A subgroup of ankylosing spondylitis associated with HLA-B7 in American blacks. In a study of 34 American black patients with primary ankylosing spondylitis, 18 were found to be HLA-B27-negative. Of these, 10 possessed HLA-B7 (55.6%) compared to 23.7% of 59 B27-negative black controls (P less than 0.025, relative risk = 4). On comparing these 10 B7-positive patients (group I) with 16 B27-positive black patients (group II), a difference in mean age at onset of disease was found: 33.6 years in group I and 22.2 years in group II (P less than 0.005). In addition, a family history of ankylosing spondylitis was absent in group I patients but present in 6 patients in group II (P = 0.034). These findings indicate an association between HLA-B7 and ankylosing spondylitis in American blacks and suggest that these patients who lack B27 but possess B7 represent a subgroup of patients with this disease.


The infectious etiology of rheumatoid arthritis. New considerations. Several possible mechanisms of chronic inflammatory arthritis that might be initiated by infectious agents are discussed. Some recent information on mycoplasma infections, long-term virus infections, and shed bacterial components provides the bases for new experimental approaches. Currently, evidence of involvement of mycoplasma or viral agents in rheumatoid arthritis is tenuous. Chronic peptidoglycan-immune-complex formation is a consideration that has been discussed, but only recently pursued in depth. It may well be that experimental studies on the infectious etiology of rheumatoid arthritis will be revitalized through an appreciation of the bacterial antigen load in the gastrointestinal tract. The perimental vehicles for testing this possibility are available and should be directly applicable at the clinical level.


The specific suppression of contact sensitivity. The ability of 2,4,6 trinitrobenzenesulfonic acid (TNBS)-induced regulatory cells to modulate the generation of contact sensitivity (CS) to picryl chloride has been studied. We have confirmed that such suppressor cells can modulate CS and have extended these observations to demonstrate that suppressor factors, liberated by suppressor cells in supernatants, can limit the generation of CS. We have also confirmed that hapten-coupled membrane products can induce tolerance to PCl. The investigation of the properties of the suppressor factors has revealed that they are hapten-specific and bear H2 coded specificities.


Association of systemic lupus erythematosus and SLE-like syndromes with hereditary and acquired complement deficiency states. The most prominent association of rheumatic diseases with hereditary complement deficiency is systemic lupus erythematosus (SLE) and discoid lupus erythematosus with homozygous C2 deficiency in females. The lupus disease in these patients differ from classic lupus in 1) the increased incidence of discoid lesions, 2) the low incidence of renal disease, 3) the low or absent titers of antibodies to native DNA, and 4) the infrequent finding of immunoglobulin and complement in skin lesions. The strong positive linkage disequilibrium between C2 deficiency and HLA genes raises the possibility that genes other than those determining C2 levels may have the primary role in determining predisposition to disease in these patients. However, the finding of similar diseases in certain patients with hereditary angioedema and SLE-related syndrome who have acquired deficiency of the early components of complement supports a primary role of the C2 deficiency gene in predisposing to lupus disease in these patients.


Epidemiology of anti-lymphocyte antibodies in systemic lupus erythematosus. Lymphocytotoxic antibodies (LCTA) occur in 80% of SLE patients and 40% to 60% of their asymptomatic relatives. They occur equally in consanguineous and nonconsanguineous relatives, are more common with close proband contact, and vary directly with LCTA activity in the proband. LCTA are also increased in laboratory personnel handling SLE blood. Anti-RNA antibodies are found in SLE patients and consanguineous relatives. Both antibodies are primarily IgM in relatives but are IgM and IgG in SLE patients.


Effect of clofibrate on in vivo triglyceride production and clearance in genetically hyperlipemic rats. Seventeen hyperlipemic and 17 normolipemic littermate Zucker rats were treated with clofibrate or normal saline to determine the effect of this drug upon hepatic triglyceride (TG) production and peripheral TG disposal. Peripheral Intralipid clearance was not different in hyperlipemic rats relative to control animals. Hyperlipemic animals demonstrated abnormally elevated TG production in the fasted state which was not corrected with clofibrate administration. Following treatment, peripheral Intralipid clearance was increased 100% in hyperlipemic rats, but unchanged in normolipemic animals. These observations provide further evidence that the predominant lipid reducing action of clofibrate is manifested only in the hyperlipemic state, and predominantly upon peripheral lipid disposal.


Incorporation of labelled glucose and glycerol into phospholipids and triglycerides by rat and rabbit aorta. [1-14C]Glycerol and [U1-14C]glucose were incorporated into aortic phospholipids and triglycerides by isolated rat and rabbit aorta. The major portion of radioactivity was found in the glycerol moiety of triglycerides and phospholipids. Within the range of concentrations studied the rate of incorporation of [1-14C]glycerol was dependent on substrate concentration, whereas the rate of incorporation of [U1-14C]glucose was not. The distribution of radioactivity in the glycerophosphatides was found mainly in phosphatidylserine and phosphatidic acid when [1-14C]glycerol was used as substrate. On the other hand, the major portion of radioactivity was found in phosphatidylcholine when [U1-14C]glucose was the substrate. The incorporation of [1-14C]glycerol and [U1-14C]glucose into aortic triglycerides and phospholipids was markedly increased by aortae obtained from rabbits fed an atherogenic diet as compared to aortae from control animals. Results from double-labeled glycerol containing both [2-3H]glycerol and [1-14C]glycerol indicated that the biosynthesis of triglycerides and phospholipids in the rat aorta did not use phosphatidic acid from the same pool.


Clofibrate retardation of naturally-occurring arteriosclerosis in repeatedly-bred male and female rats. Repeatedly-bred, male and female Sprague-Dawley rats which develop hyperglycemia, hyperlipidemia, hypertension, and arteriosclerosis spontaneously were killed at sequential time intervals, i.e., when the females had completed 1, 2, 3 and 4 pregnancies. The control breeders received no treatment; the experimental animals were given 113 mg of clofibrate/100 g of b.w., subcutaneously, daily, 5 times per week. Clofibrate-treated breeders manifested reduction in blood pressure and in the incidence and severity of arterial disease characteristic of repeatedly-bred rats. The aortic lesions of the clofibrate-treated breeders showed attenuation of the usual severe ground substance alterations, the degenerative changes in connective tissue elements, e.g., fibrosis and elastosis, and absence of calcification and cartilaginous metaplasia. Clofibrate-treated breeders did not show any unusual elevation in serum enzymes, e.g., CPK, SGOT, SGPT and LDH, or significant reduction of their hyperlipidemia. They manifested a definite reduction in adrenocortical and medullary histopathology and their circulating corticosterone levels were subnormal compared to non-treated breeders. It is suggested that the protective effect of clofibrate was mediated through its ability to block normal adrenal steroidogenic pathways rather than through its antilipemic action.


The role of hemodynamic factors in arterial wall thickening in the rat. In a study on the pathogenesis of arterial wall thickening, hemodynamic factors in the common carotid artery of the rat were experimentally altered with an autograft. The relationship between flow pattern and wall-thickening was examined in a half-ring bypass model with an induced stenosis, using both flow-visualization in a corresponding in vitro model circuit and observation of the wall by microscopy. Wall-thickening was found in the neighborhood of bifurcations, junctions and curved segments, which corresponded to regions of low-shear in the flow-field. Marked histologic changes in the wall were observed in the post-stenotic segments where the flow field was very disturbed. Histologic changes in the arterial wall correlated well with flow patterns.


Protective effects of clofibrate on isoproterenol-induced myocardial infarction in arteriosclerotic and non-arteriosclerotic rats. Male and female, arteriosclerotic and non-arteriosclerotic rats were treated with the anti-lipemic agent, clofibrate, for 8 days and then subjected to an acute myocardial infarction by injecting them with two large doses of isoproterenol spaced 24 hours apart. The animals were killed at sequential time intervals during the acute necrosis and early repair phases of myocardial infarction. Pre-treatment with clofibrate caused a definite improvement in survival, less shock and prostration, and ECG evidence of little or no ischemia. Increased SGOT levels, hepatic lipid and necrosis were indicative of advanced liver damage. Although clofibrate-treated animals showed little change in serum lipids during the acute cardiac necrosis phase, they were hyperglycemic and showed the greatest increase in BUN levels. Clofibrate-treated animals had higher serum corticosterone levels than those given isoproterenol alone. Despite superior survival rates, both the arteriosclerotic and non-arteriosclerotic, clofibrate-treated animals exhibited equally severe histopathologic evidence of myocardial damage. It is suggested that the protective effect of prophylactic treatment with clofibrate against isoproterenol-induced myocardial infarction in rats may be due to its ability to change corticosterone levels in the circulation.


Hypothesis on microangiopathy of cutaneous capillaries. Microangiopathy is a more or less pronounced PAS deposit-located exterior to the endothelial cells of the lymphatics and the basal membrane of the capillaries. This lesion, found in various normal and pathological states, has generated numerous pathogenic hypotheses. The presence of microangiopathy in 5 groups of 50 subjects representing five different clinical conditions, subjects over 60 years old or less than 40, diabetics, latent diabetics or patients with severe coronary heart disease, together with microscopic and/or ultrastructural lesions of the connective tissue (fibroblasts, collagen and elastic fibers, ground substance) has enabled us to propose a pathogenic hypothesis applicable to any microangiopathy. The initial change, hereditary or acquired, would be fibroblastic or interstitial. It would be characterized by the accumulation of glycoproteins, proteoglyacans and soluble collagen in the interstitium. Incomplete drainage of these macromolecules would occur around the blood and lymphatic capillaries and manifest itself by a PAS deposit, the hallmark of the microangiopathy.


Coronary degeneration in sexually mature rainbow and steelhead trout, Salmo gairdneri. Correlation of coronary arteriosclerosis with sexual maturation was found in rainbow and steelhead trout. All sexually mature nonanadromous rainbow, precocial male steelhead, and adult spawning steelhead had myointimal hyperplasia but it was infrequent in immature juveniles. Lesion severity in adult rainbow and precocial steelhead approach that of spawning steelhead.


Distortion of endothelial repair. The effect of hypercholesterolaemia on regeneration of aortic endothelium following injury by endotoxin. A scanning electron microscope study. Five young male New Zealand White rabbits were fed a semi-synthetic diet containing 0.2% cholesterol for 2 weeks and a control group of 5 animals was fed a normal stock diet. All animals were then injected intravenously with a single dose of endotoxin from Serratia marcescens (200 microgram/kg body weight) and continued on their respective diets for a further 4 weeks. The aortas were then stained with silver nitrate and fixed under pressure for Scanning Electron Microscopy (SEM). Argyrophilic endothelial cells were present in both groups of animals 4 weeks after endotoxin injections. In the cholesterol-fed animals, however, these cells were often covered with pits and craters. These findings suggest that the hypercholesterolaemia may affect the regeneration of arterial endothelial cells.


