolute rates of change in fibre sizes differed considerably between subjects, but the rates of change relative to the rate of gain of total body-weight (expressed as % recovery or % expected weight-for height (Nelson, 1975)) were similar between subjects after the initial growth spurt. The pattern of recovery appeared to differ between older and younger subjects. 4. Fibre sizes correlated with body-weight but not with age in the malnourished subjects. A significant correlation between fibre areas and either weight or age was observed during rehabilitation and after clinical recovery. 5. Fibre sizes of clinically-recovered subjects (mean age, 13.8 months; weight, 8.7 kg) were only approximately 60% of that for a well-nourished 6-month-old control subject (6.4 kg). These results suggest that a longer period of time is required for fibres to reach their expected size. Therefore, when the child has regained body-weight to that of a normal child of the same height, his muscles have not yet recovered and his body composition is abnormal.


The measurement of food iron absorption in man. A methodological study on the measurement of dietary non-haem-Fe absorption when the subjects have a free choice of food items.

1. The present study was considered as a first step to develop a method to measure food iron absorption from realistic common meals prepared and consumed by the subjects themselves in their own homes. The absorption of Fe from the meals was measured by means of the extrinsic-tag method modified to allow for a free choice of food items. 2. The mean Fe intake was 2.79 mg and the mean Fe absorption approximately 0.30 mg. The Fe status of the subjects corresponded to 31.5% absorption from a reference dose solution containing 3 mg elemental Fe as ferrous ascorbate. The variation in food Fe absorption obtained in this field study was found to be of the same magnitude as that obtained in studies performed under more controlled conditions as in the laboratory. 3. The conclusion was drawn that the proposed method could be used in realistic field studies aimed at, for example, explaining or preventing a high prevalence of deficiency. A suffciently long run-in period and a careful instruction of the subjects was considered essential for the design of future field studies.


Iron status in a general practice and its relationship to morbidity.

1. An epidemiological study of iron status in general practice has been undertaken. An age-sex register was established and a 10% stratified random sample (194 males and 220 females) of the patients above the age of 15 years was studied. A full blood count, serum Fe concentration, transferrin saturation and serum ferritin concentration were measured and correlated with clinical features associated with Fe deficiency. 2. The geometric mean for serum ferritin for males was 77 micrograms/l (20-196 micrograms/l) and for females 37 micrograms/l (8--177 micrograms/l). A low serum ferritin (male less than 30 micrograms/l, female less than 20 micrograms/l) in the absence of anaemia was found in fifteen males and thirty-five females. In this group forty patients had clinical features which are aetiologically associated with Fe deficiency. The incidence of similar features in a control group of thirty-three patients was seven. 3. It is suggested that this is indirect evidence that serum ferritin concentration is the most sensitive monitor of Fe status enabling the detection of pre-anaemic Fe deficiency.


The utilization of chopped and pelleted lucerne (Medicago sativa) by growing lambs.

1. The efficiency of utilization of the dietary energy and nitrogen contained in a dried lucerne (Medicago sativa cv. Chartainvilliers) given either chopped (CL) or ground (1.96 mm screen) and pelleted (PL), was measured in a comparative slaughter experiment. Growing lambs were given equal amounts of digestible energy in the chopped or pelleted form at each of the three planes of nutrition for a period of 100 d. 2. The initial energy, fat and protein content of both the carcass and the total body of the test lambs was estimated from regression equations between fasted (18 h) live weight and these components, derived from a group of twenty-three comparable lambs. The final energy, fat and protein content of the test lambs was determined directly by chemical analyses. 3. The metabolizable energy (ME) content of the diets was derived at each plane of nutrition from measured faecal and urinary losses and estimated methane losses. The depression in ME content with grinding and pelleting the dried lucerne was small (CL 8.69 MJ/kg dry matter (DM), PL 8.42 MG/kg DM). 4. The efficiency of utilization of the ME of the dried lucerne for growth and fattening was higher (P less than 0.01) when given in the ground pelleted form (0.533), than in the chopped form (0.284). The net energy value of the PL (3.5 MJ/kg DM) was higher than that of CL (2.2 MJ/kg DM). 5. Thus lambs fed on PL grew faster and had a higher caracass weight gain, carcass protein and fat retention than lambs fed on CL. The composition of the carcass was not altered by the physical processing treatment. 6. Digestion studies with these same CL and PL diets had shown that grinding and pelleting depressed digestion in the forestomachs and increased digestion in the small intestine compared with the chopped form. The increased efficiency of utilization of the gross energy and ME and the higher net energy value of PL was attributed primarily to a change in the site of digestion within the alimentary tract. Associated with this change was a higher value for absorbed amino acids : absorbed energy and an increased apparent absorption of methionine for lambs fed on PL. The difference in the energy costs of eating and ruminating the CL and PL was small.


