Changes in the kinetic behaviour of threonine transport into Trypanosoma brucei elicited by variation in hydrogen ion concentration

1. The dependence of V and V/K(m) for threonine transport into Trypanosoma brucei upon the external concentration of H(+) was studied. 2. Two ionizing groups, the alpha-amino group of the substrate and a group at the substrate-binding site of the carrier, were found to influence the observed kinetic behaviour of transport. 3. The pK of the group at the substrate-binding site on the free carrier was found to be 6.95 at 30 degrees C and to be temperature-dependent; its heat of ionization was -63.8kJ, which is outside the range for most proton dissociations and suggests a significant contribution from some other source, possibly the remainder of the carrier or the membrane environment. 4. Binding of substrate caused the pK of its alpha-amino group to shift to a higher value, whereas that of the carrier group shifted to a lower value (6.65 at 30 degrees C). 5. The ionic interaction between substrate and carrier appeared to be involved in the stabilizing of the protonated substrate and the species of the carrier-substrate complex required for the membrane-translocation step. 6. The same ionic species of carrier-substrate complex is required for both substrate dissociation and translocation of the substrate through the membrane. 7. H(+) symport or antiport did not occur during threonine uptake.     

Properties of soluble somatostatin-binding protein

A soluble somatostatin-binding protein was detected in the cytosol fractions of various rat, human and bovine tissues. Maximum binding occurred at pH8.0-8.5 and was Ca(2+)-dependent. The specific binding of somatostatin per 10mug of cytosol protein from 12 rat tissues ranged between 36 and 15%, and 3% for peripheral blood cells. There was also substantial binding in cytosol from human anterior pituitary and liver, and bovine anterior pituitary. The specific binding in rat and human plasma in the presence of EDTA was only 1%. Gel filtration suggested a molecular weight of approx. 80000 for the somatostatin-binding protein from several sources. Exposure of the binding protein to trypsin eliminates somatostatin-binding activity but ribonuclease and deoxyribonuclease have no effect. The binding protein is thermolabile, ethanol-precipitable, and not completely specific for somatostatin. Bound (125)I-labelled [Tyr(1)]somatostatin is not easily displaced by excess of unlabelled somatostatin. The effects of dithiothreitol and mercaptoethanol on the binding of (125)I-labelled [Tyr(1)]somatostatin to the binding protein suggests that binding involves two sequential steps, first loose binding, then disulphide linkage. Since semipurified somatostatin-binding protein causes a dose-related inhibition of the binding of (125)I-labelled [Tyr(1)]somatostatin in radioimmunoassays for somatostatin, estimates of somatostatin content of tissue extracts by radioimmunoassay in some cases may be spuriously high. It is not yet clear whether the binding protein is a true cytosol protein or an easily solubilized membrane protein.     

Fatty acid biosynthesis by a particulate preparation from germinating pea

1. Fatty acid synthesis was studied in microsomal preparations from germinating pea (Pisum sativum). 2. The preparations synthesized a mixture of saturated fatty acids up to a chain length of C(24) from [(14)C]malonyl-CoA. 3. Whereas hexadecanoic acid was made de novo, octadecanoic acid and icosanoic acid were synthesized by elongation. 4. The products formed during [(14)C]malonyl-CoA incubation were analysed, and unesterified fatty acids and polar lipids were found to be major products. [(14)C]Palmitic acid represented a high percentage of the acyl-carrier protein esters, whereas (14)C-labelled very-long-chain fatty acids were mainly present as unesterified fatty acids. CoA esters were minor products. 5. The addition of exogenous lipids to the incubation system usually resulted in stimulation of [(14)C]malonyl-CoA incorporation into fatty acids. The greatest stimulation was obtained with dipalmitoyl phosphatidylcholine. Both exogenous palmitic acid and dipalmitoyl phosphatidylcholine increased the amount of [(14)C]-stearic acid synthesized, relative to [(14)C]palmitic acid. Addition of stearic acid increased the amount of [(14)C]icosanoic acid formed. 6. [(14)C]Stearic acid was elongated more effectively to icosanoic acid than [(14)C]stearoyl-CoA, and its conversion was not decreased by addition of unlabelled stearoyl-CoA. 7. Incorporation of [(14)C]malonyl-CoA into fatty acids was markedly decreased by iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid). Palmitate elongation was sensitive to arsenite addition, and stearate elongation to the presence of Triton X-100 or fluoride. The action of fluoride was not, apparently, due to chelation. 8. The microsomal preparations differed from soluble fractions from germinating pea in (a) synthesizing very-long-chain fatty acids, (b) not utilizing exogenous palmitate-acyl-carrier protein as a substrate for palmitate elongation and (c) having fatty acid synthesis stimulated by the addition of certain complex lipids.     

The association of the sulphogalactosylglycerolipid of rat brain with myelination

The sulphogalactosylglycerolipid of rat brain is closely associated with the process of myelination, as demonstrated by the following observations. 1. The lipid is barely detectable in rat brain before 10 days of age, accumulates rapidly between age 10 and 25 days, and remains relatively constant in amount (between 0.3 and 0.4mumol per brain) thereafter into adult life. 2. The activity of adenosine 3'-phosphate 5'-sulphatophosphate-galactosyldiacylglycerol sulphotransferase is almost absent before 10 days of age, attains a maximum at age 20 days, and slowly decreases thereafter with increasing age. This developmental pattern correlates well with that of other myelin-specific metabolites. 3. Both the concentration of the sulphogalactosylglycerolipid and the activity of sulphotransferase are greatly decreased in the non-myelinating jimpy mouse. 4. The myelin fraction of rat brain contains most of the sulphogalactosylglycerolipid. The lipid occurs in a diacyl and an alkylacyl form. Determinations of the relative amount of each type in brain showed about a 1:1 mixture in both 21-day-old and adult rats. Rats injected with H(2) (35)SO(4) at 20 days of age lost (35)S from the diacyl form at a higher rate than from the alkylacyl compound over a 21-day period. These data suggest that the diacyl form has a higher turnover than the alkylacyl derivative. The percentage of the total sulpholipid content of brain contributed by the sulphogalactosylglycerolipid is 16% in 21-day-old rats and 8.4% in adult rats.     