The effect of bezafibrate on the fibrinolytic enzyme system and the drug interaction with racemic phenprocoumon. The influence of a new hypolipidaemic agent, bezafibrate, on anticoagulant requirements and fibrinolysis was studied in 15 patients with hyperlipidaemia on long-term treatment with racemic phenprocoumon. Our results suggest a dose-dependent augmentation of the anticoagulant response to the coumarin drug. Treatment with bezafibrate at 450 and 600 mg daily required a reduction of the phenprocoumon dose by 18.5 and 33.5%, respectively. Correspondingly, the serum level of phenprocoumon decreased by 11.6 and 35.3%. No evidence for an altered drug elimination of racemic phenprocoumon could be found during treatment with bezafibrate. The results support the hypothesis that bezafibrate and analogous hypolipidaemic drugs enhance the response to oral anticoagulant drugs by increasing the affinity of the receptor site for coumarins or the rate of degradation of the vitamin-K-dependent clotting factors. The investigation of the fibrinolytic enzyme system demonstrated an increase of the fibrinolytic activity by enhancing the activity of the plasminogen activator. The lysis time for euglobulin clot was reduced significantly, plasma fibrinogen only moderately. The antiplasmin activity could not be altered substantially by a decrease of alpha1-antitrypsin and a slight increase of alpha2-macroglobulin. In contrast with the inhibition of platelet function the effect of bezafibrate on the fibrinolytic enzyme system showed no dose dependence.


The antiocclusive effect of coronary dilatation with age. Human coronary arteries were perfusion-fixed; sectioned and their external and lumenal circumferences measured by microscopic planimetry. They were found to dilate with increasing age, and this change seems to be more a degenerative process than a response to increasing heart weight. It is inferred that a moderate degree of coronary dilatation compensates for the tendency of atherosclerosis to occlude the lumen. Absence of any coronary dilatation might be hazardous in that the stenosing effects of atherosclerosis would be enhanced. By contrast over-dilatation (ectasia) is dangerous in that it causes a reduced flow-rate and, hence, promotes thrombosis.


Unscheduled DNA synthesis induced by procarcinogens in suspensions and primary cultures of hepatocytes on collagen membranes. Unscheduled DNA synthesis was induced by procarcinogens in freshly isolated suspensions and primary cultures (6 days old) of hepatocytes on collagen membranes. Incorporation of [3H]thymidine in the presence of hydroxyurea was used to measure unscheduled DNA synthesis. When hepatocellular DNA was isolated on cesium chloride gradients, significant levels of unscheduled DNA synthesis were measured. Similar concentrations of procarcinogens elicited higher levels of unscheduled DNA synthesis in hepatocellular suspensions than in primary cultures. The results demonstrate that hepatocytes cultured on collagen membranes can metabolize chemical carcinogens. Suspensions of freshly isolated hepatocytes, however, are more active in procarcinogen metabolism than are those of primary cultures. The selective advantages of the two systems of hepatocytes can be utilized for the establishment of short-term in vitro screening systems of mutagens and carcinogens.


Studies on subunit structure and evidence that ligandin is a heterodimer. Several lines of evidence indicate that ligandin consists of two different subunits. The protein dissociates into two components that are detected by electrophoresis in a discontinuous sodium dodecyl sulfate system, or in acid-urea gels, and by isoelectric focusing in the presence of urea. The apparent molecular weights of the two polypeptides are 25,000 and 22,000. Alkylated or succinylated ligandins also exhibit subunit heterogeneity and resolved into two bands in these electrophoretic systems. Cross-linked ligandin showed only one band in sodium dodecyl sulfate-gel electrophoresis indicating that the two subunits are part of a heterodimeric protein rather than monomers of two different proteins. No dansylated terminal amino acids were detected suggesting that the NH2-terminal residues of both chains are blocked. One mole of arginine or phenylalanine was released per mole of ligandin after digestion with carboxypeptidase B or A, respectively. Tryptic maps of succinylated ligandin were consistent with identical disposition of arginine residues in both chains, but several additional tryptic peptides were obtained with native ligandin as compared to the predicted number if both subunits were identical. These observations are consistent with the possibility that both subunits contain common sequences and that a small peptide of about 25 to 30 amino acid residues is cleaved from the COOH-terminal of the larger subunit to produce the smaller subunit.


178-Nucleotide sequence surrounding the cos site of bacteriophage lambda DNA. A nucleotide sequence of 61 nucleotides at the left end and 117 nucleotides at the right end of DNA from bacteriophage lambdacI857Sam7 was determined by the Maxam and Gilbert method. A perfect inverted repeat sequence of 10 nucleotides is near the left end, and one of 15 nucleotides is near the right end. DNA from another closely related lambda strain, lambdacI857prm116Sam7, has about 10% divergence in the sequence of the first 110 nucleotides at the right end and has a 17-member perfect inverted repeat sequence.


Rate-limiting factors in urate synthesis and gluconeogenesis in avian liver. 1. Urate synthesis and other metabolic characteristics of isolated chicken hepatocytes were studied. 2. The distinction is made between immediate precursors of the purine ring (glycine, glutamine, aspartate, formyltetrahydrofolate, bicarbonate) and ultimate precursors from which the immediate precursors are formed in the liver. 3. In hepatocytes from well-fed chickens the rate of urate synthesis was not greatly increased by the addition of amino acids or NH(4)Cl, but in hepatocytes from 72h-starved chickens the rate was much increased when alanine or asparagine was added as the only substrate. Other amino acids, when added alone, did not affect the rate. The exceptional effect of alanine and asparagine is due to the ready formation of the immediate precursors. 4. Conditions are described under which glutamine, serine, glycine plus formate, ribose and glucose increased the rate of urate synthesis. 5. At 1mm-NH(4)Cl (a concentration not much higher than that of blood plasma) the rate of urate synthesis in the presence of lactate was increased, but higher concentrations inhibited urate synthesis in the presence of lactate or alanine; with alanine even 1mm-NH(4)Cl was inhibitory. 6. Glucose synthesis from lactate, alanine or dihydroxyacetone was also inhibited by 1mm-NH(4)Cl. 7. NH(4)Cl inhibition of urate and glucose synthesis was paralleled by an increased rate of glutamine synthesis. Thus in the presence of NH(4)Cl the gluconeogenic precursors are diverted from the pathway of gluconeogenesis to that of glutamate and glutamine synthesis. This implies that the synthesis of these amino acids is the primary process in the detoxication of ammonia in the avian liver. 8. Urate synthesis, like urea synthesis, can be looked on as a cyclic process with either phosphoribosyl pyrophosphate or ribose acting as the carrier on which the purine ring is assembled. 9. The energy requirements of urate synthesis depend on whether phosphoribosyl pyrophosphate is regenerated from IMP by pyrophosphorylase or by phosphorylation and pyrophosphorylation of ribose. It is 6 or 9 pyrophosphate bonds of ATP respectively.


[Measurement of spinal cord blood flow in rabbits: central gray matter flow and CO2 reactivity (author's transl)]. Spinal cord blood flow (SCBF) was measured in 20 rabbits anesthetized with sodium pentobarbital. The platinum electrodes (0.3 mm in diameter) were inserted into the central gray matter of the thoracic spinal cord (Th8-10). In all animals, blood pressure and pAO2 remained constantly in the normal range. The pACO2 was changed by ventilation. Blood gases were checked both before and after each spinal cord blood flow measurement. The results are as follows. 1. Seventy-three percent of the clearances recorded from the gray matter were monoexponential and in the white matter almost all were monoexponential. 2. The mean values of gray matter SCBF of all animals was 38.3 +/- 1.85 ml/100 g/min (SE) at normo-capnea and the white matter was 19.5 +/- 1.33 ml/100 g/min. 3. An almost linear relationship exists between the pACO2 and SCBF. The values were 0.62 ml/100 g/min/torr in the gray matter and 0.38 ml/100 g/min/torr in the white matter.


[Differences of behavior of DSCT neurons in decerebrate and barbiturate anesthetized cats (author's transl)]. Responses of dorsal spinocerebellar tract (DSCT) neurons to random electrical stimulation of peripheral nerves of the hindleg in decerebrate cats were studied using cross-correlation analysis of the output spike train. The spontaneous background discharge in barbiturate's cats had longer mean interspike interval than that in decerebrate cats, but there was no difference of the coefficient of variation in the two groups. This suggests that barbiturate has no effect on raising of the electrical threshold of the axon, but prolonging effect on interspike interval. Comparison of the conduction velocity showed to be slower in barbiturate's cats, so barbiturate reduces the conduction rate of the axon. The DSCT responses to the electrical stimulation of peripheral nerve in decerebrate cats were recorded in 60%, while 85% in barbiturate's cats. The reasons for this difference are that the DSCT  cells in decerebrate cats are tonically inhibited from the pontine and bulbar reticular formation and the function of brain-stem reticular formation is suppressed by the barbiturate. Type 2 response in decerebrate cats, which represents reduced excitability of the DSCT cells, had inhibition with shorter initial peak latency and duration as compared to that in barbiturate's cats. This confirms the result of Eccles et al. that barbiturate in moderate dosage increases and prolongs the presynaptic inhibition.


[Intracranial saccular aneurysms in the first three decades of life (author's transl)]. An analysis of surgically-treated 1,000 intracranial saccular aneurysms in our clinic during a period from June, 1961 to September, 1975, revealed 39 cases in patients under the age of 29 years. Some characteristics were emphasized in this studies. The sex incidence was male predominant, which exhibited a ratio of 4 to 1 for the ages below 20 years and 2.1 to 1 for the ages below 30 years. In sites distribution, there were striking incidence of aneurysms of the internal carotid termination, on the contrary, lower incidence of aneurysms of the middle cerebral artery, comparing with the cases in all ages. The common complication of cerebral infarction secondary to the vasopasm associated with rupture of the aneurysm occured less often than in adults cases. The surgical results of 39 cases in the first three decades of life, consisted of 29 excellent cases, 5 good, 4 poor and 1 death, have been as good or better than in the cases of all ages. Furthermore, the results of follow-up studies, which were obtained from 33 cases out of 38 survival cases, revealed 31 cases with full working and no deterioration.