The amino acid requirements of the preruminant calf.

1. Ten calves (50--58 kg live weight) were given a diet consisting of diluted whole milk, wheat gluten and supplemented with appropriate nutrients including amino acids but deficient in lysine. The lysine reequirements of these calves, which were growing at approximately 0.25 kg/d, were estimated from responses to lysine supplementation of this diet. From plasma urea, plasma lysine, nitrogen retention and apparent digestibility of N responses the estimated lysine requirements were 8.5, 7.5, 7.2 and 7.6 g/d respectively. 2. From the mean lysine requirement (7.8 g/d) and the ratio, lysine: otheressential amino acids in carcasses of similar calves the estimated requirements were (g/d): methionine 2.1, cystine 1.6, threonine 4.9, valine 4.8, isoleucine 3.4, leucine 8.4, tyrosine 3.0, phenylalanine 4.4, histidine 3.0, arginine 8.5, tryptophan 1.0.


The optimal amino acid supplementation of barley for the growing pig. 1. Response of nitrogen metabolism to progressive supplementation.

1. In five experiments, involving 142 female pigs weighing on average 33 kg, estimates were made of the amounts of essential amino acids which minimized urinary N excretion when diets with barley as the only source of protein were given at the rate of 120 g/kg0.75 per d. 2. With additions of lysine (4.0 g/kg diet) and threonine, (1.2 g/kg diet) to barley urinary N excretion decreased from 0.91 to 0.36 g/kg0.75 per d, corresponding to an increase in biological value (bv) from 0.51 to 0.86. 3. With these additions of lysine and threonine, there were no responses to additions of tryptophan, methionine or isoleucine, or to further additions of lysine or threonine, but addition of histidine significantly reduced N excretion. 4. No optimal addition of histidine could be determined; the mean rate of N excretion after addition of histidine (not less than 0.3 g/kg diet) was 0.27 g/kg0.75 per d, corresponding to a BV of 0.93. 5. There was a variation between pigs from different litters in their responses to added histidine. Those with low rates of N excretion on the unsupplemented diet did not respond to additions of histidine, but those with high rates did. 6. It is concluded that additions of only three amino acids can greatly improve the nutritive value of barley protein for the growing pig and that the amino acid composition of the supplemented protein closely approaches the ideal; it is also similar to whole-body tissue protein.


The amino acid supplementation of barley for the growing pig. 2. Optimal additions of lysine and threonine for growth.

1. Thirty-three diets were made by adding to ground barley combinations of L-lysine (0--6.0 g/kg) and L-threonine (0--3.0 g/kg), together with vitamins and minerals. Each was given to two female and two castrated male pigs during their growth from 25 to 60 kg. 2. Growth rate increased from 0.36 kg/d to a maximum of 0.65 kg/d with additions of 3.8 g L-lysine/kg and 1.8 g L-threonine/kg; these values and the maximum gains achieved were slightly higher for castrates than for females. Minimum values for food conversion ratio of 2.84 for castrates and 3.06 for females were achieved with similar amino acid additions to those giving fastest growth. 3. Carcass fat, estimated by sepcific gravity, was least with the addition of 5.9 g L-lysine/kg, but continued to decrease up to the highest threonine concentrations. Backfat thickness was also reduced by amino acid additions but failed to reach a minimum.


Protein quality in cereals and pulses. 1. Application of microbiological and other in vitro methods in the evaluation of rice (Oryza sativa L.), sorghum (Sorghum vulgare Pers.), barley and field beans (Vicia faba L.).

1. The Streptococcus zymogenes assay procedure was modified and used in the estimation of available methionine and relative nutritional value in rice (Oryza sativa L.), sorghum (Sorghum vulgare Pers.), barley and field beans (Vicia faba L.). The results were assessed in relation to the content of tannins and 'dye-binding lysine', and to published information on the nutritional quality of some of the test samples. 2. In grain of ten varieties of sorghum, for which other workers had reported a correlation (r -0.82; P less than 0.01) between tannin content and amino acid digestibility in chicks, the available methionine content ranged from 6.3 to 17.7 g/kg protein (nitrogen x 6.25) and was highly correlated with tannin content (r -0.97; P less than 0.001). The content of total methionine and dye-binding lysine varied little between varieties and was not related to tannin content. 3. In nine samples of rice the availability of methionine was uniformly high, in accordance with literature values for true digestibility of the N. 4. In field beans the presence of tammins in the seed coat was associated with a significant (P less than 0.001) reduction in the availability of methionine. In eleven tannin-free varieties the coefficient of availability averaged 0.791, compared with 0.685 in eleven tannin-containing varieties. The standard deviation associated with both values was 0.04. There was no such difference between the corresponding dye-binding lysine values. 5. Thirty-three samples of barley were examined, representing fourteen varieties, four of which had been grown at several widely different latitudes in England and Sweden. There was little difference in methionine content between samples. The average availability coefficient for eighteen samples grown in England was 0.995+/-0.044, and for fifteen varieties grown in Sweden it was 0.851+/-0.042. The difference was not associated with any difference in tannin content, which was uniformly low. The findings contradict published evidence for marked increase in tannin content with increase in geographical latitude of cultivation, associated with decrease in digestibility of N.


