Ferrokinetics and erythropoiesis in man: an evaluation of ferrokinetic measurements.

The plasma iron clearance half-time and plasma iron turnover have usually been interpreted as measures of total erythroid activity and the red-cell utilization of 59Fe has been equated with effective red-cell production. Erythrocyte iron turnover has sometimes been calculated as the product of plasma iron turnover and percentage utilization. We have assessed these measurements as estimates of erythroid activity by comparing them with total marrow iron turnover and red-cell iron turnover determined by the method of Ricketts et al (1975) in 10 normal subjects and 51 patients with an uncomplicated haematological disorder. The results show that the plasma iron clearance half-time does not reflect erythroid activity and that plasma iron turnover can be particularly misleading in patients with reduced marrow activity. Red-cell utilization and erythrocyte iron turnover give a distorted reflection of effective erythropoiesis except in patients with erythroid hypoplasia. Marrow iron turnover and red-cell iron turnover provide more realistic and generally applicable assessments of the degree and effectiveness of erythroid activity.


Aplastic anaemia. Histological investigation of methacrylate embedded bone marrow biopsy specimens; correlation with survival after conventional treatment in 15 adult patients.

Aplastic anaemia is a rare disease with a poor prognosis: 40% of the patients die within 6 months after diagnosis. The clinical condition and laboratory data show only a poor correlation with the chance of survival for longer than 6 months. Conventional bone marrow biopsy investigations did not have any predictive value, but methyl-methacrylate embedded biopsy material from 15 patients given only supportive care showed histological features considered important for the assessment of the prognosis. In 3 micron methacrylate sections non-myeloid cells, like lymphocytes, could be readily distinguished from erythropoietic cells. Three types of inflammatory infiltrate were easily distinguished according to amount and distribution. Seven of the eight patients with marked infiltration (Grade III) died within 6 months after biopsy, but none of the patients with a sparse infiltrate (Grade I), including five patients in remission. In six of 10 patients, who were not in remission, histological data permitted correction of the clinically estimated prognosis.


Factor-VIII activity and antigen, platelet count and biochemical changes after adrenoceptor stimulation.

Adrenaline, isoprenaline and salbutamol were administered by intravenous infusion to human subjects. Isoprenaline was covered with practolol in an attempt to reduce the unpleasantness of the circulatory effects. Changes were recorded in pulse rate and blood pressure, and in blood levels of factors V, VIII, X, XI, and XII, platelet count, lactate, pyruvate, potassium and free fatty acids. Factor VIII was studied by clotting assays, by reactions with two rabbit antisera and two human antibodies, and by desulphated agarose chromatography. At the rate at which they were adiminstered, all three drugs increased the pulse rate by 20-40 beats/min. Factor VIII rose c. 2.5 X with adrenaline but only c. 1.5 X with isoprenaline and salbutamol; but other clotting factors did not alter. Chromatography provided no evidence of a change in the size of the molecule carrying factor-VIII clotting activity. The rate of clearance of the heightened plasma activity could not be shown to differ from that of "ordinary" factor VIII infused into haemophiliacs. The platelet count rose after adrenaline, fell after salbutamol and did not change significantly after isoprenaline. Among the biochemical responses, the only significant difference between the drugs was that lactate rose after adrenaline and salbutamol but did not change after isoprenaline. The rise in factor-VIII clotting activity after adrenaline is considered to represent a real increase in blood concentration, presumably by release of additional factor VIII from stores. The evidence suggests that this could be classified as a beta2 effect; and that the quantity which can be released is unrelated to the current plasma level. The rise in platelet count produced by adrenaline may be the resultant of an alpha-mediated rise due to contraction of the exchangeable splenic pool and a beta2-mediated fall, the alpha effect predominating.


Congenital dyserythropoietic anaemia, types I and II: aberrant pattern of erythrocyte membrane proteins in CDA II, as revealed by two-dimensional polyacrylamide gel electrophoresis.

The protein composition of the erythrocyte membrane of two patients with CDA I and four patients with CDA II has been investigated by two-dimensional polyacrylamide gel electrophoresis. The two-dimensional patterns of erythrocyte membrane proteins of the patients with CDA were compared with the well-established pattern of normal individuals. No alterations could be detected in the patients with CDA I, but a striking deviation from normal was observed in all patients with CDA II. The major aberrations of the erythrocyte membrane proteins in CDA II are the lack of protein B1.1, the drastically increased concentration of protein B1.4 and the presence of a new component, C2'. These results are discussed in relation to the known morphological and immunological abnormalities of the erythrocyte membrane in CDA II.


Effect of hypertransfusion on granulopoiesis in bone marrow depression: studies in the irradiated mouse.

The response of granulopoietic activity in bone marrow depression to a reduction in the demand for erythropoiesis has been examined by means of hypertransfusing C-57B mice which had been exposed to sublethal whole body irradiation. Multiple intraperitoneal injections of 0.5 ml packed red cells from irradiated donors were employed to maintain the haematocrit sufficiently above the normal range to produce significant suppression of erythropoietic activity for the duration of the bone marrow depression. This was associated with elevation above control values of 32--102% in the blood granulocyte count, 22--78% in total cells of the granulocytic series per femur, and up to 44% in total agar colony forming units per femur. Restoration of essentially normal values occurred 13 d after irradiation in contrast to 17--18 d in controls. Single transfusions which produced less suppression of erythroblast numbers per femur resulted in an intermediate degree of improvement in these parameters. Such changes in the granulocyte compartment indicate improved granulopoietic capacity in the hypertransfused group. It is suggested that this effect reflects increased production of granulocyte progenitors due to the reduction in competing demands on the compromised multipotential stem cell compartment for progenitors of the erythroid series. The findings raise the possibility that hypertransfusion might be capable of producing a beneficial effect on granulopoiesis in human bone marrow depression.


Biphasic thrombopoietic response to severe hypobaric hypoxia.

Thrombopoiesis has been studied during and after an 11 d exposure to discontinuous hypobaric hypoxia. Exposure of rats to 0.4 atmospheres for 16--17 h daily initially caused an increase in platelet count which reached a peak of 1.5 times baseline on days 4 and 5. This thrombocytosis was followed by a decrease in platelets to a nadir of 50--60% of baseline on days 12 and 13. That thrombocytosis results from increased platelet production is supported by increased [35S]sulphate incorporation into platelets and increased megakaryocyte size and turnover. The thrombocytopenia with continued hypoxia seems to result from decreased platelet production since 51Cr-platelet survival was normal while megakaryocyte concentration was decreased to one-half that of untreated controls. These observations suggest that differentiation of precursors into megakaryocytes was decreased during the thrombocytopenic period, although the fewer remaining megakaryocytes appeared stimulated because of their larger size and increased [3H]thymidine labelling. Thus, hypobaric hypoxia had a biphasic effect on thrombopoiesis with increased platelet production in the first few days of exposure followed by subnormal production.


Acquired dysfibrinogenaemia in acute and chronic liver disease.

Plasma from patients with both acute and chronic liver disease has been examined for evidence of acquired dysfibrinogenaemia, using electrophoretic methods and coagulation tests. An examination of isolated fibrins upon SDS polyacryamide gel electrophoresis failed to demonstrate any molecular or structural defect associated with the polypeptide chains of the patients' fibrinogen or fibrinogen derivatives produced by thrombin or plasmin. However, purified fibrin monomers isolated from plasma using both Reptilase and thrombin exhibited delayed polymerization rates and the occurrence of acquired dysfibrinogenaemia in liver disease is therefore confirmed.


Mucosal iron-binding proteins in mice with X-linked anaemia.

The mouse with X-linked anaemia [sla] has a defect in iron absorption which can be temporarily reversee by feeding a low iron diet. Duodenal non-haem iron was significantly higher in the sla than in the normal mouse on an iron supplemented diet but non-haem iron was reduced to minute amounts when the mice were fed a low iron diet. Gel chromatography on Sephadex G-200 of th partial-free supernatant of pooled mucosal homogenates revealed the presence of three proteins binding 59Fe. Fraction I [mol wt 450 000] resembled ferritin and was present in both normal and sla mice fed an iron supplemented diet. Fraction II [mol wt 78 ooo] eluted in a similar position to transferrin and was evident in both normal and sla mice fed an iron deficient diet. Fraction III [mol wt less than 15 000] contained equivalent amounts of radioiron in normal and sla mice fed the iron deficient diet, whereas this fraction contained less radioactivity in sla animals in two of three experiments in which the animals were fed an iron supplemented diet. The iron transport defect in sla mice does not appear to reside in the iron-binding proteins in the supernatant fraction of the intestinal mucosa and the cause of the defect in iron absorption remains to be elucidated.


Splenic red cell pooling in hairy cell leukaemia.

The splenic red cell volume has been measured directly by an isotope method with quantitative scanning in 10 patients with leukaemic reticuloendotheliosis (hairy cell leukaemia). The volume ranged between 211 and 726 ml (mean 410 ml, SD 158) and this constituted 15--48% (mean 28.1%, SD 9.5) of the total circulating red cell volume. This is an exceptionally large pool when compared with that found in myeloproliferative and lymphoproliferative disorders with the same degree of splenomegaly. It is consistent with the histological features which show marked red cell accumulation in the splenic cord areas. The red cell pooling in the spleen thus appears to be a significant factor in the anaemia and there was fairly good correlation between the percentage of improvement in the anaemia and the percentage of red cell volume contained in the spleen. By direct measurement of the splenic red cell pool, it is possible to predict the extent to which splenectomy will benefit the anaemia and this may also provide an indirect measure of the extent of bone marrow dysfunction in the causation of the anaemia.


Evidence for rapid loss of newly synthesized haemoglobin S molecules in sickle cell anaemia and sickle cell trait.

The present study indicates that newly completed haemoglobin S molecules rather than free betas-chains are preferentially bound to the reticulocyte stroma of individuals with sickle cell trait and sickle cell anaemia. Reticulocytes from indivdiuals with HbAA, AS and SS were incubated with [3H]eucine from 1.25 min to 120 min. Unlike the stroma-free haemolysates, the stroma of all individuals contained an excess of labelled beta-chains relative to alpha-chains after short incubation times. In haemoglobin AA and AS individuals, the stromal betaA radioactivity was 1--2% of the total cellular betaA radioactivity. In haemoglobin AS and SS individuals, the stromal betaS radioactivity was 3--5% and 10--20% of the total cellular betaS radioactivity, respectively. All of the stroma beta-chain radioactivity was associated with completed haemoglobin molecules. Because of the unlabelled free alpha-chain pool found in reticulocytes, after short incubation times newly completed haemoglobin molecules have predominantly labelled beta-chains and unlabelled alpha-chains. These findings suggest that part of the discrepancy between the stroma and stroma-free haemolysate alpha/beta radioactivities seen in HbAS and HbSS individuals may result from normal labelling kinetics. A pulse chase experiment performed on an individual with HbSS revealed that comleted HbS molecules, in addition to being associated with the stroma, were lost from the cell.