[The relationship between the rupturing of intracranial aneurysm and season, weather and patient's acitivity (author's transl)]. Some physical and mental stresses have been notices as the exciting factors for the occurrence of cerebrovascular diseases. However, little has been known about them of ruptured intracranial aneurysms. From this point of view, the correlation between the several physical strains and the occurrence of ruptured intracranial aneurysm were studied in this report. This study was based on 920 cases of ruptured intracranial saccular aneurysm which were verified by direct aneurysmal surgery. As for the season, the occurrence of ruptured intracranial aneurysms was apt to be frequent in winter and infrequent in summer. No relation between the onsets and the meteorological fronts could be found. The incidence during sleep was about 6%. This incidence was thought to be rather lower considering the fact that one-third of our lives is spent in sleep. Lifting and bending, defecation and/or urination and emotional strain were among the commonest environmental events associated with the onset. Based on these results, wheather or season, which is mild physical stress, can in no way be considered a great cause of rupturing of intracranial aneurysm, but it should seen that patient's activities or events have some influences in rupturing of aneurysm to a great extent.


[Normal pressure hydrocephalus. Part 1. Dynamic study of CSF circulation in patients with normal pressure hydrocephalus (author's transl)]. To clarify the pathogenesis of normal pressure hydrocephalus (NPH), quantitative measurement of CSF dynamics was attempted using RI techniques. In this study results concerning RI ventriculography and RI transfer test from CSF to plasma were reported. "Barrier ratio" and regional cerebral blood flow study in NPH will be reported elsewhere. Fifty-three patients with chronic communicating hydrocephalus wer devided into two groups. Thus, twenty-seven cases were diagnosed as NPH according to clinical symptoms and signs, and others were considered as mere chronic communicating hydrocephalus without any NPH characteristics. Results are as follows: 1) 169Yb--DTPA ventriculography; In NPH group, there was longer retention of RI injected into lateral ventricle, and RI activity was not detected in the cisterna magna 60 minutes after the injection. These findings show remarkable delay of CSF flow in NPH patients. 2) Transfer test of 169Yb--DTPA from CSF to plasma; Immediately after the RI injection, Transfer ratio of RI activity from CSF to plasma was measured. It was revealed that intraventricular RI was more rapidly transfered to plasma in NPH group compared with in the control group, suggesting accelerated trans-ependymal absorption of RI in NPH group.


[Radioisotope cisternographic study on intracranial arachnoid cyst (author's transl)]. The dynamics of the CSF circulation in six cases of intracranial arachnoid cysts was examined by RI cisternography suing 0.5 to 1.0 mCi of 169Yb DTPA or 50 to 100 microCi of 131I HSA injected into the lumbar subarachnoid space. Serial scintigrams were obtained with rectilineal scintillation scanner at 2, 4, 6, 24 and 48 hours after injection. The communication of the cavity of arachnoid cyst and subarachnoid space was recognized in all cases. The cysts were best visualized at 24 hours in most cases. Four patterns of the entry and stasis of RI in cysts were observed as follows, 1) rapid filling of RI into the cyst and delayed clearance, 2) both rapid filling and clearance, 3) slow filling and delayed clearance, 4) no filling.


[Dynamic aspect of brain edema in cases of acute carbon-monoxide poisoning (author's transl)]. Eleven cases of acute carbon-monoxide poisoning in various stages were observed with the computerized tomography for 17 times and analysed their correlation between intracranial lesion and their clinical courses. Five of 11 cases revealed characterististic pathologic findings in CT scan, however complete recovery of consciousness was observed in only 4 of 6 cases with normal CT findings. In early stage of acute carbon-monoxide poisoning, CT scans showed typical diffuse subcortical low absorption with no obvious ventricular dilatation. The entity of this typical finding was supposed to be the so called "vasogenic edema" attributed to increased extracellular fluid caused by increased permeability of vessels due to hypoxic process of the poisoning. While this diffuse subcortical edema was subsiding gradually in about 2 weeks, progressive brain atrophy was supervening and resulted finally in severe dilatation of the ventricular system. In spite of two exceptional cases of vegetative state with normal CT scan in early stage seemed suggestive of poor prognosis in most of cases. Pathogenesis of subcortical brain edema and the effect of hyperbaric therapy in the cases of acute carbon monoxide poisoning were also discussed.


[Bulbar reticular control on DSCT activity in decerebrate cats (author's transl)]. Effects of bulbar reticular tetanic stimulation on the responses of dorsal spinocerebellar tract (DSCT) units were studied in the decerebrate cat. The conditioning stimulation of lateral or paramedian reticular nucleus was found to reduce polysynaptic activation of DSCT cells (type 3 response) selectively without affecting either the spontaneous background discharge or the monosynaptic activation (type 1 response) or inhibition (type 2 response) by afferent inputs from the peripheral nerves of hindleg. These results show that the descending inhibition to the polysynaptic activation of DSCT cells occurs presynaptically by reticulospinal pathways from brain-stem reticular formation.


Diuretic-induced hypokalaemia, pancuronium neuromuscular blockade and its antagonism by neostigmine. The effect of hypokalaemia on a neuromuscular blockade induced by pancuronium and its antagonism by neostigmine was studied in the cat anterior tibialis-peroneal nerve preparation using the constant infusion of pancuronium technique. Hypokalaemia was induced by chronic administration of chlorothiazide. The infusion rate of pancuronium required to maintain a 90% depression of twitch tension was reduced from 0.72 +/- 0.06 microgram kg-1 min-1 in the cats with a normal serum concentration of potassium (K+ = 4.4 +/- 0.2 mmol litre-1; n = 7) to 0.41 +/- 0.07 microgram kg-1 min-1 in the hypokalaemic cats (K+ = 2.3 +/- 0.1 mmol litre-1; n = 8). The dose of neostigmine necessary for 50% antagonism of the pancuronium-induced depression of twitch tension (ED50) was 10.0 microgram kg-1 in the cats with a normal potassium concentration and 18.5 microgram kg-1 in hypokalaemic cats. We conclude that hypokalaemia decreases the dose of pancuronium required for neuromuscular blockade and increased the dose of neotigmine required for antagonism of the block.


Quantitative analysis of the effects of volatile anaesthetics on cortical electroencephalographic activity. Methoxyflurane, halothane and enflurane were compared in respect of their ability to modify cortical activity in Sprague-Dawley rats with chronically implanted electrodes. An analytical instrumentation system for analysis of the electroencephalogram (e.e.g), equipped with a memory unit and capable of obtaining on-line analysis of time histograms of the e.e.g. was used. Methoxyflurane was found to be four times as potent as enflurane, and 2.6 times as potent as halothane 15 to 25 min after the commencement of anesthesia. Halothane was found to be 1.5 times as potent as enflurane.


Nitrous oxide and neutrophil chemotaxis in man. Inhalation of 60% nitrous oxide in oxygen by seven adult male volunteers for 60 min was found to increase significantly polymorphonuclear leuocyte (PMN) chemotaxis, but not to influence total white or PMN blood cell counts. Also, the addition or morphine 0.2 mg kg-1 i.v. during the breathing of nitrous oxide did not alter any variable. These results indicate that nitrous oxide and nitrous oxide incombination with morphine do not depress PMN chemotaxis.


Gastric emptying following hysterectomy with extradural analgesia. Using the rate of absorption of paracetamol following oral administration of the drug, gastric emptying was measured in 21 patients following hysterectomy. Gastric emptying was inhibited markedly in patients receiving narcotic analgesia after operation, but only a moderate delay was observed in patients undergoing extradural analgesia.


Effect of segmental extradural analgesia on placental blood flow during normal labour. Placental blood flow was measured during the first stage of normal labour using a xenon-133 clearance technique before and after segmental extradural analgesia. Analgesia was produced with 0.5% plain bupivacaine in eight patients and with 0.5% bupivacaine--adrenaline in 10 patients. Segmental extradural analgesia with a small dose (20 mg) of bupivacaine did not change placental blood flow significantly. The addition of adrenaline 20 microgram produced no effect.


Effect of anaesthesia for delivery on the weight of infants during the first 5 days of life. Two hundred and forty-one neonates who had been delivered vaginally were selected randomly. The control group consisted of infants delivered without anaesthesia. In the study groups either extradural (mepivacaine, lignocaine or bupivacaine), spinal or general anaesthesia was administered. Each of the six groups was divided into breast- and bottle-fed subgroups. The results showed no significant difference in weight changes among the various groups. The bottle-fed subgroup showed less weight loss than the breast-fed subgroup (P less than 0.05). There was no interaction between method of feeding and type of anaesthesia.


Intermittent mandatory ventilation during anaesthesia using the Manley Servovent ventilator. The lungs of 25 patients were ventilated with intermittent mandatory ventilation (IMV) during anaesthesia using the Manley Servovent Model MS. This ventilatory mode is especially suitable for prolonged operations in which there is no need for muscle relaxation. While incorporating the advantages of spontaneous and mechanical ventilation, it is superior to both in selected cases. The Manley Servovent Model MS ventilator is capable of delivering IMV without modification, using a single source of gas.


Intubation lesions of the larynx.

Intubation lesions of the larynx. One thousand patients were examined immediately after extubation of the trachea at the end of anaesthesia. Severe lesions of the larynx were detected in 62 patients (6.2%). The lesions encountered included haematoma, laceration of the mucous membrane, laceration of the muscle and subluxation of the arytenoid cartilage. In spite of early diagnosis and adequate treatment, it was not possible to avoid an effect on the voice in nine patients.


Comparative study of the effects of oral and i.m. atropine and hyoscine in volunteers. The effects of equivalent doses of atropine and hyoscine following oral and i.m. administration were assessed on salivary secretion, heart rate, arterial pressure, body temperature, pupillary size, near-point of vision and sweat-gland activity. The ratio of oral to i.m. doses of atropine on heart rate and salivary secretion appears to be 2:1 and that of hyoscine on salivary secretion about 5-6:1. Following oral administration the effects on the eye are minimal even after the highest doses of the two drugs, while the decrease in salivation is adequate.


A comparison of buprenorphine and pethidine for immediate postoperative pain relief by the i.v. route. A comparison was made of the relief of pain after operation, obtained following the i.v. administration of buprenophrine and pethidine in 60 patients with lower abdominal incisions. No difference could be detected between the maximum analgesia produced by eigher drug, but analgesia following buprenorphine appeared to last about four times as long as that following pethidine. When the drugs were compared on  a "dose per body weight" basis the results supported a ratio of potency in the order of 1 : 200 in favour of buprenorphine. Vomiting, drowsiness and dizziness were less frequent following buprenorphine, but a similar frequency of nausea was observed with both agents. Marked miosis occurred 5--10 min after the i.v. injection of buprenorphine, but no serious side-effects were observed with either drug. The i.v. injection of buprenorphine, but no serious side-effects were observed with either drug. The i.v. administration of buprenorphine appeared to be effective in the management of pain after operation in patients with lower abdominal incisions.