Effect of dietary composition and cold exposure on non-shivering thermogenesis in young pigs and its alteration by the beta-blocker propranolol.

1. Young pigs were fed on three diets consecutively, each diet being given for 1 week. The diets were given in random order as (g pig feed/kg body-weight): (a) 20, (b) 60, (c) 20 plus a supplement with the energy equivalent of 40 g pig feed/kg. The supplements included desiccated coconut, fish meal and glucose. 2. At the end of each week resting metabolic rate, beginning 12--14 h after feeding, was measured overnight using an open-circuit respiration chamber at thermoneutrality. 3. The oxygen consumption of pigs on the 60 g/kg diet was always higher than on the 20 g/kg diet. The addition of desiccated coconut, or fish meal also increased metabolic rate; whereas with added glucose, O2 consumption tended to be even lower than on 20 g/kg alone. 4. The administration of the beta-blocker propranolol to pigs on ad lib, food intake reduced the rate of overnight resting O2 consumption, measured from 10 until 20 h after feeding, by 12%, but it had no effect on O2 consumption when the intake was 20 g feed/kg. Exposure to mild cold (15 degrees) caused an increase in O2 consumption and this was reduced by 14% after injection of propranolol.


Responses to post-ruminal infusions of casein and arginine, and to dietary protein supplements in lactating goats.

1. In Expt 1 a study was made in goats of responses in terms of milk production, nitrogen utilization and plasma amino acids to abomasas infusions of casein (45 g/d) in goats given 2.5 kg/d of a ration containing crude protein (N x 6.25) at 109 (L1) or 146 (H1) g/kg. 2. In Expt 2 a study was made in goats of responses in terms of milk production, nitrogen utilization, plasma amino acids and growth hormone levels to abomasal infusions of casein (45 g/d) or arginine (25 g/d) in goats given 2.3 kg/d of a ration containing crude protein (N x 6.25) at 104 g/kg (L2). These observations were made also in goats given a ration containing crude protein at 136 g/kg (H2). 3. Milk production in Expt 1 was 2.75, 2.45 and 2.76 kg/d on L1+casein, H1 and H1+casein treatments respectively, the response to casein infusion being significant (P less than 0.05). Milk production in Expt 2 was 1.90, 2.04, 1.96 and 1.96 kg/d on L1, L2+casein, L2+arginine and H2 treatments respectively, and the differences were not significant. 4. Total N intake in Expt 1 was 49, 58 and 64 g/d on L1+casein, H1 and H1+casein treatments respectively. Faecal N was similar on the three treatments (14 g/d), urinary N was 15, 23 and 30 g/d and milk N was 14, 12 and 14 g/d on the respective treatments. Total N intake in Expt 2 was 33, 40, 43 and 44 g/d on L2, L2+casein, L2+arginine and H2 treatments respectively. Faecal N was similar on the four treatments (12 g/d), urinary N was 7, 10, 13 and 14 g/d and milk N was 9, 9, 8 and 8 g/d on the respective treatments. 5. The concentration of indispensable amino acids in plasma was increased by casein infusion in both experiments. It was 1279, 825 and 1133 micrometers/l on L1+casein, H1 and H1+casein treatments respectively in Expt 1, and 1081, 1582, 1055 and 1163 micrometers/l on L2, L2+casein, L2+arginine and H2 treatments respectively in Expt 2. 6. The concentration of arginine in plasma was doubled 1 h after the onset of arginine infusion in Expt 2, Growth hormone levels in plasma were not increased when arginine levels rose following arginine infusion. and did not differ between treatments. 7. The results of the two experiments showed that the stimulatory effect on milk production of intra-abomasal infusion of casein was not reproduced by increasing the dietary intake of protein or by infusing arginine. The results of the second experiment showed that abomasal infusion of arginine did not stimulate production of growth hormone and that growth hormone apparently was not implicated in the effects of casein infusion on milk production.