Abnormal folate polyglutamate ratios in untreated pernicious anaemia corrected by therapy.

The relative amounts of folate compounds having 1--3--, 4--, 5-- and 6--glutamic acid residues in the molecule were measured in red blood cells. The results were expressed in relation to methyltetrahydrofolate pentaglutamate (5-glutamic acid residues) which is the analogue present in highest concentration. When the concentration of pentaglutamate is given a value of 100 the relative concentration of folates with 1--3--, 4--, 5-- and 6--glutamic acid residues in control subjects are 48, 48, 100 and 50 respectively. In folate deficiency there is a fall in the relative amount of the short chain glutamic acid chain compounds in addition to having an overall fall in the amount of folate present, the values for 1--3, 4, 5 and 6 chain folates being 16, 14, 100 and 39. By contrast, in untreated pernicious anaemia there is an accumulation of the short chain folates as well as an overall fall in polyglutamate concentration and the relative concentrations were 72, 44, 100 and 30. Folate polyglutamate appears to be the active coenzyme and the reltive amounts of glutamic acid residues may serve to regulate the rate of enzyme activity. In pernicious anaemia the amount of enzyme is reduced and on this hypothesis the regulatory function impaired.


Clinical course and survival after elective splenectomy in 19 patients with primary myelofibrosis.

Nineteen patients with primary myelofibrosis and myeloid metaplasia (MMM), who fulfilled well-defined criteria, underwent elective splenectomy as soon as the diagnosis was confirmed. Nine patients developed postoperative complications such as intra-abdominal bleeding (three), rupture of the inferior vena cava (one), ascites (two), Australia antigen-positive hepatitis (three), mesenteric artery thrombosis (one) and pneumonia (one). One patient died within a month of the operation due to secondary intra-abdominal infection. The mean age of the patients at splenectomy was 56 years and the mean duration of their disease 2.4 years. The median actuarial survival after operation was 51 months. Although the series of patients is small, it seems that splenectomy did not have an adverse effect on life expectancy. The haematological status and the quality of life improved after splenectomy in 17 of 19 patients. The results warrant a further trial with elective splenectomy in an early stage of MMM.


The conformation of fibrinogen and fibrin: an electron microscopy study.

A mica adsorption technique was used to prepare fibrinogen and early fibrin polymers for examination in the transmission electron microscope. Globular particles 10 +/- 3 nm in diameter were observed and a model for the process of aggregation of these globules into fibrous units is proposed. A comparison is made between the results obtained by the mica adsorption technique and those obtained by conventional preparation techniques involving fixation and staining.


Specific interaction of HLA antibodies (eluates) with washed platelets.

The mechanism of complement-independent action of HLA-A2 antibodies (eluates) on washed platelets was investigated. HLA-specific alteration was confirmed by serological (platelet micro-complement fixation), morphological (platelet spreading) and functional parameters (platelet aggregation, inhibition of collagen-induced platelet aggregation, [14C]serotonin release). In the presence of fibrinogen and calcium ions, HLA antibodies induced instantaneous platelet aggregation and release. Although no morphological (spreading) and functional changes (collagen-induded aggregation) were seen, these platelets did not aggregate or release when fibrinogen was subsequently added. When platelets--in the presence of fibrinogen--were incubated with antibody concentrations too low to induce platelet aggregation or release, specific reduction of platelet reactivity was observed by subsequent collagen aggregation. HLA-specific action of antibodies on washed platelets was inhibited by apyrase and acetyl-salicylic acid, indicating an active participation of platelets in HLA antibody-induced platelet alteration.


A circadian variation of melphalan (L-phenylalanine nitrogen mustard) toxicity to murine bone marrow: relevance to cancer treatment protocols.

The toxicity of Melphalan to murine bone marrow was assessed by automated Coulter counts of femoral marrow nucleated cells. A significant dose-response slope (p less than 0.001) was shown and also a significant variation along the 24 h scale. With food available ad libitum and light from 06.00 to 18.00 hours, the minimum marrow depression occurred around 16.00 hours. Extrapolating these findings to human chemotherapy it would appear that prescription of the drug during the early part of the activity span (usually breakfast) will minimize marrow depression. This differs from a human study in which Melphalan was given at 'bedtime' as an adjuvant to mastectomy in breast carcinoma and in which there was leucopenia at some stage of treatment in all 103 patients. It is recommended that in future cancer therapy protocols test for circadian variability of white cell depression by varying treatment times systematically along the 24 h scale.


Management of hyperprolactinaemic amenorrhoea.

Results of treatment of 52 patients with amenorrhoea associated with hyperprolactinaemia are presented. All patients had a detailed radiological examination of the pituitary fossa, including lateral tomography in every patient and air encephalography in those in whom a pituitary tumour was suspected. There were 17 patients with untreated pituitary tumours, 5 patients with previously treated pituitary tumours and persisting hyperprolactinaemia, and 30 patients with normal pituitary radiology. Patients with pituitary tumours were treated either by transsphenoidal or transfrontal surgical extirpation of the tumour, followed, if necessary, by external irradiation and/or bromocriptine, Four patients were treated with external irradiation as primary therapy, and three patients who did not wish to conceive were treated with bromocriptine as primary therapy. Patients with normal radiological appearances were treated with bromocriptine as primary treatment. Ovulatory menstrual cycles developed in 42 patients and there were 19 pregnancies. Those ovulating but not conceiving had adequate nonendocrine factors to account for the disparity. Failure of response was seen in 10 patients and was due to inadequate fall of prolactin in response to surgery (2 patients), external irradiation (3 patients) and bromocriptine (1 patient), and gonadotrophin deficiency which developed after surgery in 3 patients but was present pre-operatively in 1. The relative merits of treatment by surgery, external irradiation and bromocriptine are discussed and a policy of treatment outlined.


Plasma levels of pregnancy-specific beta 1-glycoprotein in complicated pregnancies.

Maternal plasma levels of the trophoblast product, pregnancy-specific beta 1-glycoprotein (PS beta G), have been measured both in normal pregnancies and in pregnancies complicated by pre-eclampsia and/or fetal growth retardation. PS beta G levels correlate significantly with placental and fetal weight and fall below the normal range in about 60 per cent of all patients with fetal growth retardation. PS beta G measurements appear to give a considerably better indication of fetal size than measurements of either human placental lactogen (HPL) or plasma oestriol.


Vaginal administration of a single dose of 16, 16 dimethyl prostaglandin E2 p-benzaldehyde semicarbazone ester for pre-operative cervical dilatation in first trimester nulliparae.

A single pessary containing 0-5 mg 16, 16 dimethyl prostaglandin E2 p-benzaldehyde semicarbazone ester was used for cervical dilatation prior to vacuum aspiration in 124 first trimester nulliparae. Five hours after prostaglandin administration the cervix had dilated to 8 mm or more in 87 patients (70 per cent). The uterus was evacuated in these patients without mechanical dilatation of the cervix. In the remaining 37 patients the cervix had become soft and dilated 5 to 7 mm and further mechanical dilatation could be carried out easily. Side effects were minor and included vomiting in two patients, diarrhoea in one and transient pyrexia in two patients. There were no complications and no damage to the cervix or uterus during evacuation.


Fetal heart rate monitoring during caesarean section.

The fetal heart rate was monitored during 30 deliveries by Caesarean section. General anaesthesia, but not epidural analgesia, caused a loss of beat-to-beat variation, but appeared to have no other adverse effects on fetal heart rate. The loss of beat-to-beat variation occurred after intravenous thiopentone and was most marked when atropine was used as premedication.


Treosulfan (dihydroxybusulphan) in the management of ovarian carcinoma.

Of 36 patients with ovarian carcinoma treated with treosulfan (dihydroxybusulphan) 30-5 per cent had a complete response for a mean of 19-0 months and 33-3 per cent had a partial response. Treosulfan may have a role in the management of ascites associated with ovarian neoplasm.


Ileo-uterine fistula as a complication of myomectomy. Case report.

A patient is described who developed an ileo-uterine fistula after myomectomy. The fistula caused no symptoms and was discovered at hysterosalpingography for infertility.


Intraocular gentamicin as intraoperative prophylaxis in South India eye camps.

The incidence of bacterial endophthalmitis has been 3-6% after cataract extraction in the eye camps of South India from 1961-75. During this time 50 791 cases were operated using systemic and topical chloramphenicol prophylaxis. This study evaluated the prophylactic intracameral injection of 50 microng of gentamicin in eye camp cataract extractions. Only 6 of 1626 patients treated with intracameral gentamicin at operation developed bacterial endophthalmitis, an incidence of 0-37%.


The association of rubeosis iridis with endothelialisation of the anterior chamber: report of a clinical case with histopathological review of 16 additional cases.

A patient is reported in whom the clinical observation of extension of the corneal endothelium and Descemet's membrane in association with rubeosis iridis was confirmed by histopathological examination. In a study of eyes which had been removed and demonstrated rubeosis iridis and secondary glaucoma in 16 additional eyes revealed extension of the corneal endothelium and Descemet's membrane on to the anterior iris surface. In all instances the transition occurred at the pseudoangle formed by the attachment of peripheral anterior synechiae. The suggestion is made that the fibrovascular membrane of rubeosis iridis is the stimulus which excites the extension of the endothelium on to the iris surface. The association between rubeosis iridis and extension of the corneal endothelium and Descemet's membrane on to the iris has not been previously appreciated.


Preliminary report on effect of retinal panphotocoagulation on rubeosis iridis and neovascular glaucoma.

Eight eyes with central retinal vein occlusion one eye with old central retinal artery occlusion complicated by rubeosis iridis or increased intraocular pressure, or both; and one eye with diabetic retinopathy and rubeosis iridis were treated by retinal panphotocoagulation. Vision did not improve but intraocular pressure was lower and iris neovascularisation regressed in most cases, supporting the hypothesis that retinal hypoxia is a cause of iris neovascularisation and suggesting that retinal panphotocoagulation has a potential prophylactic and therapeutic role in rubeosis iridis and early neovascular glaucoma.


Nitrogen retention in the pig.

I. Published results have been used to study the relationships between nitrogen retention (NR), body-weight (W) and N intake in the pig. 2. The general decrease in maximal NR (g/d per kg W0.75) with increasing W (kg) was curvilinear for values of W from 1-5 to 45: NR = 3.324--0.098 W + 0.001 W2; and rectilinear for values of W from 45 to 165: NR = 1.252--0.006 W. Values for protein requirements derived from these equations agreed closely with published estimates. 3. The slopes of the curves for NR (g/d per kg W0.75) v. N intake (g/d) decreased as W (kg) increased from about 2.5 to 190. After extrapolation to a proposed common intercept on the NR axis of--150 mg N/d per kg W0.75, regression analysis of the intercepts of these curves on the N-intake axis v. W gave an estimate of N requirements for maintenance of 246 + 19 mg/d per kg W0.75. 4. The results also indicated that at low N intakes net protein utilization (N retention + total obligatory N losses divided by N intake) was essentially independent of W, whereas the gross efficiency of N utilization (NR divided by N intake) was influenced by both W and N intake.