True anaphylaxis to suxamethonium chloride. A case report. A severe anaphylactic reaction to suxamethonium chloride is reported. The patient had received suxamethonium uneventfully on four previous occasions. Previous sensitization and immunological studies indicated that this was a classical type I hypersensitivity reaction.


A rapid-response u.v. halothane meter.

A rapid-response u.v. halothane meter. The design and performance characteristics of an instrument for monitoring halothane concentrations which operates on the principle of the absorption of u.v. light are described. The innovation is that the entire exhaled breath passes through the sample cell, enabling the breath-by-breath halothane concentration to be measured accurately and instantaneously. Stablized power supplies, solid-state circuitry and filtering make  the instrument stable and selective. A valve prevents rebreathing of gas exposed previously to u.v. light in the sample cell. Inspired and end-tidal concentrations are displayed digitally and an analog output is available also. Zero and gain drift were negligible after an initial settling period, and interference from other respiratory gases was not detectable. The monitor is suitable for use during anaesthesia for adults and children.


Sensitivity and accuracy of the visual analogue scale: a psycho-physical classroom experiment. 1 Twenty-seven intelligent volunteers took part in a classroom experiment on two occasions to assess subjectively, ordinally related volumes of sound (offered in random sequence), using the visual analogue scale. 2 It was simple to use and largely acceptable. Big differences were significant by parametric tests but small ones were sometimes significant only with ordinal ones. Within-subject comparisons were more accurate and more sensitive than those between-subjects. The t-test was very robust. 3 Five out of 49 results were erroneously significant; they remained so no matter how the data were handled. It was concluded that this was due to a shift in either perception, cognition or scoring between the two sessions. 4 Arcsine transformations made little difference. The mechanism of these is fully discussed. A conversion to proportional scores resulted in very much improved sensitivity. 5 It is recommended that authors using the visual analogue scale: have valid reason in their own setting for using a transformation; present the distribution, or at least the medians and ranges of their raw scores; also use a simple but different measure so as to demonstrate internal consistency between them.


Plasma concentrations of isosorbide dinitrate and its metabolites after chronic high oral dosage in man. 1 We have previously reported that vasodilator headache due to isosorbide dinitrate (ISDN) can be circumvented by using small 'priming' doses for an induction period of 1-2 weeks, after which it is possible to increase to dose rapidly to 360-720 mg, daily without recurrence of headache and without toxicity. The present study corroborates this earlier finding. 2. Chronic oral administration of doses of ISDN of this order of magnitude results in prolonged high plasma concentrations of the parent compound, as well as higher levels of the metabolites 2-ISMN and 5-ISMN. 3. It is our thesis that chronic high oral dosage of ISDN saturates the intrahepatic biotransformation process, and allows high concentrations of ISDN and its metabolites to enter the systemic circulation.


An intradermal study of the local anaesthetic and vascular effects of the isomers of bupivacaine. 1 L(-)- and D(+)-bupivacaine in eight different concentrations from 0.06 to 7.69 mmol/l (0.002--0.25%) and physiological saline were given intradermally to seventeen volunteers, using a double-blind technique. 2 Local colour changes were observed as 'nil', 'pink' or 'pale' and analgesia to pinprick was assessed every 10 min to give an estimate of apparent in vivo potency and duration of action (50% recovery). 3 Both isomers were vasodilator in all cases at 7.69 mmol/l, but the incidence of vasodilatation waned with decreasing concentration, more rapidly with L(-)-than with D(+)-bupivacaine. 4 Only L(-)-bupivacaine showed a vasoconstrictor effect. This was maximal at a concentration of 0.48 mmol/l, when the incidence of pallor was 92%. 5 L(-)-bupivacaine had a longer duration of analgesic action than the (D+)-isomer from 0.48-3.84 mmol/l; this was reflected in a higher apparent in vivo potency.


Compliance with anticonvulsant therapy in a hospital clinic and in the community. 1 Seizure control, saliva anticonvulsant concentration, prescribing habits and compliance with anticonvulsant medication have been compared in 86 epileptic subjects attending either a specialist hospital clinic or general practice surgeries. 2 Of all subjects experiencing recurrent seizures 70% had saliva concentrations of phenytoin below the range equivalent to the 'therapeutic range' of plasma concentration. Mean saliva phenytoin concentrations did not differ significantly between the two treatment settings and were low largely because of the low mean dosage prescribed. 3 Eleven subjects in all had no detectable phenytoin in their and could clearly be identified as noncompliant. Freedom from seizures appeared to predispose to poor compliance in these subjects as well as among those admitting repeated omission of doses.


Comparison of digoxin and medigoxin in normal subjects. 1 The properties of a recently introduced digitalis glycoside, 4-beta-methyl digoxin (medigoxin) were compared to those of a standard digoxin preparation. Using a radioimmunoassay (RIA) technique, serial plasma levels were recorded for 8 h following a single oral dose in five fasting volunteer subjects, and urinary glycoside elimination was measured for 4 consecutive days after dosage by use of a modification of the RIA method. 2 It was found that this RIA was suitable for plasma level measurement of both digoxin and midigoxin by reference to appropriate standard curves. Comparison of the plasma level profiles of these two drugs showed that medigoxin was very rapidly absorbed with peak levels occurring within 15--30 min, while digoxin produced peak levels after 45--75 min. The area under the plasma level-time curve produced by medigoxin was also consistently greater than that produced by digoxin, even though the medigoxin dose used was smaller. Quantitative comparison of these areas after adjustment to compensate for differing doses showed that medigoxin is considerably more biologically available than digoxin under study conditions (ratio 1.6 +/- 0.25:1), and comparison of quantitative urinary elimination suggested that medigoxin is eliminated in the urine to a lesser extent than digoxin and therefore it undergoes more metabolism and/or hepato-biliary elimination.


Heparin pharmacokinetics: increased requirements in pulmonary embolism. Heparin disappearance after injection and plasma levels during continuous infusion were studied in normal subjects and patients with thrombophlebitis, pulmonary embolism, renal failure, and liver failure. Heparin removal in normal subjects after 75 u/kg was nearly linear with a clearance of 0.64 ml/min/kg, SD +/- 0.11. Clearance varied inversely with dose. Heparin clearance in pulmonary embolism (0.80 ml/min/kg +/- 0.23) was significantly accelerated compared both to normals (P less than 0.005) and to thrombophlebitis patients (0.55 ml/min/kg +/- 0.19, P less than 0.01); the disappearance was more curvilinear in thrombophlebitis and pulmonary embolism than in normal subjects (P less than 0.025). Continuous infusion heparin requirements were greater in pulmonary embolism than in thrombophlebitis, in accordance with pharmacokinetic predictions. The pattern and rate of disappearance in renal disease was similar to normal subjects; in liver disease clearance was accelerated (0.86 ml/min/kg +/- 0.28) and disappearance curvilinear. Because of accelerated clearance, the initial dose of heparin in pulmonary embolism should be greater (25 u/kg/h) than in thrombophlebitis (10-15 u/kg/h). Variability within patient groups necessitates some laboratory control of dosage.


Microheterogeneity of human antithrombin III. Antithrombin purified from normal human plasma has been separated into two fractions by isoelectric focusing in a pH 4-6 gradient. These fractions were homogeneous by polyacrylamide gel electrophoresis, had similar amino acid composition and the same specific activity. Both of them cross reacted with antiserum against antithrombin. They were found to contain different amounts of sialic acid and aminosugars. After neuraminidase treatment only a single, homogeneous peak was found by isoelectric focusing--with unchanged antithrombin activity--suggesting that the microheterogeneity is due to a difference in glycosylation.


Characteristic surface morphology of human and murine myeloma cells: a scanning and transmission electron microscopic study. Cells from cultured human and murine myeloma cell lines and circulating leukaemic plasma cells from four patients with generalized myeloma were studied by transmission and scanning electron microscopy and time-lapse cinematography. Both circulating and cultured cells exhibited consistent surface architectures and varying numbers of prominent blebs of different sizes were seen, in addition to microvilli. The presence of surface blebs appears to be a characteristic feature for secreting and non-secreting myeloma cells.


Erythrocyte populations in pyruvate kinase deficiency anaemia following splenectomy. I. Cell morphology. Light and scanning electron microscopic examination of fresh erythrocytes from the blood of four patients splenectomized for severe pyruvate kinase (PK) deficiency haemolytic anaemia has revealed three morphologically different cell populations. Most striking in these patients was the presence of crenated red cells (30-50% of total) which fit current criteria for definition as echinocytes, but also with unusual surface retraction compatible with the existence of intracellular shrinkage, a phenomenon known to occur after incubation of PK deficient red cells in vitro. Additional cell types include immature reticulocytes (7-8% of total) and thin macrocytic discocytes (45-65% of total) none of which are considered specific for the disease. These morphological findings are in sharp contrast with the paucity of red cell shape abnormalities usually described in PK deficient patients before splenectomy and may provide insight into the mechanisms leading to accelerated erythrocyte destruction in this disease.


Erythrocyte populations in pyruvate kinase deficiency anaemia following splenectomy. II. Cell deformability. The deformability of three previously described morphological populations of erythrocytes from splenectomized pyruvate kinase (PK) deficient patients was assessed using both Nucleopore filtration and micropipette techniques. Mean deformability was decreased in fresh cells from all patients studied. Selective measurements indicated that immature lobulated reticulocytes in each patient were the least deformable cells, followed by bizarre echinocytes characterized by a hyperviscous behaviour, and by thin macrocytic discocytes whose values were closest to those of control erythrocytes. We conclude that poorly deformable reticulocytes in these patients, after initial trapping in the spleen, are unable to return to the circulation because of their inordinate dependence on mitochondrial oxydative phosphorylation for maintenance of a sufficient intracellular concentration of adenosine triphosphate (ATP); hence their rapid and selective destruction. Splenectomy therefore may be beneficial by allowing such cells to mature more slowly in the circulation of these patients until they develop crenation and hyperviscosity, two manifestations of profound ATP depletion possibly leading to destruction by the liver.


Coloboma of optic nerve with overlay of peripapillary retina. An eye that had the clinical appearance of a tumour of the nerve head was found on histological examination to have a congenitally large scleral canal with secondary buckling of the overlying retina.