Availability of lysine in protein concentrates as determined by the slope-ratio assay with growing pigs and rats and by chemical techniques.

1. A slope-ratio assay was developed with growing pigs to determine the availability of lysine in five protein concentrates. The basal diet contained 5.2 g lysine/kg and six levels of lysine, in 500 mg/kg increments, were used to determine the pig's response to standard lysine. The protein concentrates were incorporated into the basal diet to provide five levels of total lysine, again in 500 mg/kg increments, at the expense of wheat starch. A daily feeding scale based on live weight was used to ensure similar nutrient intakes. Pigs were fed at three-hourly intervals to ensure the utilization of free amino acids in the diets. Four pigs were allotted to each dose level and response was assessed over the 20--45 kg growth phase. 2. Potency estimates for available lysine in the five protein concentrates varied, depending on whether live-weight gain or carcass gain was used as the criterion of response. Carcass gain was considered more appropriate as it was not influenced by variation in gut fill. Availability of lysine in the five proteins, using carcass gain/d were (proportion of total) cottonseed meal 0.39,fish meal 0.89, meat-and-bone meal 0.50, skim-milk powder 0.88 and soya-bean meal 0.87. 3. Rat slope-ratio assay results for available lysine in the five protein concentrates were in general agreement with those from the pigs. In contrast, the differences in available lysine were not detected by the chemical Silcock available-lysine test (Roach et al. 1967) nor by the direct fluorodinitrobenzene procedure (Carpenter, 1960).


Secondary fermentation in the runen of a sheep given a diet based on molasses.

1. The extent of conversion of acetate-carbon to carbon dioxide in the rumen of a 40 kg wether consuming 1 kg molasses/d was estimated using isotope-tracer-dilution techniques. 2. There was a high rate of conversion of acetate to CO2 (6.0 g C/d) in the rumen. There were high concentrations in the rumen of Methanosarcina approximately 6 x 10(9)/ml which represents a significant proportion of the rumen bacterial biomass. These organisms are usually found in mud and sludge and are capable of oxidizing acetate. 3. The most likely explanation of these results was that there was an extensive secondary or sludge-type fermentation occurring in the rumen which results in volatile fatty acids being converted to CO2 and methane. In similar studies with sheep given lucerne (Medicago sativa) diets, conversion of acetate-C to CO2 within the rumen was not evident. 4. It is suggested that a major effect of the presence of secondary fermentation processes in the rumen may be to reduce availability of energy nutrients to the animal, and to alter the ratio protein:energy in the absorbed nutrients.


Superoxide dismutase (EC 1.15.1.1), zinc status and ethanol consumption in maternal and foetal rat livers.

1. The activity of superoxide dismutase (EC 1.15.1.1) was not greatly affected by zinc deficiency in maternal or foetal (20 d) rat livers, although in the latter tissue it did appear to be slightly raised by Zn depletion when compared with pair-fed control animals. 2. Enzyme activity was significantly higher in livers from all foetuses after administration of aqueous ethanol at 100 or 200 ml/l to the dams during pregnancy. 3. Plasma Zn levels were significantly increased in Zn-deficient dams after ingestion of alcohol during pregnancy.


Radiochemical synthesis and photochemical properties of the uncoupler 2-azido-4-nitrophenol, a versatile photoaffinity labeling reagent.

2-Amino-4-nitrophenol was tritiated in an acid-catalyzed hydrogen exchange reaction. Radioactive 2-azido-4-nitrophenol with a specific radioactivity up to 21 mCi/mmol was synthesized from 2-amino-4-nitrophenol by diazotization and azide coupling. The photochemical properties of the uncoupler, 2-azido-4-nitrophenol, were studied as free solute and as ligand bound to uncoupler binding sites in bovine serum albumin and mitochondria. Based on product analyses, irradiation of free or bound 2-azido-4-nitrophenolate with visible light results in the formation of nitrene intermediates with a singlet to triplet ratio of 6:1 to 9:1. 2-Azido-4-nitrophenolate and bovine serum albumin form a strong 1:1 complex (KD = 0.7 micron) which can be converted into a photoproduct with a covalent bond between the label and the protein. The acid dissociation constant of the protein-bound 2-amino-4-nitrophenol moiety is strongly pH dependent. Photoaffinity labeling of mitochondria by 2-azido-4-nitrophenolate follows a pattern expected from equilibrium binding studies using normal and lipid-depleted particles: polypeptides were found to bear 90-95% of the radioactive label, and 5-10% of the latter was bound to phospholipids. Two polypeptides (approximately 56 000 and 31 000 daltons) were associated with 60% of the label, indicating a high degree of specific photochemical labeling.