Fat mobilization in Pietrain and Large White pigs.

I. Two experiments were done with Pietrain and Large White pigs (about 50 kg body-weight) to determine whether fat mobilization is enhanced in the stress-sensitive Pietrains (which also produce pale, soft, exudative (PSE) meat). 2. In Expt I, plasma glucose and insulin concentrations and free fatty acid (FFA) concentrations and composition were measured in five Pietrains and five Large Whites after feeding, during an infusion of norepinephrine (2.5 microng/kg body-weight per min), 16 and 21 h after the withdrawal of food and following insulin administration (0.3 IU/kg body-weight). The entry rate of oleic acid was measured 4 h after feeding. Body composition and longissimus dorsi pH measurements were made on killing the pigs 4 d after the experiment. 3. After feeding, the Pietrans tended to have lower concentrations of glucose and insulin in plasma compared with the Large Whites, and higher concentrations of FFA. The Pietrans also had a faster entry rate of oleic acid into body tissues. In fasting (16 h) these differences in hormone and metabolite concentrations were also present and relationships between them and body development could be detected. Pietrains had higher concentrations of FFA and lower concentrations of insulin than Large Whites at a particular stage of the development of fat and muscle (subcutaneous fat weight divided by longissimus muscle weight). 4. The fatty acid composition of FFA resembled that of the backfat triglycerides in fasting and during norepinephrine infusion but not in the fed state. In particular the proportion of fatty acid 18:I was low in the fed pigs and that of 18:2 was high. The contrast in FFA composition between the fed and stimulated state was greater in the Large Whites. 5. The Pietrains were less sensitive to the antilipolytic action of insulin. Glucose and FFA concentrations were similar in both breeds during the norepinephrine infusion although insulin concentrations were increased to a larger extent in Large Whites at the termination of the infusion. 6. In Expt 2, anaesthetized Pietrain and Large White pigs were given constant infusions of norepinephrine alone (2.5 microng/kg body-weight per min) and norepinephrine + propranolol (2, 10 and 20 microng/kg body-weight per min) or phentolamine (2 and 10 microng/kg body-weight per min). The aim was to determine whether the breeds differed in their sensitivity to norepinephrine when conscious responses to the hormone were prevented. 7. When norepinephrine alone was infused, fat mobilization was greater in the Pietrain pigs and glucose concentrations were greater in the Large Whites. Propranolol markedly reduced the lipolytic action of norepinephrine, particularly in the Pietrain pigs studied, and phentolamine reduced its glycogenolytic action, particularly in the Large White pig. 8...


Nitrogen retention in rats fed on diets enriched with arginine and glycine. 1. Improved N retention after trauma.

1. Nitrogen retention was measured in adult rats (250-350 g) subjected to the trauma of hind-leg fracture and given diets with or without arginine plus glycine supplementation. Observations were also recorded on excretion of creatine, creatinine, allantoin, and orotic acid. Liver and skeletal muscle transaminase activities were also determined. 2. When traumatized rats weighing approximately 250 g were given a diet with 200 g casein/kg, supplemented with 20 g arginine and 10 g glycine/kg (EC diet) or a casein diet made isonitrogenous with the EC diet by addition of aspartic acid (C diet), a 60-70% increase in N retention was observed for the first 5 d post-injury for animals consuming the EC diet. A soya-bean (S) diet, isonitrogenous to the diet containing 20% casein, supplemented with arginine and glycine was as effective as the EC diet in promoting significantly better N retention of traumatized rats (350 g) in comparison to rats given the C diet. 3. When the dietary casein content was reduced to 100 g/kg, supplements of 10 g arginine and 5 g glycine or 20 g arginine and 10 g glycine/kg did not improve N retention. It is suggested that both protein quality and protein quantity are important following injury. 4. An increased excretion of creatine was observed in traumatized rats given the high-protein diets supplemented with arginine and glycine. No consistent changes were noted for urine creatinine. 5. 5. Urine allantoin levels remained stable after leg-fracture in rats consuming either the C or EC diets. Differences in the levels of urine orotic acid were found during both the pre- and post-injury periods in rats given the C, EC or S diets. 6. The mechanisms responsible for the improved N retention of traumatized rats consuming the high-protein diets with supplements of arginine and glycine may be related to the role of arginine both as a constituent of muscle tissue and as an intermediate in the urea cycle. 7. In traumatized rats fed the C or EC diets, liver transaminase activity increased whereas the transaminase activity in skeletal muscle decreased. These results support the recent concept that the increased excretion of N following injury arises from diminished reutilization of amino acids by muscle tissue without an acute increase in the rate of muscle catabolism.


Effect of diet on the metabolism of labelled tocopherol in sheep.

1. Eighteen crossbred wethers were allotted at random (six per treatment) to each of the following diets: (1) maize-silage; (2) grass-silage; and (3) chopped hay. After 6 months a single oral dose of D-alpha-[5-Me-3H]tocopherol was given to each sheep on these three treatments. 2. Blood plasma, rumen liquor and urine radioactivity were measured for 4 d and, at the end of this period, the animals were killed and tissue distribution of 3H was determined. 3. Maize-silage generally contained less alpha-tocopherol than grass-silage or hay. Tissue uptake of 3H was greater on maize-silage than other diets. 4. In muscle, spleen and liver, tocopherol concentrations were lower in the maize-silage than the grass-silage fed animals. 5. A tendency to higher uptake of radioactivity was recorded at all times in the plasma and its lipid extract of sheep fed on maize-silage than those fed on grass-silage or hay. 6. Urine clearance of radioactivity tended to be higher in animals fed on the maize-silage than those fed on grass-silage or hay. This difference of magnitude in urinary excretion was probably related to the rate of metabolism of the ingested radiotocopherol.


Concurrent studies of the flow of digesta in the duodenum and of exocrine pancreatic secretion in calves. 5. The effect of giving milk once and twice daily, and of weaning.

1. The effects of giving milk once or twice daily (Expt 1) and of weaning (Expt 2) on the flow rate of digesta through the duodenum and on pancreatic secretion were studied in four and two Ayrshire calves respectively. The calves were prepared with duodenal re-entrant and pancreatic sac cannulas. 2. In Expt 1, when whole milk was offered ad lib. once daily, the calves ingested 141 g/kg live weight (46 g dry matter (DM)/kg live weight 0.75) at a single meal. The subsequent pattern of flow and total recovery of polyethylene glycol from the duodenum suggested that none of the ingested milk passed into the rumeno-reticulum. As the total daily quantity of milk ingested when the calves were fed once and twice daily was similar, it was concluded that abomasal distension is unlikely to be the sole factor limiting milk intake in the preruminant calf. 3. With twice-daily feeding, there were no differences in the pattern or total flow of fluid, electrolytes, nitrogen or fat through the duodenum of the calves following the 09.00 and 21.00 hours meals. The pattern of flow of duodenal and pancreatic fluids and the concentration of electrolytes, N and fat were markedly different when the calves were fed once or twice daily. The patterns of flow of fluid and the concentration of electrolytes in the duodenal digesta reflected the frequency of feeding and the size of the meal and the consequent balance between feed and endogenous components of the digesta. The quantity of the apparent endogenous secretion and pancreatic secretion was markedly less when the calves were fed once daily. 4. In Expt 2, the two Ayshire calves were given whole milk twice daily (diet MM), whole milk once daily with concentrates (diet MC), concentrates alone (diet CC) or dried grass alone (diet DG). The calves consumed 46, 49, 45 and 51 g DM/kg live weight 0.75 when given diets MM, MC, CC and DG respectively. 5. The twice-daily fluctuations in the flow and concentration of fluid, electrolytes, N and fat in the duodenal digesta and the pancreatic fluid observed when diet MM was given were replaced by relatively constant flow rates and composition when diet CC or DG was given. 6. Over the 24 h experimental period 97, 70, 50 and 58% of the DM and 112, 98, 99 and 84% of the N in the feed passed through the duodenum of calves when given diets MM, MC, CC and DG respectively. 7. When dry food was given, the rate of pancreatic fluid secretion  was markedly lower (11.3 and 13.5 ml/kg live weight for diets CC and DG respectively) than when diet MM (19.7 ml/kg live weight) was given.


Megadosage of ascorbic acid in an Antarctic expedition.

1. No difference in health was observed between men on megadosage of ascorbic acid and controls during the year of an Antarctic expedition. 2. All men appeared to have a satisfactory intake of ascorbic acid throughout the year. 3. There was a statistically significant decrease in excretion of ascorbic acid by men on megadosage over the year, and by the controls. 4. The decline in excretion by the control group may be explained by dietary change, but the decline in those on megadosage may be due to altered handling of ascorbic acid by the body. 5. No complications due to megadosage of ascorbic acid were observed.


Effect of environmental temperature and food intake on the distribution of fat in growing hairless mice.

1. The fat content of the skin and of the skin-free carcass was measured in young, growing hairless mice about 4-8 weeks old kept at either 22 degrees (cool environment, i.e. below their critical temperature) or at 33 degrees (warm environment; i.e. within their thermoneutral range). The food intake of groups of the mice reared at each temperature was restricted to between 77 and 89% of that of a litter-mate fed ab lib. 2. In all the mice, whether fed ad lib. or on a restricted intake, those reared in the warm environment contained about 1-5 times as much fat as those reared in the cool environment. At both temperatures and feeding levels, approximately 25% of the total body fat was present in the skin. 3. Each mouse reared at 33 degrees on an ad lib. regimen reached about the same plateau weight in the same period as its litter-mate reared at 22 degrees. However its food intake during this period of growth was only about half that of the mouse reared at the lower temperature, so its food conversion ratio (dry food intake:body-weight gain) was twice as efficient. 4. It is concluded that neither the growth rate nor the distribution of fat within the body of the growing mouse can be influenced by rearing the animals at these different environmental temperatures. Rearing at the higher temperature, however, both decreases food intake and also promotes deposition of more fat within the body.


Scope and mechanism of carbohydrase action: stereospecific hydration of D-glucal catalyzed by alpha- and beta-glucosidase.