Pathophysiology of diabetic retinopathy. After a brief analysis of the pathological picture of diabetic retinopathy, of which only the topographical distribution of the vascular lesions appears to be specific, the results obtained with 2 new methods of study of the retinal circulation, are presented. These methods are vitreous fluorophotometry and fluorometric determination of segmental retinal blood flow. Vitreous fluorophotometry has shown that a disturbance of the blood-retinal barrier, possibly functional, appears in diabetic eyes before any lesion is clinically visible in the fundus, and that there is a close correlation between the severity of the vascular lesions and higher vitreous fluorophotometry readings. Blood flow studies have shown that in diabetes the retinal blood flow increases markedly with progress of background retinopathy, decreasing finally where proliferative retinopathy, with marked arteriolar narrowing, is present. On the basis of these findings a working hypothesis for the pathogenesis of diabetic retinopathy is presented.


Some observations about retinal vascular-neuronal interrelationships. The relationship between the intraretinal vessels of the human fundus and the surrounding neuronal tissue is discussed with regard to various disease states. Present evidence suggests that primary retinal neuronal death does not lead to secondary retinal capillary death. Primary retinal capillary non-perfusion generally leads to concomitant retinal neuronal degeneration, but if the occlusion takes place over a long period of time the surrounding neural retina may survive. It is suggested that many retinal degenerative changes, including retinal detachment, are due to primary retinal vascular alterations.


Radiographic abnormalities in eyes with retinoblastoma and other disorders. The importance of radiographic examinations of pathological ocular material is stressed. Characteristic ocular radiodensities are observed with retinoblastomas, calcified cataracts, senile scleral plaques, intraocular ossification, and a variety of radiodense foreign bodies. Radiographs supplement other techniques for the documentation of ocular abnormalities and in certain instances may be the ideal method. They also permit the precise localisation of radiodense foreign bodies that need to be removed prior to the processing of tissue for microscopic examination. In certain situations valuable data can be obtained by x-ray examinations of embedded material. Retinoblastomas have an extremely high incidence of radiodensities with a characteristics appearance. This finding stresses the clinical importance of utilising and developing clinically applicable techniques for the detection of calcification in patients with suspected retinoblastomas.


Amoebic keratitis: a clinicopathological case report. A 22-year-old farmer with recurrent corneal erosions and keratoiritis underwent a penetrating keratoplasty. Investigations carried out on the corneal disc revealed an amoebic keratitis, which is considered to be a very rare condition.


Senile scleral plaques and senile scleromalacia. A case of senile scleral plaques is reported in which expulsion of a calcified plaque anterior to the insertion of the medial horizontal rectus muscle gave rise to senile scleromalacia. Histopathological examination revealed a second calcified plaque at the site of the contralateral senile scleral plaque in this eye. This case indicates that senile scleromalacia is a scleral disease sui generis which occurs by expulsion of a calcified plaque in advanced cases of senile scleral plaques. Senile scleromalacia has not previously been described as a clinical entity.


Lymphocytic tumours of indeterminate nature: a 5-year follow-up of 98 conjunctival and orbital lesions. Ninety-eight adnexal lymphocytic tumours of indeterminate nature have been followed up for at least 5 years. Both non-disseminating and disseminating tumours have either very similar or identical histopathological features. It is not possible to make a definitive diagnosis when the lesions are first seen, and a conclusion can be reached only after a prolonged follow-up. The nature of the non-disseminating tumours is unknown.


Pit-like changes of the optic nerve head in open-angle glaucoma. Six patients with open-angle glaucoma and acquired pit-like changes in the optic nerve head are presented. In 1 patient evolution of the pit-like defect is documented. In all 6 patients progression of associated visual field deficits is described. It is suggested that such pit-like changes in selected patients with glaucoma may not represent congenital lesions but rather local, progressive nerve head disease, occurring particularly in response to raised intraocular pressure. The management of patients with optic nerve head pitting and the pathogenesis of glaucomatous optic neuropathy are discussed with respect to this observation.


Contractile protein antigens in the cells of malignant melanoma of the choroid and their diagnostic significance. Sera from patients with proved and suspected melanoma of the uvea and from patients with innocuous uveal naevi were found by immunohistological techniques to have an affinity for the cytoplasm of malignant choroidal melanoma cells in an appreciable proportion of cases. The responsible antibodies were subsequently shown to react with smooth muscle protein in a variety of tissues and not to be melanoma-specific. It was also shown by electron microscopy that microfilaments consistent with contractile protein are a feature of the cytoplasm of malignant choroidal melanoma cells. The latter observations, which do not appear to have been reported previously, mean that false positives are to be expected if immunodiagnostic techniques are used for the recognition of malignant disease of the uvea, especially as smooth muscle antibodies can occur in non-neoplastic ocular diseases.


Multicentric primary malignant melanomas of the choroid: two separate malignant melanomas of the choroid and two uveal naevi in one eye. Two completely separate malignant melanomas of the choroid occurred in 1 eye containing 2 additional uveal naevi. Review of the literature disclosed 7 further patients with unilateral multicentric malignant melanomas of the choroid and 11 bilateral multicentric malignant melanomas of the uvea. General aspects of multicentric primary neoplasia are discussed and an interpretation is attempted.


Ocular hypertension and systemic responses to the water-drinking test. Osmolal, electrolyte, and other responses in peripheral blood were recorded after water loading of patients with ocular hypertension and chronic simple glaucoma, and with a small group of younger normal subjects. The osmolal responses of the ocular hypertensive group were significantly greater than for the glaucoma group, and further differences in plasma potassium concentration were also found. Drinking habits may exert a significant influence in ocular hypertension. The relative contributions to the diagnostic category of the variables recorded was evaluated by discriminant analysis, and the application of this retrospective approach to the assessment of new patients is discussed.


Influence of starches from different sources on protein utilization in rats. 1. The role of the starch component of cereals and legumes on the utilization of casein-protein was studied. 2. In comparison with maize starch the legume and potato starches caused a significant lowering of net protein utilization (NPU). And this reduction in NPU could be partially restored by cooking the diets before they were fed to the rats. 3. It is suggested that in NPU studies involving rats the experimental diet should always be cooked so that the conclusions drawn may be applicable to human diets.


Dietarily-induced changes in voluntary ethanol consumption and ethanol metabolism in the rat. 1. The voluntary ethanol consumption, ethanol elimination rate and blood acetaldehyde level after intraperiotoneal injection of ethanol were studied in rats receiving diets with highly imbalanced proportions of dietary protein, carbohydrate and fat. 2. The rats, which received the low-protein diet containing 0.05 of the total energy as protein, 0.80 as carbohydrate and o.15 as fat, drank only approximately half as much ethanol as did the control group, which received 0.30 of its total food energy from protein, 0.55 from carbohydrate and 0.15 from fat. Ethanol elimination rate in the low-protein group was decreased and the blood acetaldehyde level after ethanol injection was markedly increased. 3. On the high-fat diet, which contained 0.30 of the total energy from protein, 0.05 from carbohydrate and 0.65 from fat, the rats drank significantly more ethanol than did the rats on the control diet; their ethanol elimination rate was decreased but their blood acetaldehyde level was not affected. 4. In conclusion, the strong decrease in voluntary ethanol drinking by the low-protein group may have been caused by the increased acetaldehyde accumulation in the blood, but a particularly low blood acetaldehyde level was not one of the factors inducing excessive ethanol drinking in the high-fat group.


The estimation of whole-body zinc and Zn turnover in rheumatoid and osteoarthritis using 65Zn tracer. 1. A method of estimating whole-body zinc, and Zn balances using a two-compartment model in combination with whole-body counting of 65Zn, is described. The method is applied to patients with rheumatoid and osteoarthritis. 2. The results suggested that there was not a wide variation in whole-body Zn and Zn turnover in individuals with these two diseases and no clearcut difference between patients with one or the other.


Lysosomal stability, superoxide dismutase and zinc deficiency in regenerating rat liver. 1. Lysosomal stability was slightly increased in regenerating livers from rats receiving a zinc-deficient (less than 0.5 mg/kg) diet for 10 d before surgery. 2. The activity of superoxide dismutase (EC 1.15.1.1) was significantly reduced in the same tissues. 3. The results do not support the view that increased lysosomal fragility represents a major biochemical consequence of nutritional Zn deficiency in animals.


Retention of fluoride from diets containing materials produced during aluminium smelting. 1. Using male rats, the availability of fluoride from five by products of the aluminium smelting industry was tested. This was done by considering the balance between fluoride administered in controlled diets and the total fluoride content of waste products eliminated over an experimental period of 1 week. 2. Results indicated that yields of available fluoride expressed as percentage weight of original material were cryolite (Na3ALF6), 45.9%; sodium fluoride (NaF), 41.7%; aluminium fluoride (AFL3.H20), 9.1%; mist eliminator grid solids (with CaF2), 9.4%; reclaimed alumina with adsorbed F, 0.27%. 3. Of the various tissues analysed for fluoride content, only the kidney and femur showed any significant correlation with the amounts of fluoride absorbed or retained.


Transfer of urea from the blood to the rumen of sheep. 1. The rate of transfer of plasma urea-nitrogen to rumen ammonia was measured by infusion of 15NH4Cl and [15N]urea into sheep given brome grass (Bromus inermis) or lucerne (Medicago sativa) pellets. Urea was infused into the rumen or abomasum of two sheep given brome grass in order to increase the concentration of rumen ammonia. 2. From 6.2 to 9.8 g/d of plasma urea-N were transferred to the rumen of sheep given brome grass pellets and a measurement of 1.3 g nitrogen/d was obtained for a sheep given lucerne pellets. When urea was infused into the rumen of sheep given brome grass pellets the transfer was only 2.8--3.7 g N/d. 3. There was a significant negative correlation between the rate of transfer of plasma urea-N to the rumen and the concentration of rumen ammonia.


The effect of dihomo-gamma-linolenic acid (20: 3,n-6) on the composition of phospholipid fatty acids in the liver of rats deficient in essential fatty acids. 1. Rats were fed on either a diet deficient in essential fatty acid (EFA) or one supplemented with dihomo-gamma-linolenic acid (20:3,n-6) at levels that represented 0.25, 0.5, 1.0 and 2.0% of the dietary energy. 2. Supplementation of the diet of EFA-deficient animals with 20:3,n-6 reversed most of the fatty acid changes induced in the liver phospholipid fraction. 3 The EFA potency of 20:3,n-6 was found to be similar to that of gamma-linolenic acid (18:3,n-6) which has been shown to be higher than that of linoleic acid (18:2,n-6).