Folylpoly-gamma-glutamates as cosubstrates of 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase.

N10-Formyltetrahydropteroylpoly-gamma-glutamates (N10-formyl-H4PteGlun) having n = 1, 3, 4, 5, 6, and 7 glutamyl residues have been tested as cosubstrates of the purine biosynthesis enzyme 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase (AICAR transformylase) of chicken liver. The cosubstrates were synthesized by solid-phase synthesis, reduced catalytically, and formylated; a purified enzyme preparation was used and assayed spectrophotometrically following the deltaOD at 298 nm resulting from conversion of the formylated folate to the free tetrahydro form. Km values and Vm values determined at saturating concentrations of AICAR and at 25 and 150 mM KC1 were used to calculate the relative specificity constants Vm/Km for the N10-formyl-H4PteGlun. At physiologic [K+] (150 mM) they were 1.0, 52, 250, 93, 120, and 59 and at the lower (25 mM) [K+] the relative specificity constants were 1.0, 64, 78,34, 48, and 37 for n = 1, 3, 4, 5, 6, and 7, respectively. The poly-gamma-glutamates are clearly the preferred cosubstrates, particularly when tested at physiologic [K+]. The maximal relative specificity constant observed with N10-formyl-H4PteGlu4 supports the hypothesis that regulation of certain pathways of one-carbon metabolism may operate via alterations of the poly-gamma-glutamyl chain length. No inhibition by the unnatural (d) isomers of the N10-formyl-H4PteGlun was observed.


Rotational relaxation times of 1,6-diphenyl-1,3,5-hexatriene in phospholipids isolated from LM cell membranes. Effects of phospholipid polar head-group and fatty acid composition.

Phospholipids were isolated from mitochondrial, microsomal, and plasma membranes of LM cells and fractionated into individual phospholipid classes on silicic acid columns. The fatty acid composition and the rotational relaxation time of 1,6-diphenyl-1,3,5-hexatriene (DPH) were determined for each phospholipid class. Sphingomyelin was the only phospholipid isolated from LM cell membranes that showed a phase transition within the temperature range investigated, 5-40 degrees C. The rotational relaxation times for DPH were lowest in phosphatidylcholine in all the membrane fractions. Phosphatidylcholine isolated from the three membrane fractions of choline-supplemented cells had similar rotational relaxation times and phosphatidylcholine isolated from microsomal membranes of linoleate-supplemented cells had lower rotational relaxation times. The results indicate that the differences in the rotational relaxation times of DPH between mitochondrial, microsomal, and plasma membrane phospholipids could be explained primarily by differences in the polar head-group composition, while differences in the fatty acid composition had only a minor effect. This provides a basis for understanding how the different lipid components in these cells contribute to membrane fluidity.


Histon-histone interactions within chromatin. Preliminary location of multiple contact sites between histones 2A, 2B, and 4.

The contact-site cross-linkers tetranitromethane, UV light, formaldehyde, and a monofunctional imido ester have been used to generate a collection of histone-histone dimers and trimers from nuclei and chromatin. Four different H2B-H4 dimers have been isolated. Preliminary CNBr peptide mapping has shown that all are cross-linked at different positions that are apparently clustered within the C-terminal regions of these histones. Similarily, two different H2A-H2B dimers and two different H2A-H2B-H4 trimers have been partially characterized. The data suggest a functional map for H2B in which the N-terminal third interacts with DNA, the middle third interacts with H2A, and the C-terminal third interacts with H4. We hope, by pursuing this type of analysis, to develop a detailed understanding of each histone-histone binding interaction through saturation cross-linking of the binding sites.


Specific histone-histone contacts are ruptured when nucleosomes unfold at low ionic strength.

The ordered unfolding of the nucleosome core within chromatin at low ionic strengths has been studied. The results show that, when nuclei are lysed gently in solutions of very low ionic strength, their constituent nucleosomes rupture at a major H2B-H4 binding site but remain unperturbed at the site of the H2A-H2B interaction. These conclusions are based on data which show that at least four separate but closely spaced H2B-H4 contacts, identifiable by contact-site cross-linking in intact nuclei, are broken when nuclei are suspended in very dilute buffers. Appropriate controls on purified nucleosomes monomers demonstrate that the H2B-H4 contacts being broken are indeed intranucleosomal. Sedimentation of nucleosomes in the ultracentrifuge at various salt concentrations reveals that a significant conformational transition occurs in the range of ionic strength over which the H2B-H4 binding site ruptures.


