Transport of L-methionine in human diploid fibroblast strain WI38. The transport of L-methionine in human diploid fibroblast strain WI38 was investigated. The uptake of L-methionine was measured in sparse cell cultures in a simple balanced salt solution buffered with either Tris.HCl of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Similar results were obtained with these two buffers. Cultures were allowed to equilibrate with the buffered saline before transport was measured. The presence of glucose in the buffered saline results in a slight reduction in the initial rate of transport for the first 2 h of equilibration in buffered saline. L-Methionine is actively transported in WI38 by saturable, chemicallly specific mechanisms which are temperature, pH and, in part Na+ dependent, and are reactive with both L- and D-stereoisomers. Kinetic analysis of initial rates of transport at substrate concentrations from 0.0005 to 100 mM indicated the presence of two saturable transport systems. System 1 has an apparent KM of 21.7 micrometer and an apparent V of 3.57 nmol/mg per min. System 2 has an apparent KM of 547 micrometer and an apparent V of 22.6 nmol/mg per min. Kinetic analysis of initial rates of transport in Na+-free media or after treatment with ouabain suggested that system 1 is Na+ independent and that system 2 is Na+ dependent. Preloading of cells with unlabeled L-methionine greatly increases the initial rate of uptake. Efflux of transported methionine is temperature dependent, and is greatly increased in the presence of unlabeled L- or D-methionine or L-phenylalanine, but not in the presence of L-arginine. L-Methionine transport is strongly inhibited by other neutral amino acids, and is very weakly inhibited by dibasic amino acids, dicarboxylic amino acids, proline or glycine.


Characteristics of proline transport into R3230AC mammary tumor cells. Cells separated by enzyme treatment of the R3230AC mammary carcinoma were used to characterize the entry of proline. These cells showed minimal changes in cell viability and intracellular volume and were found to be suitable for transport studies, since the vi of proline was maintained for at least 4 h when cells were stored at 37 or 4 degrees C, or when transport was measured in the presence or absence of Na+. Proline was acitvely transported by these tumor cells, reaching a distribution ratio ([proline] intracellular/[proline] extracellular) of 20 after 2 h. Proline entry consisted of two processes, one saturable (carrier mediated) and the other, non-saturable. The carrier-mediated entry, Km - 0.83 mM and V = 151.10(-5) mumol/min per 5.10(6) cells, was Na+-dependent, sensitive to pH and metabolic inhibitors, and completely inhibited by alpha-(methylamino)-isobutyric acid (Ki = 0.34 mM). Proline entry in the absence of Na+ was 20% that in the presence of Na+ and was found to be due to a non-saturable process, since (a) vi of proline uptake in the absence of Na+ increases linearly with increasing proline concentration and (b) was not suppressed by either 20 mM alpha-(methyl-amino)-isobutyric acid, 50 mM glycine +20 mM phenylalanine, or 50 mM serine +20 mM phenylalanine when proline uptake was measured in the presence or absence of Na+. Therefore, under the conditions studied, we conclude that proline transport appears to be restricted to the A (alanine-preferring) system. Furthermore, these cells should provide a suitable model to study the effect of hormonal manipulations on the amino acid transport process.


Induction of reverse flow of Na+ through the active transport pathway in toad urinary bladder. When active Na+ transport across the toad urinary bladder was abolished by ouabain, a 'reversed' short circuit current could be induced by an Na+ concentration gradient. This reversed current was increased by vasopressin and inhibited by amiloride and appears to represent net Na+ movement 'backwards' through epithelial cells which normally participate in active Na+ transport across the bladder.


Self-association of human erythrocyte glycophorin A. Appearance of low mobility bands on sodium dodecyl sulfate gels. We have examined the self-association of glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, using sodium dodecyl sulfate (SDS) polyacrylamide gels and circular dichroism. Pure glycophorin A has a tendency to form multiple bands on SDS gels at positions of higher apparent molecular weight than the PAS 1 and PAS 2 bands previously seen. These high molecular weight bands do not have mobilities corresponding to integral polymers of PAS 1 and PAS 2. Circular dichroism spectra of solutions giving rise to these bands or to PAS 1 and PAS 2 bands alone, indicate that these species all have essentially the same peptide conformation.