The in vitro metabolism of 25-hydroxycholecalciferol by pig kidney: effect of low dietary levels of calcium and phosphorus. 1. Homogenates of pig kidney incubated in vitro convert 25-hydroxycholecalciferol to the dihydroxymetabolites almost as efficiently as homogenates of chick kidney under similar conditions. 2. The cortex of the kidney carries out this conversion more efficiently than does the medulla. 3. Kidneys from pigs given low calcium or low phosphorus diets produce about 40% more 1,25-dihydroxycholecalciferol than those from pigs given diets containing normal levels of Ca and P.


The effects of defaunation of the rumen on the growth of cattle on low-protein high-energy diets. 1. The effects of defaunation of the rumen of cattle on low-protein diets was studied using animals given free access to a basal diet of liquid molasses and 1500 g oaten straw/head per d. These diets induced moderate numbers of protozoa in the rumen. 2. Nonyl phenol ethoxylate (trade name teric GN9) was used for defaunation; 100 g teric GN9 was found to be sufficient to eliminate protozoa from the rumen. 3. In cattle given the basal diet without bypass protein supplementation, defaunation had no effect on growth rates. Addition of 240 g of a feed pellet containing bypass protein increased growth rate significantly. Growth rates were significantly increased by 43% in cattle on the higher protein intake and where protozoa were removed. Intake of molasses was apparently stimulated by a protein supplementation but not by defaunation and this finding is discussed. 4. The results demonstrate that in cattle given a molasses-based diet, low in bypass protein, growth rates can be stimulated by defaunation without an effect on feed intake, the main effect apparently arising through an increased efficiency of utilization of feed.


Adaptations in the calcium and phosphorus metabolism of sheep in response to an intravenous infusion of Ca. 1. The effect on calcium and phosphorus metabolism of a high rate of Ca infused directly into the blood of Ca -deficient wethers, already receiving an adequate Ca intake, has been studied by a combination of balance and radioisotope techniques. 2. The rate of Ca retention, which was already high, increased only slightly during the infusion before it reached a maximum. The surplus Ca was compensated for by a decrease in the rate of absorption and an increase in the rate of urinary Ca excretion. 3. These findings support the theory that at maximum retention the rate of Ca absorption becomes regulated according to the rate at which Ca can be stored in bone. 4. results suggest that the decreased absorption was due to a decrease in the rate of active absorption and that the low rate remaining was due to diffusion. 5. The rate of P retention was increased by the Ca infusion, possibly as a result of the increased Ca retention.


An evaluation of total lysine as a predictor of lysine status in protein concentrates for growing pigs. 1. Eight protein concentrates were compared on a total lysine basis in lysine-deficient diets for pigs during the 20--45 kg growth phase. Each diet was also supplemented with free lysine to verify that lysine was the first limiting amino acid. 2. With fish meal, skim-milk powder, rapeseed meal and soya-bean meal growth rates and feed conversion efficiencies were similar and superior (P less than 0.05) to those produced with cottonseed meal, two meat meals and sunflower meal. 3. The response to added free lysine confirmed that lysine was the first limiting amino acid in all diets except skim milk, where the growth response was not significant (P less than 0.05). 4. The growth response of the pigs indicated that the availability of lysine in cottonseed meal, the two meat meals and sunflower meal was reduced by approximately 60% compared to that in the other protein concentrates. 5. Rat bio-assay estimates of the availability of lysine in the protein concentrates were in general agreement with those calculated from the response in pigs. There was little relationship between the Silcock estimates for lysine availability for the cottonseed, meat meals or sunflower meal relative to either the rat bio-assay estimates or the response of pigs.


Differences in the proportions of branched-chain fatty acids in subcutaneous triacylglycerols of barley-fed ruminants. 1. The fatty acids of the triacylglycerols of subcutaneous adipose tissue of cattle, sheep, goats and red deer (Cervus elaphus) which consumed either herbage or a barley-rich diet were analysed for their content of branched-chain components. 2. Whereas the consumption of the barley-rich diet by sheep and goats was associated with the occurrence of relatively high proportions of branched-chain fatty acids, it was not in cattle and red deer.


Quantitative selenium metabolism in normal New Zealand women. 1. Quantitative selenium metabolism has been studied in normal young New Zealand women by measuring total Se intake and urinary and faecal Se output, and by using values for absorption, excretion and turnover of 75Se determined after administration of[75Se]selenomethionine or [75Se]selenite. 2. In a period of 14 d when a normal ad lib. diet was being consumed, mean dietary Se for four women was 24.2 microgram/d, mean urinary Se was 13.1 microgram/d and mean faecal Se was 10.8 microgram/d; mean Se balance during this time was + 0.3 microgram/d. 3. Intestinal absorption of food Se was 0.76--0.83 of intake (mean 0.79). 4. Whole-body Se was calculated in three different ways; (a) using the specific activity of urinary Se and retained whole-body 75Se; (b) using plasma Se and the occupancy of 75Se in whole-body and plasma; (c) using absorbed food Se and the occupancy of absorbed 75Se in whole-body. 5. Whole-body Se calculated from measurements obtained following the administration of [75Se]selenomethionine was 4.7--10.0 mg (mean 6.9) using method (a), 4.1--7.2 mg (mean 5.2) using method (b) and 4.3--8.9 mg (mean 6.2) using method (c). 6. Whole-body Se calculated from results obtained after giving [75Se]selenite was 2.7--3.4 mg (mean 2.9) using method (a), 2.3--5.0 mg (mean 3.5) using method (b) and 2.1--3.0 mg (mean 2.6) using method (c). 7. The results of this study indicate that the minimum dietary requirement of Se for the maintenance of normal human health is probably not more than 20 microgram/d.


Haematological studies on vegans.

Haematological studies on vegans. 1. The concentrations of vitamin B12 and folate in the serum and folate in the erythrocytes were determined and full blood counts made on a series of caucasian vegans and omnivore controls. 2. The blood counts and films were normal in all the vegans and no subject had a haemoblobin concentration below the lower limit of normality. 3. Although within the normal range, male but not female vegans had lower values for erythrocyte counts and higher values for mean corpuscular volume and mean corpuscular haemoglobin than their controls regardless of whether they were taking vitamin B12 supplements or not. 4. The mean serum vitamin B12 concentration was lower in the vegans not taking vitamin B12 supplements and in those using foods supplemented with the vitamin than in the controls, but in no subject was it below 80 ng/1. 5. The serum folate concentrations were higher in the vegans than in their controls. The mean value for erythrocyte folate tended to be greater in the vegans not taking vitamin B12 supplements. No subject had an erythrocyte folate concentration of less than 100 microgram/1. 6. It is concluded that megaloblastic anaemia is very rare in caucasian vegans and that a diet consisting entirely of plant foods is generally adequate to promote normal blood formation providing it is composed of a mixture of unrefined cereals, pulses, nuts, fruit and vegetables and is supplemented with vitamin B12.


Molecular weight in detergent solution of acetylcholine receptor from Torpedo californica. Acetylcholine receptor extracted in detergent solution from the electric tissue of Torpedo californica and purified by affinity chromatography contains predominantly two molecular weight species. These have been separated by sedimentation in a sucrose density gradient, and their molecular properties have been determined by sedimentation equilibrium and sedimentation velocity measurements in the analytical ultracentrifuge. The molecular weights of these species have been determined, without prior determination of the extent of detergent bound to them, by the adjustment of solvent density with D2O so as to blank out the contribution of bound detergent to the sedimentation potential. The molecular weights of the protein moieties are 250 000 and 500 000. Since these species are identical in specific activity and polypeptide composition they are related as monomer and dimer. The hydrodynamic properties of the detergent complexes of monomer and dimer were derived from combined measurements of sedimentation equilibrium and sedimentation velocity. The S20,w'S are 8.6 S and 12.8 S and the Stokes radii are 7.3 nm and 9.5 nm. For both monomer and dimer, the ratio of the Stokes radius to the minimum possible radius for the protein-detergent complex falls outside the range of values for globular proteins.


Two-step purification of the major phosphorylated protein in reticulocyte 40S ribosomal subunits. In reticulocytes, a single ribosomal protein, S13, has been shown to be phosphorylated by the cAMP-regulated protein kinases. The 40S ribosomal subunits were phosphorylated in vitro with [gamma-32P]ATP to facilitate the identification of S13 during the two-step purification procedure. Total ribosomal protein from the 40S subunit was fractionated by phosphocellulose chromatography in urea, and S13 was purified to homogeneity by gel filtration on Sephadex G-100. The protein was identified by the radioactive phosphate, by molecular weight, and by the migration characteristics in a two-dimensional polyacrylamide gel electrophoresis system. Thin-layer electrophoresis of partial acid hydrolysates of S13 showed that more than one phosphorylated residue was present in the same oligopeptide, indicating at least some of the phosphoryl groups were clustered in the protein molecule.


Selectivity of RNA chain initiation in vitro. 3. Variables affecting initiation of transcription. The effects of salt, temperature, enzyme to DNA ratio, and heparin challenge on both total RNA synthesis and synthesis from specific promoters are examined using DNA from bacteriophages lambdacb2 and T7. Determination of synthesis from specific promoters is carried out by the fractionation and quantitation on polyethylenimine-cellulose thin-layer chromatograms of the 5'-terminal oligonucleotides produced by digestion of the RNA products with T1 RNase. The major findings of this work are that (1) lambdacb2 promoters are more salt sensitive than T7 promoters and the salt concentration affects individual promoters differently, (2) T7 promoters initiate maximally at 37 degrees C but the transition temperatures of promoters vary and may be dependent on the salt concentration, (3) increasing the enzyme to DNA ratio results in increasing initiations at the promoters on T7 DNA without causing measurable initiation at non-promoters, and (4) T7 and lambdacb2 promoters show differences in stability when challenged with heparin.


Enzymatic oligoribonucleotide synthesis with T4 RNA ligase. The substrate specificity of T4 RNA ligase has been examined to determine whether the intermolecular reaction is sufficiently general to realize its potential in the enzymatic synthesis of oligoribonucleotides of defined sequence. Reactions between a variety of acceptor molecules with 3'- and 5'-bydroxyl groups and donor molecules with 3'- and 5'-phosphates indicate that the minimal substrates are a trinucleoside diphosphate acceptor and a nucleoside 3',5'-bisphosphate donor. Increasing the chain length of either the acceptor or donnor has little effect on the rate or extent of reaction. Although the base composition of the donor has only a small effect on the reaction rate, the presence of uridine in the acceptor greatly reduces the amount of product formed. The presence of a phosphate on the 3' terminus of the donor molecule permits a unique intermolecular product with a 5'-hydroxyl and a 3'-phosphate. By enzymatically either adding a 5'-phosphate or removing the 3'-phosphate, a new donor or acceptor is prepared so synthesis of an oligomer chain can proceed in either direction. With the simplicity of this enzymatic pathway and the rather broad substrate specificity of T4 RNA ligase, a convenient method for the synthesis of oligoribonucleotides is established.