Mitochondrial degeneration after organic phosphate poisoning in prosimian primates

Summary The degenerative reaction of mitochondria to tricresylphosphate (TCP) poisoning in spinal ganglion cells of Slow Loris (Nycticebus coucang coucang) were studied with the electron microscope. In neurones of animals treated with TCP, mitochondria display various stages of alterations which confirm mitochondrial involvement in TCP poisoning. The role of degenerated mitochondria in the formation of neuronal lipofuscin is discussed. It is suggested that the lipofuscin granule is a metabolic product inherently related to mitochondrial degeneration, irrespective of the primary cause: ageing or intoxication.Fellow of the Deutscher Akademischer Austauschdienst on study leave from the Medical Faculty, University of SingaporeA grant from the Deutsche Forschungsgemeinschaft (Gl. 28/20) is gratefully acknowledgedThe skilfull technical assistance of Mr. Tajuddin b.M. Ali, Mr. P. Gopal, Mr. R. Dungan and Mrs. C. Weinrichter is gratefully acknowledged     

Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Contact hypersensitivity (CH) to 2,4-dinitro-1-fluorobenzene (DNFB) was induced in guinea pigs and mice by DNFB skin application. Development of CH was suppressed in both species either by cyclophosphamide (CY) treatment after sensitization or by single intravenous injection of dinitrobenzene-sulfonate (DNBS) before sensitization (hapten-induced tolerance). Additional treatment schedules were employed in guinea pigs, with the following results: Suppression of CH by injection of DNBS concomitant with sensitization; abrogation of hapten-induced tolerance by administration of CY before sensitization; and potentiation of CH skin reactivity by administration of CY before sensitization.Pretreatment by two injections of the methanol extraction residue (MER) tubercle bacillus fraction restored significantly the ability of CY treated animals to respond to DNFB sensitization. In contrast, administration of MER either by one injection before sensitization, concomitant with DNFB, or after sensitization did not prevent immunosuppression by CY.MER treatment was not effective in reversing hapten-induced tolerance in mice, and had only an occasional effect on this process in guinea pigs. Abrogation of hapten-induced tolerance and potentiation of DNFB sensitization by CY in guinea pigs were also not influenced by MER treatment.Supported by Contract NO1-CM-12127 from the NCI and by research grants from Concern Foundation, Inc., the Lautenberg Endowment, the National Council for Research and Development, Israel, and the GSF Munich, Germany, and the Leukemia Research Foundation, Inc.     

Myocardial myocytolysis following vagal stimulation in squirrel monkey: A case report

This report describes a case of premature ventricular contractions and electrocardiographic signs of myocardial infarction which developed following stimulation of the left vagus nerve in an awake squirrel monkey. Post mortem examination revealed myocardial myocytolysis.     

Rat brain aryl acylamidase: multiple forms and inhibition effects of LSD, serotonin and related compounds

At least two forms of aryl acylamidase (E.C.3.5.1.13, AAA) were separated from rat brain extracts by ammonium sulfate precipitation (33–60% saturation) and subsequent Bio-Gel column chromatography. Fraction AAA-1 showed pH optimum at 7.5 whereas AAA-2 showed a pH optimum at 5.5 AAA-1 activity was markedly inhibited at pH 7.5 by d-LSD and 2-Br-LSD, moderately inhibited by 5-HT and slightly inhibited by tryptamine but it was not affected by 1-LSD, at 0.1 mM concentration. AAA-2 was only moderately inhibited at pH 5.5 by d-LSD and 2-Br-LSD but not affected by 1-LSD, 5-HT or tryptamine at the same concentrations. Catecholamines and their structurally related drugs had no significant effects on either enzyme activity. Kinetic studies with AAA-1 indicated competitive inhibition by d-LSD with a K_i value of 4.90 ± 0.61 μM.     

Biosynthesis of pterocarpan phytoalexins in Trifolium pratense

Feeding experiments have demonstrated that 7,2′-dihydroxy-4′-methoxy-isoflavone-[¹⁴C-Me] and -isoflavanone-[¹⁴C-Me] are extremely efficient precursors of the phytoalexin demethylhomopterocarpin in Cu²⁺-treated red clover seedlings. Neither of these compounds, nor demethylhomopterocarpin-[¹⁴C-Me], was incorporated into a second pterocarpan phytoalexin, maackiain. 3-Hydroxy-9-methoxypterocarp-6a-ene-[¹⁴C-Me] was a poor precursor of both pterocarpans. A biosynthetic pathway to demethylhomopterocarpin via 2′-hydroxylation of formononetin (7-hydroxy-4′-methoxyisoflavone) and subsequent reduction to the isoflavanone is proposed. The conversion of this isoflavanone into the pterocarpan may involve the corresponding isoflavanol and a carbonium ion intermediate. The branch-point to maackiain is probably at the formononetin stage. The presence of two coumestans, 9-O-methylcoumestrol and medicagol, previously unreported in red clover, is demonstrated. Biosynthetic implications are discussed.