The planar distributions of surface proteins and intramembrane particles in Acholeplasma laidlawii are differentially affected by the physical state of membrane lipids. We have studied the influence of changes in lipid organization on the planar distribution of two classes of membrane proteins: integral proteins which have amino groups exposed to labelling at the membrane surface by the biotinavidin-ferritin procedure, and those proteins which penetrate the lipid bilayer sufficiently to be seen as intramembranous particles by freeze-fracture electron-microscopy. When the membranes are examined at temperatures below the lipid phase transition, the first class is dispersed and the second patched. At temperatures in the middle of the transition range, both classes are patched. At temperatures just above the phase transition the first class is dispersed and the second patched, and at temperatures well above the transition both classes are dispersed. Freeze-etch studies of avidin-ferritin-labeled membranes confirmed that the distribution seen by the labeling and the freeze-fracture techniques coexist in single membranes. Thus, there exist two distinct classes of membrane proteins with differential organizational responses to the lipid state.


The conformation of the polar group of lysophosphatidylcholine in H2O: conformational changes induced by polyvalent cations. The conformation of the polar group of egg lysophosphatidylcholine and 1-myristoyl-sn-glycer-3-phosphorylcholine present as micelles in aqueous solution has been studied using NMR methods. In the absence of polyvalent cations the preferred conformation derived from spin-spin coupling constants is similar, but not identical, to that of phosphatidylethanolamine in the crystal structure (cf. Hitchcock, P.B., Mason, R., Thomas, K.M. and Shipley, G.F. (1974) Proc. Natl. Acad. Sci. U.S. 71, 3036--3040). The presence of lanthanides induces a conformational change involving primarily the phosphorylcholine group, e.g. torsion angle alpha5 changes from an all gauche to an approximate trans disposition. The gauche leads to trans transitions observed with torsion angles alpha3 and alpha5 produce a more extended orientation of the polar group (relative to the hydrocarbon chain axis). In the presence of lanthanides the conformation of lysophosphatidylcholine is very similar to that of the diacyl phosphatidylcholines observed in fully hydrated bilayers (cf. Hauser, H., Phillips, M.C., Levine, B.A. and Williams, R.J.P. (1976) Nature 261, 390--394) with the P-N vector at an angle of about 45 degrees to the bilayer.


Characterization of mixed micelles of phospholipids of various classes and a synthetic, homogeneous analogue of the nonionic detergent Triton X-100 containing nine oxyethylene groups. The synthesis and high-pressure liquid chromatographic purification of the homogenous nonionic surfactant p-(1,1,3,3-tetramethylbutyl)phenoxynonaoxyethylene glycol (OPE-9) in quantities suitable for membrane solubilization studies is reported. Micelles of OPE-9 and mixed micelles of OPE-9 with dimyristoyl and dipalmitoyl phosphatidylcholine as well as phosphatidylserine, phosphatidylethanolamine, lysophosphatidylcholine, sphingomyelin, and palmitic acid were characterized by column chromatography on 6% agarose. It was found that at 28 degrees C OPE-9 micelles have a Stokes' radius of 32 A, giving a molecular weight for a spherical micells of about half that of micelles of the polydisperse nonionic surfactant Triton X-100 under the same conditions. The micelle size is temperature dependent: at 40 degrees C the OPE-9 micelles have a Stokes' radius of 44 A, giving a molecular weight for a spherical micelle of about twice that of the OPE-9 micelles at 28 degrees C. The size of the mixed micelles varies linearly (as measured by Kav) with the mole fraction of phospholipid. The mixed micelle size was found to be relatively independent of the absolute concentration of surfactant over a four-fold range if the mole fraction of phospholipid is kept constant. The usefulness of the OPE-9/phospholipid mixed micelle system for lipolytic enzyme substrates and membrane-related studies is considered.


A study of intracellular transport of secretory glycoproteins in rat liver. To study the transport of secretory glycoproteins in the endoplasmic reticulum of rat liver, the distribution of nascent glycoproteins in the membrane and luminal fraction of rough and smooth microsomes has been examined after a short-time incorporation of radioactive glucosamine in vivo. 50--60% of the radioactivity was associated with the membranes of rough and smooth microsomes, whereas about 10% of the serum albumin was found in the same fractions. The relative amount of radioactivity in the membranes was the same whether the luminal content of the microsomal vesicles was released by sonication, French press, Triton X-100, Brij 35 or sodium deoxycholate. The distribution of labeled glycoproteins between the membrane and luminal fraction of rough and smooth microsomes did not change during the time interval of 15--120 min after administration of the isotope. The similarity of the labeling patterns obtained after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated that the same set of glycoproteins were located in the lumen and the membrane of rough and smooth microsomes. A specific precipitation of nascent glycoproteins from both the membrane and luminal fractions of rough and smooth microsomes were obtained with rabbit antiserum against rat serum. The nascent glycoproteins associated with the membranes were not released by high ionic strength or treatment with mercaptoethanol. A slow exchange between [14C]glucosamine-labeled glycoproteins in the lumen and membrane fraction was, however, found.