A unique demonstration is presented of the capacity of glycosidases to create anomeric configuration de novo. Purifed Candida tropicalis alpha-glucosidase and sweet almond beta-glucosidase have been found to attack the same substrate, D-glucal, and to convert this unusual glycosyl substrate (which lacks alpha or beta anomeric configuration) to 2-deoxy-alpha-(or beta-) D-glucose, respectively. The stereospecificity of the hydration reaction catalyzed by each enzyme in D2O was revealed by the use of high-resolution (270 MHz) 1H magnetic resonance spectroscopy. The alpha-glucosidase caused a specific axial protonation (deuteration) of D-glucal at C-2, and formation of 2-deoxy-alpha-D-[2(a)-2H]glucose. The beta-glucosidase catalyzed an oppositely directed axial protonation at C-2 and formation of 2-deoxy-beta-D-[2(e)-2H]glucose. These results are not accounted for by the generally accepted mechanisms of carbohydrase action derived from studies with glycosidically linked substrates alone. D-Glucal apparently binds to the enzymes with essentially the same overall orientation as the D-glucosyl moiety of glycosidically linked substrates (with the double bond of D-glucal lying essentially in the plane of the similarly bound D-glucosyl group). Thus, the alpha-glucosidase evidently protonates D-glucal from above the double bond and alpha-D-glucosidic substrates from below the glycosidic oxygen; beta-glucosidase apparently protonates D-glucal from below the double bond and beta-D-glucosides from above the glycosidic oxygen. A detailed mechanism is proposed for the hydration of D-glucal by each enzyme, involving an incipient glycosyl carbonium ion and assuming the presence at the active site of two carboxyl groups arranged to account for catalysis of glycosylations from glycosidically linked substrates. That D-glucal serves as a glycosyl substrate for these enzymes strongly supports the concept that glycosidases and glycosyltransferases are catalysts of glycosylation (i.e., glycosylases), since this concept does not make the usual assumption that carbohydrases are restricted to acting on substrates having a glycosidic bond and either alph- or beta-anomeric configuration.


Deoxyribonucleic acid chain growth and organization of replicating units in HeLa cells.

A method for studying DNA chain growth and chromosomal organization of replicons in HeLa cells has been developed. DNA replication is initiated with bromodeoxyuridine followed by pulse labeling of active replicons with [3H]thymidine and growth of chains for finite intervals in unlabeled thymidine. Photolysis of the bromodeoxyuridine-DNA leader with 313-nm light releases the newly replicated chains that are then analyzed by sedimentation in alkaline sucrose gradients. This method of analysis provides data on the rate of chain growth, the bidirectionality of replication, and the distribution of the active replicons at specific intervals in the S period. Applying this method to cells caused to synthesize DNA at a lowered temperature (27 degrees C) or with protein synthesis restricted by cycloheximide revealed that the immediate reduction in the rate of DNA replication in both instances was due to a decreased rate of chain growth without derangement of the overall process.


Optical activity and conformation of cobra neurotoxin.

Cobra neurotoxin from Formosan cobra (Naja naja atra) venom is a compact globular protein having an intrinsic viscosity of 4.5 mL/g. The protein is stable in 7.5 M urea but can be denatured in 4.1 M guanidine hydrochloride or at elevated temperature (above 70 degrees C). Its conformation remains virtually the same in solvents of lower polarity than water such as 1,2-ethanediol or a mixed solvent of 1-propanol-1,2-ethanediol-water (5:1:1 by volume). The circular dichroism spectrum is "atypical" in water in that the peptide chromophores show a small negative circular dichroic (CD) band at 215 nm, a large positive one at 199 nm, and another large negative one below 190 nm. The CD pattern resembles to some extent that of a beta form but differs in both positions and magnitudes from the latter. It agrees qualitatively with the theoretical calculations of the reverse beta bends, suggesting that cobra toxin contains a considerable amount of beta turns and possibly a mixture of beta form and beta turns.


Evidence for topographical analogy between methionine-enkephalin and morphine derivatives.

Analogues of the endogenous opiate-receptor ligand [5-methionine]enkephalin (H-Tyr-Gly-Gly-Phe-Met-OH) were designed and synthesized for the purpose of testing the proposed similarity in spatial structure between this peptide and morphine derivatives. In the bioassay (inhibition of electrically induced contractions of the mouse vas deferens) [1-O-methyltyrosine,5-methionine]enkephalin, [1-N-methyltyrosine,5-methionine]enkephalin, [4-tryptophan,5-methionine]enkephalin, and [5-methionine sulfoxide]enkephalin possess, respectively, 0.4, 21, 27, and 67% activity of [5-methionine]enkephalin. These morphinomimetic activities correlate well with the opiate receptor affinities determined by displacement of [3H]naloxone in a guinea pig brain membrane preparation. The effects of O-methylation of the tyrosine residue and N-methylation of the terminal amino group on biological activity and receptor affinity support the hypothesis that the latter two moieties in the peptide correspond to the phenol group and the tertiary nitrogen, respectively, in morphine. Determination of the efficiency of energy transfer from tyrosine in position 1 to tryptophan in position 4 in [4-tryptophan,5-methionine]enkephalin from both tyrosine fluorescence quenching and relative enhancement of tryptophan fluorescence by means of a modified procedure permitted the calculation of an average intramolecular tyrosine-tryptophan separation of 10.0 +/- 1.1 A. Inspection of CPK models showed excellent agreement between this value and both the intrafluorophore distance in the 4 leads to 1 and 5 leads to 2 hydrogen bonded betaI-bend models of [4-tryptophan,5-methionine]enkephalin (9-11 A) and the phenol-phenyl separation in the potent morphine derivative 7alpha-(1(R)-hydroxy-1-methyl-3-phenylpropyl)-6,14-endo-ethenotetrahydrooripavine (8-10.5 A). The ensemble of these findings suggests an analogous topography for [5-methionine]enkephalin and morphine-oripavine derivatives.


Association products and conformations of salt-dissociated and acid-extracted histones. A two-phase procedure for isolating salt-dissociated histones.

We present an extremely rapid and efficient method for the separation of salt-dissociated histones from DNA in which the macromolecular components of chicken erythrocyte chromatin are partitioned in a two-phase system of the water-soluble, nonionic polymers, poly(ethylene glycol) and dextran. We have compared the association products and conformations of salt-dissociated histones purified with the two-phase procedure and histones that had been extracted with 0.4 M H2SO4. In the gel chromatography system of D. R. vander Westhuyzen and C. von Holt (1971), FEBS Lett. 14, 333-337] the association products of salt-dissociated and acid-extracted histones are indistinguishable. Furthermore, the circular dichroism spectra of histones prepared with the two methods are identical within experimental error. These results indicate that histones extracted, with sulfuric acid can adopt conformations at least very similar to those of salt-dissociated preperties of total erythrocyte histones are the same in 2 M NaCl as those of these histones bound to DNA in chromatin in 1 mM Tris-Cl (pH 7.5). This result and the studies of Weintraub et al. [Weintraub, H., Palter, K., and Van Lente, F. (1975), Cell 6, 68-110] on the patterns of tryptic digest products of histones strongly suggest that in 2 M NaCl the histones exist in conformations very similar to their conformations when bound to DNA. The concept of native histone conformations is discussed in light of our results.


Investigation of the aggregation and activation of prothrombin using quasi-elastic light scattering.

The technique of quasi-elastic light scattering was used to measure the translational diffusion coefficient, D, of purified human prothrombin in buffered aqueous solutions and to monitor for the first time the fragmentation of this protein as it is converted to thrombin. The values of D20,w, measured at two different concentrations, are 4.72 X 10(-7) CM2/S at 2MG/CM3 and 4.51 X 10(-7) CM2/S at 5MG/CM3; the corresponding molecular weights (Mw of 92 000 and 120 000), obtained by combining sedimentation velocity measurements with the diffusion data, confirm the presence of molecular aggregates of prothrombin in these solutions. These results, as well as analysis of the intensity-intensity autocorrelation functions from two-component systems with various dimer conformations, indicated the presence of end-to-end dimers in these prothrombin solutions. The values obtained for D indicate a dimer weight fraction of 0.4 to 0.5 in the 2 mg/cm3 solution and 0.6 or greater in the 5 mg/cm3 solution. The fragmentation of prothrombin was monitored in a nonphysiologic activation system, containing taipan snake venom, dihexanoylphosphatidylcholine, and CaCl2. At a temperature of 15 degrees C, conversion to thrombin proceeded very slowly and was still incomplete after 90 h. A method for determing the percentage of converted prothrombin is an activated system containing aggregates from the average value of D and light scattering data is discussed.


Association-dissociation of transcarboxylase.

Transcarboxylase consists of a central subunit to which two sets of three subunits each are attached at opposite faces. Evidence obtained by various ultracentrifugal techniques has shown that there is an equilibrium between active forms of the enzyme with six, five, four, three, two, and one subunits attached to the central subunit. Since each attached subunit contains two biotins, the biotin content of these forms varies from 12 to 2. By reactive enzyme sedimentation at pH 5.5, it has been shown that the largest form of the enzyme (with 12 biotins and a molecular weight of 1.2 X 10(6) is active and that at pH 6.8 this form of the enzyme dissociates to an enzymatically active form containing three attached subunits. This result is in accord with previous observations (Wood, H.G., Chiao, J.P., and Poto, E.M.(1977), J Biol. Chem. 252, 1490; Wrigley, N.G., Chiao, J.P., and Wood, H.G. (1977), J Biol. Chem. 252, 1500) which showed that dissociation of three of the six attache subunits occurs preferentially from one face of the central subunit and that the remaining three attached subunits are quite firmly bound to the second face of the central subunit. Multiple peaks are obtained in sedimentation velocity experiments at 60 000 rpm because the rate of sedimentation outstrips the rate of equilibration of the different forms of transcarboxylase, whereas at 30 000 rpm a single peak of the multiple form of the enzyme is observed, since equilibration of the different forms keeps abreast of the sedimentation. The rate and extent of dissociation are increased by increase in temperature. It has been shown by centrifugation under oil that the association-dissociation of the large form of the enzymes with six, five, and four attached subunits is not affected by hydrostatic pressure. In contrast, the association-dissociation of the enzyme with three and two attached subunits appears to be affected by pressure. The accumulated results indicate that the native form of the enzyme in the cytosol of the cell consists of a series of enzymatically active forms with one to six attached subunits. The predominant forms in the cytosol will depend on the concentration of the constituent subunits, the pH, the concentration of ions (particularly divalent anions), and the temperature.


Identification of retinal isomers isolated from bacteriorhodopsin.

The purple membrane of Halobacterium halobium contains the protein bacteriorhodopsin which resembles the visual pigment, rhodopsin, in many aspects. The isomeric configurations of its chromophore, retinal, were studied by a combination of methylene chloride extraction and analysis by high-pressure liquid chromatography. The light-adapted form bR570LA yields solely all-trans-retinal, while the dark-adapted form of bacteriorhodopsin, bR560DA, yields a mixture of 13-cis and all-trans with a ratio of similar to 1;1. The photointermediate M412 in a membrane modified by ether at high NaCl concentration also yields an approximately 1:1 mixture of 13-cis-and all-trans-retinals, while a similar M405 species produced by illumination in 2 M guanidine hydrochloride at high pH yields mainly 13-cis-retinal. These results indicate that the photochemical cycle of bR570LA may involve an isomerization of the retinal chromophore from the all-trans to the 13-cis form.