On the mechanism of nucleosome unfolding.

We have studied the relative stabilities to urea denaturation of histone-histone binding interactions as they occur both in chromatin and in histone complexes free in solution. We have used the two zero-length contact-site cross-linking agents, tetranitromethane and UV light, to measure the relative degree of H2B-H4 and H2A-H2B association under various conditions. The two interactions were disrupted coordinately when nuclei were treated with increasing concentrations of urea. In contrast, when histone complex in 2 M NaCl were treated with urea, the H2B-H4 interaction was found to be much less stable than the H2A-H2B interaction. We have shown previously that nucleosomes unfold at low ionic strengths such that the H2B-H4 but not the H2A-H2B interaction is broken in the process. We speculate that the preferential rupture of the H2B-H4 contact is of physiological significance.


Picosecond dynamics of tyrosine side chains in proteins.

To probe the details of small amplitude motions in proteins, a dynamical analysis of the orientation fluctuations of two tyrosine side chains in the bovine pancreatic trypsin inhibitor is presented. Detailed results are given for the time history and correlation functions obtained for the ring motion from a molecular dynamics simulation of the entire protein. It is shown that even on a picosecond time scale orientational fluctuations of +/-30 degrees from the average position occur for the tyrosine rings in the interior of the protein. It is found that the Langevin equation is applicable to the ring torsional motion, which corresponds to that of an angular harmonic oscillator with near-critical damping. Two possible microscopic models for the observed damping effects are outlined. One of these, analogous to liquid behavior, is based on kinetic theory and takes account of the collisions which occur between atoms of the protein; the other, more analogous to solid behavior, involves the coupling among a large number of harmonic oscillators. The collisional model with parameters obtained from theoretical estimates leads to good agreement with the correlation functions from the dynamic simulation. However, the dephasing of harmonic oscillations can yield similar short-time results so that a distinction between the two models is difficult. The importance of damping effects on the motions involved in conformational transitions and enzymatic reactions is discussed.


High concentration active enzyme centrifugation: analysis of active polymeric forms at up to 10 000-fold higher concentrations than with conventional methods.

This paper describes the theoretical basis, experimental technique, and experimental evaluation of a new method of analysis called "high concentration active enzyme centrifugation". It extends by up to four orders of magnitude the upper concentration limits at which the technique of "active enzyme centrifugation" can be used for analysis of enzyme structure. This new theory is largely based on certain properties of Gaussian curves which we have described in previous publications [Wei, G.J., & Deal, W.C., Jr. (1976) Anal. Biochem. 75, 113-121; Anal. Biochem. (1978) 87, 433-446]. One of the most important aspects of this development is that it extends the concentration range upward so that experiments can be performed on enzymes in the active polymeric forms corresponding to their in vivo states. Furthermore, this expansion includes the range in which most enzymes go through all their association-dissociation transitions from one polymeric form to another. Hence, the method can be used to define the various concentration-dependent transitions and also to ascertain which of the various polymeric forms of an enzyme are active, under various conditions. This method also retains the many favorable characteristics inherent in the active enzyme centrifugation technique. In studies with lactate dehydrogenase, the results from this method of band sedimentation were identical within experimental error (about 1.5%) with results from conventional boundary sedimentation velocity studies.


Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes.

Intact vesicles enriched in acetylcholine receptor from Torpedo californica electroplaque membranes can be separated from collapsed or leaky vesicles and membrane sheets on sucrose density gradients. alpha-Bungarotoxin binding in intact vesicles reveals that approximately 95% of the acetylcholine receptor containing vesicles are formed outside-out (with the synaptic membrane face exposed on the vesicle exterior). The binding data also indicated that only 5% or less of the sites for alpha-bungarotoxin binding to synaptic membranes are located on the interior, cytoplasmic face. Intact vesicles are stable to gentle pelleting and resuspension but are easily osmotically shocked. The vesicles are impermeable to sucrose and Ficoll, but glycerol readily transverses to membrane barrier. Intact vesicles provide a sealed, oriented membrane preparation for studies of vectorial acetylcholine receptor mediated processes.


Thermodynamics of glucagon aggregation.

Heats of dilution of concentrated glucagon solutions have been measured calorimetrically at 10 and 25 degrees C in 0.2 M potassium phosphate buffer of pH 10.6. Analysis of the data in terms of a monomer-trimer equilibrium gives the following thermodynamic parameters for the association reaction at 25 degrees C: delta G degrees = 7.34 kcal/mol of trimer, delta H degrees = -31.2 kcal/mol, deltaS degrees = -80 cal/(K mol), deltaCp = 430 cal/(K mol). The sensitivity of heat of dilution data to the association constant and stoichiometry of the reaction is discussed.