Synthesis of modified nucleoside 3',5'-bisphosphates and their incorporation into oligoribonucleotides with T4 RNA ligase. A simple procedure is described to prepare nucleoside 3'(2'),5'-bisphosphates from the corresponding nucleosides with the use of pyrophosphoryl chloride. This method is rapid, gives nearly quantitative yields and, most importantly, can be used for a variety of nucleosides with base and sugar modifications. Since 3',5'-bisphosphates are donors in the T4 RNA ligase reaction, a single residue can be enzymatically attached to the 3' end of oligoribonucleotides. By these procedures, five different ring-modified nucleosides and one sugar-modified nucleoside were incorporated onto the 3' end of (Ap)3C. In two cases, an additional step of synthesis with RNA ligase resulted in the modified nucleotide being located in an internal position in the oligonucleotide. Thus, a general method for the synthesis of oligoribonucleotides containing modified nucleosides is outlined. Since many of the modified nucleosides are fluorescent, oligomers containing them should be useful in a variety of physical and biochemical studies.


Interaction of aromatic residues of proteins with nucleic acids. Binding of oligopeptides to copolynucleotides of adenine and cytosine. The binding of the peptide Lys-Trp-Lys to various single-stranded copolymers of adenine and cytosine has been investigated using circular dichroism (CD) and fluorescence measurements. Two types of complexes are formed, both involving electrostatic interactions between lysyl residues and phosphate groups. The fluorescence quantum yield of the first complex is identical with that of the free peptide. The other complex involves a stacking of the polynucleotide bases with the tryptophan residue whose fluorescence is quenched. The binding of the peptide leads to a conformational change of the copolymers as shown by the CD variations. The fluorescence and CD data have been analyzed according to the model involving the two types of complexes. The values of the binding constants have been studied as a function of the cytosine content of the copolymers. Analysis of the stacking process in term of nearest neighbor frequency demonstrates that this binding is sequence dependent and is favored in AA sequence as compared with AC, CA, and CC sequence. Thus this simple tripeptide is able to distinguish between various base sequences in a single-stranded nucleic acid.


Direct microsequence analysis of polypeptides using an improved sequenator, a nonprotein carrier (polybrene), and high pressure liquid chromatography. We have combined the use of a nonprotein carrier (Polybrene), high pressure liquid chromatography, and modifications in Edman chemistry with the improvements of a commercial spinning cup sequenator suggested by Wittmann-Liebold [Wittmann-Liebold, B. (1973) Hoppe-Seyler's Z. Physiol. Chem. 354, 1415] to analyze amino acid phenylthiohydantoins obtained from automated Edman degradation of microquantities of polypeptide directly without the use of radiolabel. This approach has allowed us to determine the sequence of the N-terminal 47 residues of sperm whale myoglobin starting with 200 pmol of protein, 77 residues of an antibody light chain with 5 nmole of protein, and 54 residues of an antibody heavy chain with 8 nmol of protein. In addition, we completely sequenced a hydrophobic 14-residue peptide at the 1.5-nmol level. Our technique of direct analysis for microsamples is capable of providing routine, extende N-terminal sequence analysis for nanomole and subnanomole levels of polypeptides and protines, and it also is applicable to analysis of more classical sample quantities.


Nuclear magnetic relaxation by the manganese in aqueous suspensions of chloroplasts. Proton and oxygen-17 NMR relaxation rate (T1-1 and T2-1) data are presented for aqueous suspensions of dark-adapted chloroplasts. It is concluded from the dependence of the proton relaxation rates (PRR) upon Mn concentration that T1-1 and T2-1 are determined largely by the loosely bound Mn present in the chloroplast membranes. The frequency and temperature dependences of PRR are characteristic of Mn(II). The effects of oxidants (e.g., ferricyanide) and reductants (e.g., tetraphenylboron) on the PRR indicate that only about one-third to one-fourth of the loosely bound Mn is present in the dark-adapted chloroplasts as Mn(II), the remainder being in a higher oxidation state(s), probably Mn(III). The frequency dependence of the PRR for the chloroplast suspensions was fitted by a simplified form of the Solomon-Bloembergen-Morgan equations, and the following parameters were obtained: tauS = (1.1 +/- 0.1) X 10(-8) S; tauM = (2.2 +/- 0.2) X 10(-8) S; and B = (0.9 +/- 0.09) X 10(19). The oxygen-17 T1 and T2 data for suspensions before and after treatment with a detergent are consistent with the location of the manganese in the interior of the thylakoids. An analysis of the relaxation rates shows that the average lifetime of a water molecule inside a thylakoid is greater than 1 ms.


Mechanism of action of adenosylcobalamin: hydrogen transfer in the inactivation of diol dehydratase by glycerol. We have investigated the kinetic characteristics of the inactivation of the adenosylcobalamin-dependent enzyme propanediol dehydratase by glycerol, (RS)-1,1-dideuterioglycerol, (R)-1,1-dideuterioglycerol, and perdeuterioglycerol in the presence of 1,2-propanediol and 1,1-dideuterio-1,2-propanediol. The results imply that hydrogen (or deuterium) attached to C-1 of 1,2-propanediol participates in the inactivation process and contributes to the expression of a kinetic isotope effect on the rate of inactivation. The mechanism for this inactivation must involve the cofactor as an intermediate hydrogen carrier, presumably in the form of 5'-deoxyadenosine. Moreover, a mechanism involving a rate-determining transfer of hydrogen from an intermediate containing three equivalent hydrogens quantitatively accounts for all of the results. When diol dehydratase holoenzyme is inactivated by [1-3H]glycerol, 5'-deoxyadenosine which is enriched in tritium by a factor of 2.1 over that in glycerol can be isolated from the reaction mixture.


Binding site of a dextran-specific homogeneous IgM: thermodynamic and spectroscopic mapping by dansylated oligosaccharides. The hapten binding properties of the homogeneous mouse IgM secreted by MOPC-104E were investigated. Hapten-association constants were determined either by equilibrium and displacement equilibrium dialysis or by fluorometric titrations of the protein with the fluorescent derivatives of the haptens. For the latter type of measurements, several oligosaccharides were derivatized to the corresponding dansylhydrazones. The synthesis, generally applicable to oligosaccharides with free reducing ends, is described. Analysis of the thermodynamic parameters for the binding of 18 haptens forms the basis for proposing a model of the binding site of MOPC-104E. This model is supported and refined by results of the measurements of linear and circular polarization of the fluorescence of the dansylated haptens. The binding site is proposed to consist of a cavity with about 12-A depth, complementary to a terminal nonreducing nigerosyl group. At the entrance to this cavity, a further subsite is identified forming interactions of lower specificity with an additional glucose unit.


Electron acceptors associated with P-700 in Triton solubilized photosystem I particles from spinach chloroplasts. Flash-induced absorption changes of Triton-solubilized Photosystem I particles from spinach were studied under reducing and/or illumination conditions that serve to alter the state of bound electron acceptors. By monitoring the decay of P-700 following each of a train of flashes, we found that P-430 or components resembling it can hold 2 equivalents of electrons transferred upon successive illuminations. This requires the presence of a good electron donor, reduced phenazine methosulfate or neutral red, otherwise the back reaction of P-700+ with P-430 occurs in about 30 ms. If the two P-430 sites, designated Centers A and B, are first reduced by preilluminating flashes or chemically by dithionite under anaerobic conditions, then subsequent laser flashes generate a 250 microseconds back reaction of P-700+, which we associate with a more primary electron acceptor A2. In turn, when A2 is reduced by background (continuous) illumination in presence of neutral red and under strongly reducing conditions, laser flashes then produce a much faster (3 microseconds) back reaction at wavelengths characteristic of P-700. We associate this with another more primary electron acceptor, A1, which functions very close to P-700. The organization of these components probably corresponds to the sequence P-700-A1-A2-P-430[AB]. The relation of the optical components to acceptor species detected by EPR, by electron-spin polarization or in terms of peptide components of Photosystem I is discussed. Preliminary experiments with broken chloroplasts suggest that an analogous situation occurs there, as well.


Kinetics of ADP, ATP transport in mitochondria as studied by the quench-flow method. The kinetics in the millisecond range of ADP, ATP counterexchange in rat liver mitochondria were investigated using a quench-flow apparatus. The exchange was stopped with atractylate and the mitochondria were separated by centrifugation. 1. After correcting for leakage due to flow stress, apparent biphasic exchange kinetics were observed, with a more rapid phase within 100--200 ms, which extends to 10% of the total exchange. In general, the extent of the rapid phase increased when the translocation rate was changed under various conditions, in agreement with the model of the quench mechanism by atractylate. 2. The nature of the "rapid" phase was analyzed in the "steady" and "transient" state of the translocation and was shown to be caused by a delayed binding of atractylate due to competition with ADP. This quench delay results in a residual exchange which could explain the rapid part of the kinetics. 3. Deenergization of mitochondria by valinomycin or by uncoupler largely abolishes the rapid kinetic phase. This is explained by an increased availability of carrier sites at the outer face of the membrane to atractylate in the deenergized state, resulting in a more rapid quench. 4. The interpretation of the rapid exchange phase as a function of carrier sites accessible to atractylate quenching at the outer membrane face was simulated by a computer program based on the reorientating carrier model. With a set of rate constants, an approximate fit for the extent of quench delay with the experimental data is obtained.


Kinetics of reduction of the oxidized primary electron donor of photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation. Absorption changes (deltaA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures. In the microsecond time range the difference spectrum of deltaA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P+-700; it decays in a polyphasic manner with half-times of 17 microseconds, 210 microseconds and over 1 ms. The oxidized primary donor of Photosystem II (P+II) is not detected with a time resolution of 3 microseconds. After treatment with 3--10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P+II is observed and decays biphasically (a major phase with t1/2=20--40 microseconds, and a minor phase with t1/2 congruent to 200 microseconds), probably by reduction by an accessory electron donor. In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P+II is reduced with a half-time of 25--45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.