Estrogen photoaffinity labels. 1. Chemical and radiochemical synthesis of hexestrol diazoketone and azide derivatives; photochemical studies in solution.

Two photosensitive estrogen derivatives, hexestrol diazoketopropyl ether (5) and hexestrol azide (8a), have been synthesized in radiolabeled form, and their photochemical behavior in solution has been studied. The radiolabeled compounds were prepared in good yields according to improved synthetic procedures; they are stable and were obtained with specific activities in the range of 50-100 Ci per mmol and radiochemical purities in excess of 95%. A simpler model system, phenyl diazoketoprophyl ether, was used to study the photochemical behavior of the diazoketopropyl ether group. Direct irradiation of this compound at 254 nm in methanol led to 33% insertion product (methoxyketone) and 67% Wolff rearrangement product (ester). Irradiation of [3H]hexestrol diazoketopropyl ether (5) in methanol gives mainly nonpolar photoproducts (presumed to be the methoxy ketone and ester); however, irradiation in aqueous medium leads to large amounts of free hexestrol (52%). Photolysis of hexestrol azide (8a) in either methanol or water gives the corresponding amine in low yield as the only identifiable photoproduct.


Estrogen photoaffinity labels. 2. Reversible binding and covalent attachment of photosensitive hexestrol derivatives to the uterine estrogen receptor.

The ability of two radiolabeled, photoreactive estrogen analogues, [3H]hexestrol diazoketopropyl ether ([3H]Hex-DKP) and [3H]hexestrol azide ([3H]Hex-N3), to covalently label the uterine estrogen receptor is studied. Lamb uterine estrogen receptor preparations that have been partially purified (ammonium sulfate precipitation, Sephadex G-200 chromatography) and disaggregated by limited trypsinization can be electrophoresed on polyacrylamide gels under conditions where binding activity is preserved. This electrophoretic procedure was used to fractionate the proteins labeled by the two photoreactive estrogen analogues. Prior to photolysis, peaks of radioactivity indicating estrogen specific binding of [3H]-Hex-N3 and [3H]Hex-DKP are evident on the gels, although dissociation of the latter compound is extensive. When preparations of uterine estrogen receptor that contain the photoreactive derivatives are irradiated and then electrophoresed, reversibly labeled proteins can be distinguished from irreversibly labeled ones (covalently bonded), by extraction of the individual gel slices with organic solvents. While no irreversible binding to receptor appears to result from irradiation with [3H]Hex-DKP, irradiation with [3H]Hex-N3 does covalently label the estrogen receptor. The receptor covalently labeled with [3H]Hex-N3 has the same electrophoretic mobility as the unlabeled receptor; the covalent labeling process is estrogen-site specific, and the efficiency of labeling (15-20%) is consistent with the inactivation efficiency of Hex-N3, previously measured by an indirect assay. This is the first example of the labeling of a steroid hormone receptor by photoaffinity labeling.


Conformational properties of purine-pyrimidine and pyrimidine-purine dinucleoside monophosphates.

The detailed conformational features and dynamics of heterodinucleoside monophosphates ApU, ApC, GpU, GpC, UpA, CpA, UpG, and CpG have been studied in aqueous solution by high field nuclear magnetic resonance (NMR) spectroscopy. Analysis of the resultant NMR parameters leads to a number of discernible trends throughout the series. Thus the ribose rings of the dimers exist as equilibrium mixtures of C(2')-endo(2E) in equilibrium C(3')-endo(3E) conformers with a proclivity for the 3E pucker in most cases; the C(4')-C(5') bonds of both nucleotidyl units show significant preference (74-96%) for a gg conformation and the dominant conformer (85-89%) about C(5')-O(5') is g'g'. Orientation about the C(3')-O(3') bond is coupled to the ribose conformational equilibrium and the system exists with a bias for the 3Eg- coupled conformation in which the H(3')-C(3')-O(3')-P dihedral angle occupies the narrow range of 33-35 degrees. Dimerization, on the average, causes about 10% increase in gg and g'g' populations and the g-domain becomes increasingly populated about the C(3')-O(3') bond. The ribose equilibrium 2E in equilibrium 3E shifts in favor of 3E upon dimerization, the effect being very conspicuous for the pu-py series (similar to 40 yields 60%) and less noticeable for the py-pu systems (similar to 47 yields 58%), clearly suggesting a correlation between sequence and ribose conformational equilibrium. The temperature and dimerization data for the heterodinucleoside monophosphates show that the transition 2E yields 3E is directly related to XCN changes induced by dimerization and stacking. Analysis of the ribose coupling data shows that the percentage populations of stacked species vary from dimer to dimer with GpC displaying a maximum of 45% stacked population and UpG about 10%. However, in general, the pu-py dimers show a higher preference (27-45%) for stacked conformations than py-pu dimers (10-25%). It is proposed that the pronounced deshielding of H(5') of the 5'-nucleotidyl units upon dimerization is associated with the presence of right-handed stacks (g-g-), whereas the chemical shift trends of H(5') and H(5') of 3'-nucleotidyl units are due to the presence of left-handed stacks (g+g+) in all the dimers. In pu-py dimers, the population of the g-g- species is found to be greater than that of g+g+. Also the population of g-g- stacks in pu-py dimers is generally greater than in their corresponding matched py-pu dimers. Thus the base sequence has not only an explicit effect on the overall populations of stacked species, but also on the handedness of the stacks. The present results further confirm the interdependence of conformational bonds throughout the nucleotidyl framework.


Dependence of the deactivation reactions of photosystem II on the redox state of plastoquinone pool A varied under anaerobic conditions; Equilibria on the acceptor side of photosystem II.

Dark adapted spinach chloroplasts and Chlorella with variably reduced plastoquinone pools were given 1 or 2 saturating flashes. Under these conditions the rate of deactivation of state S2 of the oxygen evolving site of Photosystem II (B, Kok, B, Forbush, M. McGloin (1970) Photochem. Photobiol. 11, 457-475) is highly dependent on the pool redox state, undergoing a nearly 10-fold acceleration upon transforming the plastoquinone pool (A) from 100% oxidized to 90% reduced. Deactivation of state S3 is unaffected by the same variation of the pool redox state. These observations are attributed to a high concentration of Photosystem II reduced primary electron acceptor, Q-, coincident with the formation of S2 and a low concentration coincident with the formation of S3, under the conditions of highly reduced plastoquinone pool. Simultaneous determination of Q- and A2- result in an estimated equilibrium constant of 15-20 for reaction Q-B in equilibrium QB- and a value greater than 50 for equilibrium Q-B- in equilibrium QB2-, where B is the secondary electron acceptor described by B. Bouges-Bocquet ((1973) Biochim. Biophys. Acta. 314, 250-256) and B.R. Velthuys and J. Amesz ((1974) Biochim. Biophys. Acta. 333, 85-94). It is proposed that doubly reduced B becomes protonated in the last reaction.


The distribution of metabolites between spinach chloroplasts and medium during photosynthesis in vitro.

1. The formation of metabolites in the stroma compartment of isolated chloroplasts during carbon fixation, and their export to the medium, have been investigated using improved techniques. 2. Rapid separation of photosynthesising chloroplasts from the medium, accompanied by simultaneous quenching of metabolism was achieved by using silicone oil layer filtering centrifugation under illumination. Metabolites were separated by microscale ion-exchange chromatography. Quantitative determination of each metabolite was based on labelling with 32P. 3. It was found that fixed carbon was exported from the chloroplasts only as triose phosphate and phosphoglycerate, and to a minor extent, as pentose monophosphate. The main compounds accumulating in the stroma were hexose and heptose monophosphates and phosphoglycerate. A marked decrease in the concentration of inorganic phosphate in the stroma during the first 5 min of illumination was accompanied by a complementary increase in organic phosphate so that the total amount of phosphate within the chloroplasts remained constant. 4. The concentration difference for phosphoglycerate between the stroma and the medium was much higher than for triose phosphate or inorganic phosphate, although all three compounds are transported across the inner membrane of the chloroplast envelope by the same carrier. It was concluded that the efflux of phosphoglycerate was restricted.


Isolation and spectral characteristics of the photochemical reaction center of Rhodopseudomonas viridis.

A method is described for isolation of the Rhodopseudomonas viridis reaction center complex free of altered, 685 nm absorbing pigment. This improved preparation contains two c-type cytochromes in the ratio P-960: cytochrome c-558: cytochrome c-553 of 1:2:2 to 3. The near infrared spectral forms of the reduced preparation are located at 790, 832, 846, and 987 nm at 77 K; the oxidized complex absorbs at 790, 808, 829 and approx. 1310 nm. The 790 nm band is attributed to bacteriophaeophytin b and the other absorbances to bacteriochlorophyll b, The visible absorption bands may be assigned to these pigments and to the cytochromes present and, probably to a carotenoid. The presence of two bacteriochlorophyll b spectral forms in the P+-830 band suggests that exciton interactions occur among pigments in the oxidized, as well as the reduced, reaction center. Changes in the 790 and 544 nm bands upon illumination of the reaction center preparation at low redox potential may be indicative of a role for bacteriophaeophytin b in primary photochemical events.


The molecular organization of asymmetric lipid bilayers and lipid-peptide complexes.

Oriented fatty acid bilayers with asymmetric distributions of lipid head group types, hydrocarbon chain lengths, and associated polypeptides have been analyzed by a combined use of high resolution electron microscopy and X-ray diffraction techniques. The exclusion of fixatives, stains, and embedding materials has made it possible to relate unequivocally microscopic images to molecular composition. The ultrastructure of asymmetric bilayers has been determined by a novel analysis in which one half of the bilayer serves as a structural reference for the entire bilayer. Absolute electron density profiles at 7 A resolution have been computed for bilayers formed from long and short chain length lipids either segregated to opposite sides or mixed together in both sides of the bilayer. The data indicate that the two lipids self organize in a specific paired configuration. Detailed analysis of bilayers associated with poly-L-lysine shows that although this hydrophilic peptide resides near the lipid head group region, its presence alters the arrangement of the bilayer hydrocarbon chains.


Separation of plasma membrane markers by glycerol-induced blistering of muscle cells.

Glycerol (50%, w/w) was found to cause blistering of chick primary myoblast and fibroblast plasma membranes and extensive blistering of 5--6-day-old-myotube plasma plasma membranes in tissue culture. The tips of myoblasts and fibroblasts appeared to be the most sensitive portion of the plasma membrane to the blistering effect of glycerol. The glycerol-induced blistering of myotubes was reduced and delayed by brief EDTA pretreatment. Glycerol treatment (50, 15 and 8% sequentially) of myotubes was used to remove plasma membrane blisters and a plasma membrane-enriched fraction was isolated from these blisters using a modified Dextran T500-polyethylene-glycol 6000 aqueous two-phase polymer system. This fraction was found to be enriched 4.1-fold for 5'-nucleotidase activity, but not for other putative plasma membrane markers, (Na+ + K+)-ATPase activity or alpha-[125I]bungarotoxin binding material. Autoradiographs of alpha-[125I]bungarotoxin, glycerol-treated (50%, w/w) myotubes showed the plasma membrane blisters to be devoid of reduced silver grains. 5'-Nucleotidase was shown to be an ectoenzyme on myoblasts and 5-day-old myotubes and the total cellular activity was present on the cell surface. During the period of myoblast fusion and myotube formation, cell surface activity decreased to a low level while total cellular activity was elevated.