Thermodynamics of the denaturation of lysozyme in alcohol--water mixtures.

The thermal denaturation of lysozyme was studied at pH 2 in aqueous mixtures of methanol, ethanol, and 1-propanol by high sensitivity differential scanning calorimetry (DSC). The most obvious effect of alcohols was the lowering of Td, the temperature of denaturation, increasingly with higher alcohol concentration and longer alkyl chain. Both the calorimetric and van't Hoff enthalpies of denaturation initially increased and then decreased with increasing alcohol concentration, the ratio of the two enthalpies being nearly unity, 1.007 +/- 0.011, indicating the validity of the two-state approximation for the unfolding of lysozyme in these solvent systems. The reversibility of the denaturation was demonstrated by the reversibility of the DSC curves and the complete recovery of enzymic activity on cooling. The changes in heat capacity on unfolding decreased with increasing alcohol concentration for each alcohol. Experimentally determined values of denaturation temperature and of entropy and heat capacity changes were used to derive the additional thermodynamic parameters delta G degrees and delta S degrees for denaturation as a function of temperature for each alcohol--water mixture. Comparison of the thermodynamic parameters with those reported [Pfeil, W., & Privalov, P.L. (1976) Biophys. Chem. 4, 23--50] in aqueous solution at various values of pH and guanidine hydrochloride concentration showed that these latter changes have no effect on the heat capacity changes, whereas the addition of alcohols causes a sharp decrease.


Effect of thiourea on microsomal oxidation of alcohols and associated microsomal functions.

Thiourea and diethylthiourea, two compounds which react with hydroxyl radicals, inhibited NADPH-dependent microsomal oxidation of ethanol and 1-butanol. Inhibition by both compounds was more effective in the presence of the catalase inhibitor, azide. Inhibition by thiourea was noncompetitive with respect to ethanol in the absence of azide but was competitive in the presence of azide. Urea, a compound which does not react with hydroxyl radicals or H2O2, was without effect. Thiourea had no effect on NADH- and NADH-cytochrome c reductase, NADPH oxidase, and NADH- and NADPH-dependent oxygen uptake. Thiourea inhibited the activities of aniline hydroxylase and aminopyrine demethylase. Thiourea, but no other hydroxyl radical scavengers, e.g., dimethyl sulfoxide, mannitol, and benzoate, reacted directly with H202 and decreased H2O2 accumulation in the presence of azide. Therefore the actions of thiourea are complex because it can react with both hydroxyl radicals and H2O2. Differences between the actions of thiourea and those previously reported for dimethyl sulfoxide, mannitol, and benzoate, e.g., effects on drug metabolism, effectiveness of inhibition in the absence of azide, or kinetics of the inhibition, probably reflect the fact that thiourea reacts directly with H2O2 whereas the other agents do not. The current results remain consistent with the concept that microsomal oxidation of alcohols involves interactions of the alcohols with hydroxyl radicals generated from microsomal electron transfer.


Side-chain torsional potentials: effect of dipeptide, protein, and solvent environment.

Side-chain torsional potentials in the bovine pancreatic trypsin inhibitor are calculated from empirical energy functions by use of the known X-ray structure of the protein and the rigid-geometry mapping technique. The potentials are analyzed to determine the roles and relative importance of contributions from the dipeptide backbone, the protein, and the crystalline environment of solvent and other protein molecules. The structural characteristics of the side chains determine two major patterns of energy surfaces, E(X1,X2): a gamma-branched pattern and a pattern for longer, straight side chains (Arg, Lys, Glu, and Met). Most of the dipeptide potential curves and surfaces have a local minimum corresponding to the side-chain torsional angles in the X-ray structure. Addition of the protein forces sharpens and/or selects from these minima, providing very good agreement with the experimental conformation for most side chains at the surface or in the core of the protein. Inclusion of the crystalline environment produces still better results, especially for the side chains extending away from the protein. The results are discussed in terms of the details of the interactions due to the surrounding, calculated solvent-accessibility figures and the temperature factors derived from the crystallographic refinement of the pancreatic trypsin inhibitor.


Viscosity-dependent structural fluctuations in enzyme catalysis.