Erythrocyte membrane proteins. Sequential accumulation in the membrane during reticulocyte maturation. Reticulocytes of increasing maturity were separated by dextran gradient centrifugation. The accumulation in the membrane of the anion transport protein and other erythrocyte membrane proteins was studied during reticulocyte maturation by separating reticulocytes after incubation with [35S]-methionine. The incorporation of the reticulocyte membrane proteins was shown to be sequential, the anion transport protein being inserted at a very early stage in the cells' maturation.


The effect of acylcarnitines on the oxidation of branched chain alpha-keto acids in mitochondria. The oxidation of 14C-labelled branched-chain alpha-keto acids corresponding to the branched-chain amino acids valine, isoleucine and leucine has been studied in isolated mitochondria from heart, liver and skeletal muscle. 1. Heart and liver mitochondria have similar capacities to oxidize these alpha-keto acids based on protein content. Skeletal muscle mitochondria also show significant activity. 2. Half maximum rates are obtained with approximately 0.1 mM of the alpha-keto acids under optimal conditions. Added NAD and CoA had no effect on the oxidation rate, showing that endogenous mitochondrial NAD and CoA are required for the oxidation. 3. Addition of carnitine esters of fatty acids (C6--C16), succinate, pyruvate, or alpha-ketoglutarate inhibited the oxidation of the branched chain alpha-keto acids, especially in a high-energy state (no ADP added). In heart mitochondria the addition of AD (low-energy state) decreased the inhibitory effects of acylcarnitines of medium chain length or of pyruvate, and abolished the inhibitory effect of succinate. It is suggested that the oxidation rate is regulated mainly by the redox state of the mitochondria under the conditions used. 4. The results are discussed in relation to the regulation of branched-chain amino acid metabolism in the body.


Prostaglandin metabolism in rabbit kidney. Identification and properties of a novel prostaglandin 9-hydroxydehydrogenase. Prostaglandin F2alpha is metabolized in 100 000 X g supernatants of rabbit kidney by a 15-hydroxydehydrogenase and a delta13-reductase to F-series metabolites, and byt a 9-hydroxydehydrogenase to E-series compounds. The reactions were measured by radiochemical and biological assays. The 15-hydroxydehydrogenase and 9-hydroxydehydrogenase are localised specifically in renal cortex, have optimal activity at alkaline ph and are effective over a wide range of substrate concentrations. The 9-hydroxydehydrogenase oxidises the 9-alpha hydroxyl group of prostaglandin F2alpha and F1alpha but not the 9-beta hydroxyl group of prostaglandin F2beta. This enzyme is also found in rabbit stomach and ileum, but not in 8 other organs examined; the 15-hydroxydehydrogenase has a wider distribution. Unlike other 9-hydroxydehydrogenase enzymes described to date, the enzyme prepared from rabbit kidney converts prostaglandin F2alpha directly to E2 in substantial amounts; this conversion may be of importance in renal homeostasis.


On the relationship between glycerophosphoglycolipids and lipoteichoic acids in Gram-positive bacteria. I. The occurrence of phosphoglycolipids. 1. Gram-positive bacteria out of the families of Streptococcaceae, Lactobacillaceae, Micrococcaceae and Bacillaceae were investigated with respect to the occurrence and the concentration of phosphoglycolipids. 2. Phosphatidylglycolipids occur exclusively in group D Streptococci and in Streptococcus hemolyticus D-58. Phosphatidyl-alpha-kojibiosyldiacylglycerol, the prevalent species, accounts for up to 28% of the polar lipids. The related glycerophospho-phosphatidyl-alpha-kojibiosyldiacylglycerol is restricted to Streptococcus faecalis. 3. Glycerophosphoglycolipids, usually minor components, comprise thirteen compounds most of which have so far not been described. Except Micrococcus lysodeikticus all examined bacteria contained one or more glycerophosphoglycolipids. Their occurrence parallels, therefore, that of lipoteichoic acids, which supports the hypothesis of a metabolic relationship between these two membrane components.