Crystalline patterns of myelin lipids visualized by freeze fracture.

Freeze fracture of rat optic nerve reveals smooth, particle-free regions on the lammellar fracture faces of myelin when prepared by standard procedures. When the fixed, glycerin-impregnated tissue is incubated at 6 degrees C for two or more days, crystalline patterns indicative of a phase transition can be seen in the particle-free regions. The crystalline patterns can be destroyed by subsequent incubation at 37 degrees C and are not seen when the initial incubation is at room temperature or 37 degrees C. Butylated hydroxytoluene has no effect on the formation of the crystalline patterns. The time course of the formation of the crystalline patterns suggest that the rate-limiting step in the process is not the phase transition itself. We propose that the lipids associated with the particles in vivo are involved in the formation of the crystalline patterns.


Qualitative and quantitative variations of membrane lipid species in Acholeplasma laidlawii A.

In Acholeplasma laidlawii A, strain EF 22, the relative amounts of the membrane polar lipids vary as a consequence of different fatty acid supplements to the growth medium. The number of lipid species also varies; a new apolar monoglucolipid containing four fatty acid residues was present only when saturated fatty acids dominated in the growth medium. A new phosphoglucolipid, probably with a glycerophosphoryl-monoglucosyldiglyceride structure, was also found. The most pronounced variations occurred between the two dominating glucolipids, monoglucosyldiglyceride and diglucosyldiglyceride; the former being found in larger amounts when a saturated or a trans-unsaturated fatty acid was present in the medium. The amount of diglucosyldiglyceride decreased accordingly. A qualitative relationship between fatty acid properties and membrane lipid variations was established over a wide fatty acid concentration range. Incorporation of supplied fatty acids reached higher levels than normally found in other acholeplasmas. The ratio between membrane protein and lipids exhibited significant and coherent variations during growth and was to some extent influenced by the fatty acids in the medium. These changes indicate variations in lipid-protein organization in  the membranes during growth.


Molecular mechanisms of odor-sensing. II. Studies of fractions from olfactory tissue scrapings capable of sensitizing artificial lipid membranes to action of odorants.

From the preparation obtained by ultrasonic disintegration of frog olfactory epithelium scraping, a fraction capable of sensitizing artificial phospholipid membranes to the action of some chemicals assumed to be odorants in frogs was isolated. In the presence of the active fraction, the membranes respond to the addition of camphor, linalool and musk ambrette by an increased permeability to Na+ and Ca2+. The main component of the active fraction is a nucleo-protein with molecular weight of no less than 100 000.


The effect of cell membrane alteration on glucocorticoid uptake by the AtT-20/D-1 target cell.

The glucocorticoid-sensitive AtT-20/D-1 cell line was used to study cellular uptake of glucocorticoids. A previous observation that glucocorticoid uptake by these cells was temperature dependent had prompted us to postulate that glucocorticoids entered the cell by a temperature-sensitive transport process located in the cell membrane. Attempts were then made to perturb the membrane mechanism. In some of these experiments, intact cells were treated with neuraminidase or pronase. The release of sialic acid in the case of neuraminidase treatment and of sialic acid and cell surface peptides in the case of pronase treatment demonstrated that the enzymes were effective. Approx. 60% of total cellular sialic acid was released by a 15 min incubation with 20 microng/ml neuraminidase at 25 degrees C. The treated cells appeared to be viable, in that they continued to produce corticotropin at a normal rate, yet intact cell glucocorticoid binding at both 4 and 25 degrees C was only 20-30% of that of untreated cells. Treatment with pronase also caused steroid uptake at 4 and 25 degrees C to be reduced, although the extent of reduction was less than that seen following neuraminidase treatment. In other experiments, the effect of exposure of AtT-20/D-1 cells to ethanol or dimethyl sulfoxide was determined. The solvent concentrations used (0.5-10%) did not alter cell viability significantly, and the ability of the cytosol receptor to bind steroid in a cell-free preparation was unimpaired. However, incubation of intact cells with 10% (v/v) dimethyl sulfoxide or ethanol resulted in an 80-90% decrease in steroid uptake at 25 degrees C. We conclude that steroid uptake by the intact cell can be perturbed by treatments which do not affect the cytosol receptor or alter cell viability. These results support the postulate that glucocorticoids enter the AtT-20/D-1 cell by a specific membrane-associated mechanism.


Preparation of uniform haemoglobin free human erythrocyte ghosts in isotonic solution.

A method is described for the preparation of haemoglobin free human erythrocyte ghosts in isotonic solutions using dielectric breakdown technique. In this single haemolytic procedure, almost complete removal of haemoglobin (less than or equal to 0.1%) was achieved by subjecting the erythrocytes suspended in phosphate buffered, isotonic KCl solution at 0 degrees C to three consecutive electrical field pulses of 16 kV/cm in the presence of 10 mM EDTA; EDTA was used to prevent electrical haemolysis. Haemolysis is induced by subsequent dilution with isotonic and isoionic solution to lower the EDTA concentration. Haemolysis is complete after 5 min; the cells are centrifuged, washed and resuspended in a solution of the same composition and osmolarity containing 4 mM MgCl2, but no EDTA. The resealing process, carried out at 37 degrees C, was complete in about 1 h. Measurements of the size distribution of the ghost cells in the hydrodynamically focusing Coulter Counter at varying field strengths in the orifice revealed that the ghost population is nearly uniform. The mean (modal) volume of the ghost cells was 110--120 micronm3 when suspended in phosphate buffered NaCl solution. The apparent breakdown voltage was about 1.3 V.


A method for demonstrating the heterogeneity of pigeon red cell membrane vesicles based on their glycine transport activity.

A procedure is described which is capable of detecting the changes in size and/or density of small membrane vesicles resulting from solute uptake. Vesicles which have taken up solute sediment more slowly in a density gradient, the ratio of glycine uptake/vesicle-trapped space is not uniform in the vesicle population, and vesicles with higher uptake/space ratios are preferentially retarded upon centrifugation. Alanine transport activity is associated with glycine transport activity in that retardation of vesicles due to glycine uptake equally retards vesicles possessing alanine uptake activity.


The analysis of progress curves for enzyme-catalysed reactions by non-linear regression.

A procedure, based on the Gauss-Newton method for non-linear regression, has been developed to obtain enzyme kinetic constants from the analysis of progress curve data. Rules are presented which greatly simplify the derivation of the necessary equations. The method has been applied to the reactions catalysed by prephenate dehydratase, acid phosphatase and lactate dehydrogenase and has yielded values for kinetic parameters which agree well with those obtained from steady-state rate measurements.


The interaction of tetraiodofluorescein with creatine kinase.

The dye 2',4',5',7'-tetraiodofluorescein is a potent inhibitor of creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) with an apparent competitive inhibition constant with respect to MgATP2- of 2.6 - 10(-5) M. The association of the dye with the enzyme elicited a red shift in the dye's spectrum, indicative of a binding site less polar than water. The dye binds to the enzyme with an equilibrium constant of dissociation of 1.7 - 10(-5) M. MgATP or MgADP competes for the dye-binding site. Creatine binds to creatine kinase-tetraiodofluorescein complex to form a ternary complex and further causes a blue-shift in the spectrum of the bound dye. The binding of the dye to fully active creatine kinase causes conformational change that was monitored by enzyme-bound 2-mercuri-4-nitrophenol, a conformation-dependent "reporter group".


Arylsulfatase of human tissue. Studies on a form of arylsulfatase B found predominantly in brain.

The distribution of soluble arylsulfatase (aryl-sulfate sulfohydrolases, EC 3.1.6.1) in human tissues was investigated by DEAE-cellulose chromatography, All tissues examined contained arylsulfatase A and arylsulfatase B. In addition, brain singularly contained significant quantities (15-25% of total arylsulfatase) of a minor anionic arylsulfatase from designated arylsulfatase Bm, whereas only trace amounts of arylsulfatase Bm were found in liver, kidney, testis and placenta. Arylsulfatase B and arylsulfatase Bm had equal activity toward methyl-umbelliferyl sulfate, nitrocatechol sulfate and a physiological substrate UDP-N-acetylgalactosamine 4-sulfate, but both forms were inactive toward the arylsulfatase A substrates cerebroside sulfate and ascorbic acid 2-sulfate. Purified preparations of placental arylsulfatase B, brain arylsulfatase Bm, and urinary arylsulfatase A did not hydrolyze estrone sulfate, dehydroepiandrosterone sulfate or pregnenolone sulfate. The physico-chemical properties of arylsulfatase Band arylsulfatase Bm differed with respect to thermal lability, DEAE-cellulose chromatography, polyacrylamide gel electrophoresis and isoelectric focussing. In the latter technique, utilizing thin polyacrylamide slab gels, the isoelectric point for placental arylsulfatase B was 8.2, while brain arylsulfatase Bm resolved into 3 activity bands with pI values 6.8, 7.0 and 7.2. Although the physico-chemical properties differed, arylsulfatase B and arylsulfatase Bm appear to be functionally equivalent as well as generically related.


Studies on the enzymatic hydrolysis of polyglutamyl folates by chicken liver folyl poly-gamma-glutamyl carboxypeptidase. I. Intracellular localization, purification and partial characterization of the enzyme.

Intracellular distribution, purification and properties of a folyl poly-gamma-glutamyl carboxypeptidase (peptidyl-L-glutamate hydrolase, EC 3.4.12.10) from chicken liver have been investigated. The post-nuclear particulate and cell supernatant fractions showed activity. The particulate enzyme exhibited characteristics suggestive of its lysosomal origin; on solubilization, however, it cannot be distinguished from the cell cytosol activity. The bulk enzyme was purified about 80-fold to apparent homogeneity by 50--90% ammonium sulfate fractional precipitation, dialysis and chromatography on a 'mixed column' of Sephadex G-100 superimposed on CM-Sephadex C-50. The purified enzyme behaved homogeneously on Sephadex G-100 chromatography, sucrose density gradient centrifugation analyses and polyacrylamide gel electrophoresis. However, polyacrylamide gel electrophoresis in the presence of mercaptoethanol dissociated the native enzyme into two separable isoenzymic components. The enzyme exhibits two pH optima (4.1 and 5.2) and a temperature optimum of 35--40 degrees C. The reaction is linear for 20 min. The enzyme sequentially cleaves the terminal gamma-glutamyl residues of polyglutamylfolates, finally releasing a monoglutamyl end-product. An apparent Km value of 0.83 - 10(-6) M and a V of 1.50 mmol/min were determined for N5-methyltetrahydropteroyltetraglutamate with 0.20 mg enzyme protein/ml reaction. The enzyme is substantially stimulated in the presence of mercaptoethanol, Na+, Mn2+ and low concentrations of denaturing agents (urea). Citrate potently inhibits and phosphate inactivates the enzyme.