Polypeptide profiles of chlorophyll . protein complexes and thylakoid membranes of spinach chloroplasts. In addition to the major chlorophyll . protein complexes I and II, two minor chlorophyll proteins have been observed in sodium dodecyl sulfate (SDS))-polyacrylamide gels of spinach chloroplast membranes. These minor pigmented zones appeared to be derived from the light-harvesting chlorophyll a/b . protein and from the reaction centre complex of Photosystem II. Data are presented on the polypeptide profiles of purified digitonin-subschloroplast particles, with special regard to the effect of solubilization temperature and extraction of lipids. The results are compared with the SDS-polypeptide pattern of spinach thylakoids obtained under exactly the same conditions with respect to electrophoresis technique, solubilization method and presence of lipid. In addition, the effects of temperature and lipid extraction on the distinct chlorophyll . protein complexes appearing in SDS gel electrophoretograms of chloroplast membranes were studied by slicing the chlorophyll-containing regions and subjecting them to a second run with or without heating or extraction with acetone. By supplementing these data with an examination of the polypeptide composition of cytochrome f and coupling factor, it has been possible to identify most of the major chloroplast membrane polypeptides.


Intracellular location of flexirubins in Flexibacter elegans (Cytophagales). The inner and outer membranes of 2 strains of Gram-negative Flexibacter elegans, Fx e1 and Fx 3/4, could be separated on sucrose density gradients after the cells had been converted into spheroplasts, and the spheroplasts had been lysed in presence of EDTA and the detergent Brij 58. The light fraction (rho = 1.14 g . cm-3) contained the components of the respiratory chain in high concentrations, but only low amounts of the lipopolysaccharide component, 2-keto-3-deoxyoctonic acid, and was thus mainly material from the inner membrane. The heavy fraction (rho = 1.175 g . cm-3) contained only traces of respiratory chain enzymes, but the majority of the 2-keto-3-deoxyoctonic acid, and was thus mainly material from the outer membrane. The flexirubin pigments were found almost quantitatively in the latter fraction. Strain Fx 3/4 produced carotenoids in addition to flexirubins; in this case the flexirubins were located in the outer, and the carotenoids in the inner membrane.


Topography of glycoproteins in the chick synaptosomal plasma membrane. Chick brain synaptosomes or synaptic subfractions were treated with neuraminidase (EC 3.2.1.18) and/or galactose oxidase (EC 1.1.3.9) preparations in which proteolytic activity was inhibited with phenylmethanesulfonyl fluoride followed, after washing, by reductive incorporation of sodium boro[3H]hydride to identify galactose residues exposed on the synaptosomal external surface. Control experiments to demonstrate restriction of labeling to the external surface involved comparing the radioactivity in synaptoplasmic, soluble polypeptides isolated after labeled, isolated synaptoplasm and examining incorporation into fractions incubated without enzymes. Intactness of the synaptic plasma membrane after labeling was shown by trypsin digestion studies. Polypeptides were separated on sodium dodecyl sulfate polyacrylamide gels and were detected by a liquid scintillation counting procedure. Eleven major radioactive peaks were found after galactose oxidase treatment and reduction of isolated synaptic membranes. When intact synaptosomes were labeled, the same components were detected. When isolated synaptic membranes or intact synaptosomes were treated with neuraminidase before galactose oxidase treatment, three additional components were labeled. These results suggest that (a) chick synaptic membranes have a complex mixture of glycoproteins, (b) all major chick synaptic membrane glycoproteins labeled by galactose oxidase have most or all carbohydrate groups exposed at the exterior surface of the synaptosome, (c) all major, externally-disposed polypeptides of these synaptic membranes are glycoproteins.


Role of disulfide bonds in the attachment and function of large, external, transformation-sensitive glycoprotein at the cell surface. Reduction of disulfide linkages by dithiothreitol removes LETS (large, external, transformation-sensitive) protein from the cell surface. This process is dependent upon the concentration of dithiothreitol and the time and temperature of reaction. At 0 degrees C the release of LETS protein by dithiothreitol is completely blocked, but this is apparently not due to a requirement for metabolic energy. At this temperature, reduction of LETS protein is incomplete. These results suggest that intact disulfide bonds are involved in the retention of this protein on the cell surface. Furthermore, reduction of purified LETS protein interferes with its ability to confer flattened morphology and increased adhesivity when added to transformed cells. It appears, therefore, that disulfide bonds are functionally important at the cell surface.


Action of surface-active substances of biological membranes. III. Comparison of hemolytic activity of ionic and nonionic surfactants. The hemolytic action of a number of homologous series of cationic surfactants on human erythrocytes was measured. The hemolytic effects of anionic, nonionic and cationic surface-active agents are compared. The relationship which exists between the key physicochemical properties of surfactants (critical micelle concentration, hydrophile-lipophile balance) and their hemolytic capacities is discussed. The parameters required to compare the actions of various surfactants on different cellular membranes are considered in relation to the study of the correlation between the surfactant lytic effects and the features of the membrane molecular organization.


Erythrocyte lipid composition and sodium transport in human liver disease. In patients with liver disease there are usually increases in erythrocyte cholesterol and phosphatidylcholine concentrations. This increase in membrane lipid changes the shape of the erythrocyte and "spur" or "target" cells may be present. Sodium fluxes were measured in erythrocytes from 17 patients with a variety of liver diseases and from 17 normal subjects and the values related to the lipid content of the membrane. Ouabain-insensitive and ouabain-sensitive effluxes were lower in patients than in normal subjects and the reduction in ouabain-insensitive efflux was more marked. Sodium influx was also significantly lower in erythrocytes from patients than controls. Ouabain-sensitive and ouabain-insensitive effluxes and sodium influx did not correlate with the cholesterol content of erythrocytes from patients. Significant negative correlations were noted between ouabain-insensitive sodium efflux (r=--0.63, P less than 0.01), sodium influx (r=--0.61, P less than 0.01) and intracellular sodium concentration (r=--0.66, P less than 0.01) and the cholesterol : phospholipid molar ratio of the cell but there was no significant correlation between this ratio and the ouabain-sensitive sodium efflux (r=0.41, P less than 0.05). These results support the hypothesis that an altered lipid composition may affect the permeability of the erythrocyte membrane in patients with liver disease.


Association of the major coat protein of fd bacteriophage with phospholipid vesicles. The association of the major coat protein of fd bacteriophage with a phospholipid bilayer was investigated by analyzing the protein's susceptibility to proteolysis and its circular dichroism spectrum when incorporated into single-walled phospholipid vesicles. In the limits tested, this association appeared to be independent of the mass ratio of protein to lipid and of vesicle size, phospholipid composition, and method of preparation. The circular dichroism data are consistent with a similar "membrane-bound" conformation for all cases of vesicle-associated coat protein and for deoxycholate micelle-associated coat protein. Proteolysis of coat protein associated with deoxycholate micelles and with phospholipid vesicles defined the central hydrophobic core presumed to represent that portion of the protein which associates with membrane bilayers in vivo. The isolated core, which assumed a predominantly beta-type conformation in detergent solution, maintained a beta conformation when associated with a vesicle phospholipid bilayer.


A new approach in the kinetics of biological transport. The potential of reversible inhibition studies. Kinetic equations are derived for reversible inhibition of both active and facilitated transport systems for seven common experimental arrangements. It is shown that the unique features of transport kinetics may be exploited to give new kinds of information. It is also shown that that the familiar rules of enzyme kinetics, though often applied to transport, can be seriously misleading. The analysis leads to the following general conclusions: (1) A competitive mechanism frequently gives rise to non-competitive kinetics, depending on the experimental design, but a non-competitive mechanism never produces competitive kinetics. (2) Inhibition studies on exchange diffusion at equilibrium in non-active systems or in the final steady state in active systems are the only unambiguous kinetic tests to distinguish competitive from non-competitive mechanisms. (3) Substrate analogs that are bound to the carrier and transported are readily distinguished by inhibition kinetics from those not transported, even though both may rapidly enter the cell by another route. (4) Even in non-active systems competitive inhibitors commonly have far different affinities for the substrate sites on the two membranes faces: where sufficient non-polarity allows their penetration into the cell, inhibition kinetics readily establish such sideness in their action. (5) Inhibition kinetics of the mixed competitive and non-competitive type result from moderately asymmetrical binding of inhibitor at the substrate site. (6) Asymmetry is a necessary feature of active transport: hence studies of inhibition kinetics should provide important insights into its mechanism.


Uncouplers of oxidative phosphorylation. A structure-activity study of their inhibitor effect on passive chloride permeability. Uncoupling agents inhibit chloride transport in red blood cells, which is a metabolism-independent process. An analysis of the molecular requirements shows that this inhibitory activity is closely correlated with the electronic and the hydrophobic bonding properties of phenols: the more lipophilic and the more electron-attracting the substituent groups are, the greater the activity they confer on the parent molecule. A recent structure-activity study concerning various classes of reversible inhibitors of chloride transport led to the same conclusion (Motais, R. and Cousin, J.L. (1977) in International Conference on Biological Membranes: Drugs, Hormones and Membranes (Bolis, L., Hoffman, J.F. and Straub, R.W., eds.), Raven Press, New York, in the press). The effects of substituents on the activity of phenols as uncouplers have been recently examined (Stockdale, M. and Selwyn, M.J. (1971) Eur. J. Biochem. 21, 565). The comparison of these results with our data shows that uncoupling depends more on electronic properties of phenols than does choloride inhibition.


Liposomal membrane. I. Chemical damage of liposomal membranes with functional detergent. The interaction and reaction between liposomal membrane and a functional detergent, N-hexadecyl-N-(imidazol-4-yl)methyl-N,N-dimethylammonium chloride hydroperchlorate (Im-I), have been investigated in conjunction with the leakage of bromothymol blue encapsulated as a marker in the bilayers of liposomes. Im-I carries an imidazole moiety and was expected to behave as a simple lipase model. The reaction with Im-I significantly enhanced the leakage of bromothymol blue encapsulated in the egg lecithin and dipalmitoyl phosphatidylcholine liposomes. During the course of reaction with Im-I, the formation of acyl-imidazole intermediate was clearly identified, which was certainly connected with the bromothymol blue release. From various kinetic results on bromothymol blue release and acyl-imidazole formation, it has been suggested that the bromothymol blue release from liposomal bilayer may be caused by the local and instantaneous decomposition of lipids when Im-I penetrates into the bilayer. However, it has also been demonstrated that the immediate reconstruction of liposomes retains the barrier function to protect against the further release of bromothymol blue.