The effect of viscosity on the rate of catalysis of carboxypeptidase A has been tested. By use of the tripeptide carbobenzoxy-l-alanyl-l-alanyl-l-alanine [Z(L-Ala)3] as substrate, it was shown that most of the effect on the hydrolysis rate caused by the presence of 30 or 40% methanol or glycerol in aqueous solution can be ascribed to a contribution of viscosity to the catalytic rate constant, kcat. Arrhenius plots of kcat in 30 and 40% glycerol or methanol are linear and almost parallel. When the rate constants are "corrected" for the viscosity of various media, the difference between the various Arrhenius plots is considerably reduced; it vanishes, within experimental error, when the effect of the dielectric constant of the solutions is taken into account as well. It is proposed that the viscosity of the medium can influence the rate-limiting step of the enzymic reaction, which is the rate of transitions over the energy barrier preceding product formation. According to the suggested mechanism, the enzyme--substrate complex can overcome this energy barrier by viscosity-dependent structural fluctuations. The quantitative agreement between the theory and the experimental results suggests that (a) due to the temperature dependence of the viscosity of the solution, the potential energy barrier of the reaction is about 5 kcal/mol lower than the observed activation energy and (b) information about the structural flexibility of the complex can be obtained by kinetic measurements.


Accumulation of polyadenylated mRNA during liver regeneration.

Cytoplasmic and polysomal polyadenylated mRNA [poly(A)+-mRNA] increased by 120% prior to the onset of DNA synthesis during the regeneration of rat liver following partial hepatectomy. Despite this large change in cytoplasmic mRNA and an approximately 50% increase in total nuclear RNA, the amount of polyadenylated nuclear RNA increased by only 15--20% during this time. Neither the average size of nuclear or of cytoplasmic polyadenylated mRNA nor the length of their poly(adenylic acid) [poly(A)] tracts changed during liver regeneration. Polysomal poly-(A)+-mRNA increased proportionately more and at a faster rate than rRNA during the first day following partial hepatectomy. Normal livers contained a substantial proportion of cytoplasmic poly(A)+-mRNA not associated with polysomes but this proportion was not altered in 3-h regenerating liver. Thus, in regenerating liver, most preexisting cytoplasmic mRNA does not appear to be recruited into polysomes prior to the substantial increase in the amount of cytoplasmic poly(A)+-mRNA.


Cross-linking of ribosomal proteins by 4-(6-formyl-3-3-azidophenoxy)butyrimidate, a heterobifunctional, cleavable cross-linker.

For the identification of neighbor relationships between proteins in biological systems 4-(6-formyl-3-azidophenoxy)butyrimidate (FAPB-imidate), a heterobifunctional, cleavable cross-linker was synthesized. The reagent has an imido ester at one end, which is used for the attachment to amino groups of a specific protein whose environment has to be characterized. At the other end, the reagent has both an azido and an aldehyde group. The azido group can be used to cross-link the protein photochemically to a variety of chemical groups of neighboring proteins. The aldehyde group is able to cross-link the protein by reductive alkylatin to amino groups of neighboring proteins. In both cases, the cross-linker can be made radioactive with NaB3H4. the cross-linked complexes can be split at the band originating from the imidate group by treatment with ammonia. Hereby, the radioactive cross-linker remains covalently attached to the unknown neighboring protein, which can be therefore easily identified. In order to explore the usefulness of FAPB-imidate as a cross-linking agent, the compound was attached to ribosomal protein L7. With this modified L7, the existence of the well-known complex between L7 and ribosomal protein L10 could be demonstrated by the photochemical procedure. By the chemical procedure, the presence of dimers of L7 in solution could be shown.


Interaction of alpha-chymotrypsin with the fluorescent probe 1-anilinonaphthalene-8-sulfonate in solution.

The binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonate (Ans) to alpha-chymotrypsin (alpha-CHT) at pH 3.6 is accompanied by a dramatic enhancement of Ans fluorescence and a shift of the emission maximum to shorter wavelengths. Our study reveals that one Ans molecule binds to alpha-CHT at a site different from either the active site of alpha-CHT or the 2-p-toluidinylnapthalene-6-sulfonate binding site. the binding constant of Ans is about the same (10(4) M-1) at pH 3.6 and 6.4. Nanosecond fluorescence depolarization data indicate that Ans is rigidly bound to alpha-CHT. The fluorescence enhancement due to binding of Ans to alpha-CHT at low pH could be due to binding either to a hydrophobic site or to a site where local dipoles do not relax during the excited-state lifetime of Ans. As the pH is increased, fluorescence intensity of the Ans-alpha-CHT complex decreases appreciably; and the emission maximum shifts to longer wavelengths. The fluorescence decay curves exhibit a corresponding sensitivity to pH. The pH effect on the fluorescence of Ans-alpha-CHT can be interpreted in terms of a pH-dependent equilibrium between alpha-CHT conformers differing in the degree of mobility of polar residues and water molecules at the Ans binding site or structural changes in the Ans binding site.