Recovery of brain from deficiency of essential fatty acids in rats.

Rats were made visibly deficient in essential fatty acids by feeding a deficient diet and were then fed a diet containing 5.0 per cent by weight corn oil as a source of essential fatty acids. After 11-13 weeks on deficient diet the weights of the brain were 25% less than those of rats on control diets. After 5 weeks on the control diet, deficient animals had regained normal brain weight and composition and had lost the deficiency symptoms. Lipids were extracted from the brains and analysed qualitatively and quantitatively, the overall effect of essential fatty acid deficiency being a reduction in the proportion of cerebrosides and sphingomyelin, the appearance of a high proportion of eicosatrienoic acid (20:3) and the reduction of arachidonic acid (20: 4) and the other essential fatty acids. It is considered that essential fatty acid deficiency retards maturing of brains and the present data show that this effect is reversible.


Modification of fatty acid composition of rat heart lipids by feeding cod liver oil.

Modification of the fatty acid composition of cardiac phospholipids and neutral lipids was studied in rats fed a diet containing 10% cod liver oil. The results reflect the dynamic state of esterified fatty acids in neutral lipids and phospholipids of heart muscle. In cardiac neutral lipids there was a moderate but significant increase in exogenous fatty acids, 20:1(n--9), 22:1(n--11), 20:5(n--3) and 22:6(n--3), in animals fed cod liver oil, and a relative decrease in endogenous fatty acids, 16:0, 18:2(n--6 and 20:4(n--6). Increased dietary availability of 22: 6(n--3) resulted in a major increase in the content of this fatty acid in phospholipids and replacement of 18:2(n--6) and 20:4(n--6). The 22:6(n--3) was able to replace one third of 18:2(n--6): further increase in 22: 6n--3) was accompanied by a decrease in 18:0. An inverse relationship between (n--6) and (n--3) polyene fatty acids in cardiac phospholipids suggests a replacement of (n--6) acids by (n--3) fatty acids.


Effect of fasting and feeding a high-sucrose, fat-free diet on the synthesis of hepatic glycerolipids in vivo and in isolated hepatocytes.

1. The synthesis of glycerolipids from a number of radioactive precursors, such as [2-3H] glycerol, [32P]phosphate, [Me-14C]choline and [1,2-14C2]ethanolamine proceeds in enzymatically isolated hepatocytes with a specificity that agrees very well with that observed in the liver in vivo. 2. The nutritional state of the rat has a profound influence on the glycerolipid metabolism of isolated hepatocytes. Fasting strongly decreased the incorporation of glycerol via sn-glycerol 3-phosphate into triacylglycerols whereas the formation of phosphatidylcholine and, particularly, phosphatidylethanolamine was much less affected by food deprivation. Refeeding a high-sucrose, fat-free diet caused a tremendous increase in the synthesis of diacylglycerols and triacylglycerols to values exceeding those found in hepatocytes from normally fed animals. The formation of phosphatidylcholine increased more slowly and the synthesis of phosphatidylethanolamine was even depressed by refeeding a high-sucrose, fat-free diet. 3. Alteration of the nutritional state resulted in similar changes in the amounts and metabolism of hepatic glycerolipids in vivo. The formation and, consequently, the amount of diacyglycerol and triacylglycerol were decreased by fasting and increased to values above normal in rats refed a high-sucrose, fat-free diet. The formation and the amounts of phospholipids in the liver decreased slightly by fasting which was mainly due to a decrease in phosphatidylcholine. Refeeding caused a significant increase in the formation and amount of phosphatidylcholine. The amount of phosphatidylethanolamine was even further diminished by feeding a high-sucrose, fat-free diet to 48-h-fasted rats. 4. These results show that the alterations, induced in the in vivo metabolism of hepatic glycerolipids, by changes of the dietary state, are also reflected in the isolated hepatocytes. This finding strengthens the potential significance of isolated hepatocytes in studies on the regulation of hepatic lipid metabolism.


The effect of bile salts on the formation and hydrolysis of cholesterol esters by rat liver enzymes.

To determine the effects of different bile salts on the enzymic esterification of cholesterol and the hydrolysis of cholesterol esters rat liver homogenates and rat liver microsomes were incubated with varying amounts of different bile salts. Bile salts inhibited the formation of radioactive cholesterol esters in incubations of either rat liver homogenates or rat liver microsomes containing [14C]cholesterol. Chenodeoxycholate, glycochenodeoxycholate and taurochenodeoxycholate were more potent inhibitors than their comparable cholate analogues. Bile salts stimulated the hydrolysis of cholesterol esters when incubation were carried out with the liver homogenates. The dihydroxy bile salts were again more potent in this regard than the trihydroxylated bile salts. When the effects of bile salts on cholesterol ester hydrolysis were studied in in vitro incubations of hepatic microsomes a biphasic mode of acion was observed. In the absence of Na+ or K+ bile salts stimulated the hydrolysis of cholesterol oleate. However, following the addition of either Na+ or K+ to the microsomal incubations, bile salts caused an inhibition of cholesterol ester hydrolysis. Since cholesterol esterification was also inhibited under these conditions a direct inhibitory effect (not attributable to enhanced hydrolase activity) of the bile salts on the formation of cholesterol esters by the microsomes was established. Furthermore, this inhibition takes place at the transacylation step involving the fatty acyl-CoA ester and the sterol. These results suggest that bile salts can significantly alter the cholesterol-cholesterol ester profile in the liver, and furthermore, that these effects may be influenced by small changes in the intracellular environment in the region where these reactions occur.


Purification and characterization of two types of esterase from rat liver microsomes.

It was found that about 90% of the triacylglycerol lipase activity of a rat liver microsomal fraction was released by heparin treatment. The residual microsomal fraction contained about 95% of the esterase activity but little triacylglycerol lipase activity. Two kinds of esterase (esterases I and II) were purified from this residual fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, isoelectric focusing and Sephadex G-200 chromatography. The final preparations of esterases I and II, which were puri fied 70-and 140-fold, respectively, gave single protein bands on polyacrylamide gel and sodium dodecyl sulfate-gel electrophoreses. The molecular weights of esterases I and II were calculated to be about 70 000 and 160 000 by gel filtration of Sephadex G-200. Their isoelectric points were 5.82 and 6.32. Both esterases completely hydrolyzed short-chain triacylglycerols, such as tributyrylglycerol, but did not hydrolyze long-chain triacylglycerols. They preferentially hydrolyzed medium-chain-length 1-monoacylglycerols, such as 1-monocaprylylglycerol. Esterase I differed immunologically from esterase II and it was found to constitute about 30% of the total esterase activity in the microsomal fraction; esterase II constituted 50--60%. These results show the existence of two isozymes of esterase in the microsomal fraction.


Incorporation of [14c] arachidonate in pig thyroid lipids and prostaglandins.

In pig and sheep thyroid, the arachidonate content of phosphatidylinositol (14.3--17.5%) is much higher than in the other phospholipids. In the thyroid, phosphatidylinositol seems to play an important role in the biosynthesis of prostaglandins. In neutral thyroid lipids, the arachidonate content is much higher in diacylglycerols (15.8%) than in monoacylglycerols (2.9%) or triacylglycerols (4.2%). However, the most important pool of esterified arachidonate is triacylglycerols (1030 nmol of arachidonate/g tissue). Arachidonate represents a very small part of total free fatty acids measured in the presence of albumin and indomethacin (0.65% or 16.4 nmol/g tissue). Thyrotropin (50 munits/ml) causes after 1 h a 2-fold increase in the level of free arachidonate (37.3 nmol/g tissue). Pig thyroid slices rapidly take up [14C]arachidonate and incorporate it into neutral lipids and phospholipids. Specific activity of phosphatidylinositol is 3-fold higher than that of phosphatidylcholine after an hour incubation. Specific activity of diacylglycerols is 8-fold higher than that of triacylglycerols. Thyrotropin (50 munits/ml) causes a significant decrease (32--33%) of incorporation of radioactivity in lipids as compared with standard incubation. This result is compatible with the isotopic dilution of the labeled [14C]arachidonate by nonradioactive arachidonate liberated in the incubation medium under thyrotropin action. Slices and homogenates of pig thyroid weakly convert [14C]arachidonate to prostaglandins E2 and F2alpha (1--2%. Thyrotropin (50 munits/ml) always diminishes the conversion of radioactivity to prostaglandins as compared with standard incubation. This result is compatible with the above-mentioned hypothesis.


Monoethlenic C20 and C22 fatty acids in marine oil and rapeseed oil. Studies on their oxidation and on their relative ability to inhibit palmitate oxidation in heart and liver mitochondria.

1. Carnitine esters of erucic acid (22:1 n-9 cis), cetoleic acid (22:1 n-11 cis), brassidic acid (22:1 n-9 trans), gadoleic acid (20:1 n-9 cis) and oleic acid (18:1 n-9 cis) have been compared as mitochondrial substrates and as inhibitors of palmitoylcarnitine oxidation in heart and liver mitochondria. 2. Both the rate of intramitochondrial-CoA acylation and the rate of beta-oxidation decreases as the chain length increases from C18 to C22. There are no significant differences among the three C22 isomers as oxidizable substrates. 3. All the tested acylcarnitines inhibit palmitoylcarnitine oxidation. The C18 and C20 acylcarnitines inhibit by virtue of being competing substrates; i.e. the respiration is not inhibited. The C22-isomers inhibit also respiration; this shows that the inhibition of palmitolycarnitine oxidation is not compensated for by oxidation of C22-acylcarnitines. Brassidoylcarnitine inhibits the oxidation of palmitoylcarnitine and respiration less than erucoyl-and cetoleoylcarnitine. The different behaviour of the C22-isomers is probably due to the difference in their competitive properties with respect to long-chain acyl-CoA dehydrogenase. 4. All C22 acylcarnitines seem to be relatively better oxidized in the liver than in the heart mitochondria while their inhibitory effect on the usage of the radioactive palmitoylcarnitine is very similar. 5. Palmitoylcarnitine inhibits almost completely the "endogenous" formation of acetyl-CoA presumably from malate via pyruvate in the liver mitochondria while the C22-acylcarnitines cause only a partial inhibiton of this acetyl-CaO formation.


Rat intestinal glycolipids. III. Fatty acids and long chain bases of glycolipids from villus and crypt cells.

Previous studies from this laboratory have demonstrated a striking difference in rat intestinal glycolipids between differentiated villus cells and immature crypt cells. Villus cells contained proportionally greater amounts of glucosylceramide and hematoside while crypt cells were deficient in hematoside, but contained proportionally greater amounts of trihexosylceramide. In order to further elucidate possible differences between villus and crypt cell glycolipids, a study of the sphingosine and fatty acids of rat intestinal glycolipids was conducted. Villus and crypt cells were separated from rat intestine and the glycolipids purified. Fatty acids and long chain bases of the three major glycolipids (glucosylceramide, trihexosylceramide, hematoside) extracted from these cells were characterized. Phytosphingosine accounted for 63-73% of the total long chain bases in all glycolipids whether from villus or crypt cells. Hydroxy fatty acids represented 70% of total fatty acids in the glucosylceramide and in the hematoside but accounted for only 30% in the trihexosylceramide. In addition, trihexosylceramide contained a larger percentage of fatty acids with 20-carbon atoms than glucosylceramide and hematoside isolated from villus cells. These fatty acids were more concentrated in crypt cells than in villus cells glycolipids. These results suggest that hematoside and trihexosylceramide, respectively abundant in villus and in crypt cells, may be derived from a different lactosylceramide precursor and further underscore differences in villus and crypt cell glycolipid synthesis.


Glycolipid composition of human testis at different ages and the stereochemical configuration of seminolipid.

1. The sterochemical configuration of glycerol moiety of seminolipid (1-O-alkyl-2-O-beta-D-(3'-sulfo) galactopyranosylglycerol: Ishizuka, I., Suzuki, A. and Yamakawa, T. (1973). J. Biochem. 73, 77--87) from mammalian testis was determined by measurement of optical rotatory dispersion of alkylglyceryl ether moiety, establishing the final structure of the major glycolipid from mature testis of mammals as 1-O-alkyl-2-O-beta-D-(3'-sulfo)-galactopyranosyl-sn-glycerol. 2. The simple and sensitive modification of fluorometric determination of galactolipids and sphingolipids was described. This method does not require lipid extraction from silica gel of thin-layer chromatography, and allows a quantitative assessment of the sphingoglycolipids and galactosyl glycerides using a small amount of testicular tissue. 3. Various lipid classes from human testis of varying age groups were compared. The total lipid and seminolipid content was highest in adult (40-years-old) testis amounting to 39.0 mg and 158.7 nmol/g tissue, respectively. Seminolipid was not detected in infant (2 years old) or child (9 years old) and found only at the concentration of 25.3 nmol/g in the testis of aged (60--90 years of age) supporting the postulated relation of seminolipid with spermatogenesis and sexual activity. In contrast, ganglioside content was highest in the testis of aged probably reflecting fibrosis of the testis. The main ganglioside was found to be hematoside with N-acetylneuraminic acid. Gangliosides Gm1 and GD1a were also detected.


The polar lipids of group B Streptococci. II. Composition and positional distribution of fatty acids.

1. The polar lipids from group B Streptococci have been isolated. Unless overlapping fractions are worked up on each step molecular species can be lost resulting in a fatty acid composition different from the original one. 2. The lipids were shown to be 1(3),2-diacyl-3(1)-O-alpha-D-glucopyranosyl-sn-glycerol, 1(3),2-diacyl-3(1)-O-[alpha--D-glucopyranosyl-(1,2)-O-alpha-D-glucopyranosyl]-sn-glycerol, 1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol, lysylphosphatidylglycerol and 1'-3'-bis(1,2-diacyl-sn-glycero-3-phospho-glycerol. The sterochemical configuration of the phospholipids was achieved by combinations of chemical degradations. 3. The fatty acid composition of group B Streptococci lipids is qualitatively the same as in other Streptococci, but differs by a high content of stearic acid and by a low degree of unsaturation and cyclopropanization. 4. The forementioned polar lipids as well as the glucosyldiphsphatidylglycerol,, which is also found in this organism, showed a very similar composition and the same non-radom positional distribution of fatty acids. Apart from short chain fatty acids the positioning differentiates between saturated and unsaturated fatty acids: short chain and unsaturated fatty acids are accumulated at Position 2, saturated acids are preferentially linked to Position 1. 5. The uniform fatty acid make-up in all polar lipids favours the hypothesis that their diacylglycerol portions are derived from a common phosphatidic acid precursor with negligible postsynthetic rearrangements of the constituent fatty acids.


Isolation and purification of calcium-binding proteins from bovine neurohypophyses.

An acidic calcium-binding protein was isolated from the soluble fraction of the homogenate of ox neurohypophyses. The protein has a molecular weight of 35 000 and a subunit weight of 15 000. The purification procedure involved ammonium sulphate fractionation, DEAE-cellulose chromatography and gel filtration on Sephadex G-100 and Sephadex G-50. Conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated it to be a protein distinct from the S-100 protein and the soluble hormone-binding proteins (neurophysins) abundant in the neurohypophysis. This appears to be the only Ca2+-binding protein in the soluble part of the homogenate, with an apparent Kdiss for Ca2+ of 1.1 X 10(-5) M (at 22 degrees C) and a binding capacity of 2 mol of calcium per mol of protein. Two different Ca2+-binding proteins of molecular weights 16 500 and 68 000, respectively, were identified in the sodium-deoxycholate-soluble proteins from an ox neurohypophysial microsome fraction. One of them (the former) has been isolated in high purity by DEAE-cellulose chromatography and gel filtration on Sephadex G-200. This protein binds 4 mol of calcium per mol of protein with an apparent Kdiss of 1.0 X 10(-5) M (at 22 degrees C). The sodium-deoxycholate-insoluble proteins from the microsomal fraction also have Ca2+-binding components. The soluble Ca2+-binding protein has properties similar to and may be identical to Ca2+-binding proteins which have been isolated from bovine brain and have been demonstrated to be modulators of brain cyclic nucleotide phosphodiesterase and of actinomyosin ATPase. It also resembles Ca2+-binding proteins isolated from bovine adrenals and the electroplax from electrophorus electricus.


The neutralization mechanism of Vipera palaestinae neurotoxin by a purified factor from homologous serum.

The serum of Vipera palaestinae contains two separate factors which neutralize the hemorrhagic and the neurotoxic activities of its venom. The purified antineurotoxic factor shows one major band in disc electrophoresis and an 800-fold increase in specific activity. Its molecular weight is approximately 56 000 and the isoelectric point is 4.0. These data, together with the failure to form precipitin lines in immuno-diffusion tests, suggest that the antineurotoxic factor of Vipera palaestinae serum is probably an albumin-like or alpha-globulin fraction rather than an immunoglobulin fraction. In vivo experiments show that the neutralizing protein does not protect or block the neurotoxin target sites. When the  neurotoxic fraction is injected into mice intravenously at different intervals following injection of the snake serum, the neutralizing activity decreases with time. Experiments carried out in vitro show that the antineurotoxic factor is inactivated upon heating at 95 degrees C for 10 min but resists boiling when mixed with the neurotoxic component, suggesting the formation of a thermostable complex between the two components. This assumed complex dissociates at pH 2.5 and the neurotoxic activity reappears. Further investigation of the reaction with 131I-labeled neurotoxin and neurotoxin components shows that a stable complex is formed between the neutralizing protein and one of the synergistic components of the neurotoxic fraction.


Laser Raman spectra of calf thymus histones H1, H2A, and H2B.

Laser Raman spectra of the calf thymus histones H1, H2A, and H2B in aqueous solutions are presented. The amide III band in the spectrum of the very lysine-rich histone H1 in aqueous solution appears at 1245 cm-1, which is almost at the same frequency as the corresponding vibration of the ionized form of poly(L-lysine). Upon increasing the NaCl concentration to 1 M, the frequency of the amide III vibration shifts to 1250 cm-1 as a result of the formation of a more compact disordered structure of at least the N-terminal region of the protein. Changing the pH from 3 to 5 induces the same frequency shift. The amide III regions of the Raman spectra of the slightly lysine-rich histones H2A and H2B shows two bands at 1247 and 1265 cm-1 for H2A, and at 1254 and 1265 cm-1 for H2B. These doublets are attributed to vibrations involving the backbone of at least two structurally distinct parts of the histone molecules. The low frequency component is assigned to the random-coil regions of the proteins which appear to have similar conformations for H1 and H2A. The frequency of this component also suggest that the structure of the disordered regions of H2B are more compact and less extended. These conclusions confirm the conformation predictions based on the primary structures of these proteins. The high frequency component at 1265 cm-1 is assigned to the alpha-helical and rigid disordered structures of H2A and H2B, since this band increases in intensity upon addition of NaCl. The amide I' region of the histone spectra is also presented but appears to be much less sensitive to the conformation than the amide III region. The intensity of the bands due to the single bond C-C stretching modes, as well as the intensity ratio of the tyrosine Fermi doublet at 855 and 830 cm-1, are also discussed.


Thermodynamics of the isothermal interaction of beta-lactoglobulin with guanidinium chloride and urea.

A thermodynamic study of the isothermal interaction of beta-lactoglobulin with guanidinium chloride and urea has been performed. Enthalpies of interaction of the two denaturants have been obtained by calorimetric measurements, and the free energy of interaction calculated from previously determined preferential binding of denaturants. In separate dilatometric experiments the volume changes accompanying the interaction of guanidinium chloride with beta-lactoglobulin have also been determined.


Characterization of in vivo effects of dilithium carbamyl phosphate on dog hemoglobin structure and function.

The effect of oral administration of dilithium carbamyl phosphate on adult beagle dogs is dose related. The levels of carbamylation as mol valine hydantoin/mol hemoglobin tetramer reaches a plateau of 0.3-0.38 with daily doses of 100 mg/kg body weight. Related changes in oxygen binding by whole blood and hemoglobin amount to a 5-15% left shift in oxygen isotherms. After discontinuing the administration of carbamyl phosphate, the disappearance of modified hemoglobin with a return to normal oxygen binding values follows the gradual replacement of old cells by new cells in the circulation. When the level of in vivo carbamylation in dog blood is greater than 0.16, it is similar to the levels of in vitro carbamylation of hemoglobin SS which result in the interference with sickling of erythrocytes at low levelsof oxygen.


An analysis of molecular interactions involved in the assembly of tropomyosin tactoids.

Arrangements of parallel and antiparallel tropomyosin molecules are examined for residue interactions which might explain the paracrystal forms observed by electron microscopy. Molecular arrays which produce interaction maxima and corresponding computer graphics-simulated staining patterns are compared with observd electron micrographs. The best correlation of interactions with staining pattern occurs when cationic bridging of acid residues is maximized and supported by favourable ion pair interactions. In the antiparallel case, two main maxima occur and appear to correspond to divalent ion tactoids which have been previously reported. For the Cohen-Longley Mg2+ tactoid the best fit is obtained with a molecular overlap of 201 residues and an end overlap of 17-18 residues (based on a rational 287 peptide sequence). Secondary maxima correspond to other known tactoid forms. Binding of tropomyosin molecules to actin may involve hydrogen bonding to six serine residues which occur at approx. 40-residue intervals.