A phospholipid serine base exchange enzyme. A membrane bound L-serine exchange enzyme which catalyzes the exchange reaction between L-serine and phospholipid-base was solubilized and separated from the ethanolamine-exchange enzyme by Sepharose 4B and DEAE-cellulose column chromatography. The separated fraction was purified approximately 37-fold with a yield of 2--5%. This fraction did not possess ethanolamine or choline exchange activity. The optimal pH was approx. 8.0, the incorporation rate of L-serine into phospholipid was linear up to 20 min incubation time and the activity was maximum at 10 mM CaCl2. The calculated Km value for L-serine was 0.4 mM. Ethanolamine phospholipid was the most effective acceptor for L-serine incorporation, particularly ethanolamine plasmalogen. The Km values obtained were: 0.25 mM for ethanolamine plasmalogen, 0.25mM for pig liver phosphatidylethanolamine and 0.66 mM for egg yolk phosphatidylethanolamine. These observations suggest that the hydrophobic moiety in ethanolamine phospholipid, as well as the base moiety, is important for the affinity of the L-serine exchange enzyme. Neither ethanolamine nor choline inhibited the L-serine exchange activity. There was no detectable conversion of phosphatidylcholine or phosphatidylethanolamine to phosphatidic acid by the partially purified enzyme.


Phospholipid-deacylating enzymes of rat stomach mucosa. 1. Rat stomach mucosa exhibited three distinguishable phospholipid-deacylating enzyme activities: lysophospholipase, phospholipase A1 and phospholipase A2. 2. The lysophospholipase hydrolyzed 1-palmitoyl lysophosphatidylcholine to free fatty acid and glycerophosphorylcholine. This enzyme had an optimum pH of 8.0, was heat labile, did not require Ca2+ for maximum activity and was not inhibited by bile salts or buffers of high ionic strength. 3. Phospholipase A2 and phospholipase A1 deacylated dipalmitoyl phophatidylcholine to the corresponding lyso compound and free fatty acid. The specific activity of phospholipase A2 was 2--4-fold higher than that of phospholipase A1 under all the conditions tested. Both activities were enhanced 4--7.5-fold in the presence of bile salts at alkaline pH and 11-18-fold at acidic pH. 4. In the absence of bile salts, phospholipase A1 exhibited pH optima at 6.5 and 9.5 and phospholipase A2 at pH 6.5, 8.0 and 9.5. The pH optima for phospholipase A1 were shifted to pH 3.0, 6.0 and 9.0 in presence of sodium taurocholate; the activity was detected only at a single pH of 9.5 in the presence of sodium deoxycholate and at pH 10.0 in the presence of sodium glycocholate. Phospholipase A2 optimum activity was displayed at pH 3.0, 6.0 and 8.0 in presence of taurocholage, pH 7.5 and 9.0, in presence of glycocholate and only at pH 9.0 in presence of deoxycholate. 5. Ca2+ was essential for optimum activity of phospholipases A1 and A2. But phospholipase A1 lost complete activity in presence of 0.5 mM ethyleneglycolbis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) at pH 6.0, whereas phospholipase A2 lost only 50%. 6. Phospholipases A1 and A2 retained about 50% of their activities by heating at 75 degrees for 10 min. At 100 degrees, phospholipase A1 retained 22% of its activity, whereas phospholipase A2 retained only 7%.


Effect of exogenous fatty acids on the retention of phospholipid acyl groups by mouse L fibroblasts. Exogenous oleic or linoleic acid, given at a high but nontoxic level (1 mg fatty acid/day for 20 . 10(6) cells in 50 ml medium), caused substantial redistribution of the otherwise permanently retained phospholipid acyls in mouse L fibroblasts. 18--40% of the preformed phospholipid acyls were shifted to triglycerides but most returned to phospholipids when the supply of exogenous fatty acid was removed. The phospholipid acyls could be reshuttled back to triglycerides again whenever an adequate amount of exogenous fatty acid was provided. Daily changes of medium containing oleic acid bound to bovine serum albumin caused a still greater total loss of phospholipid acyls into the medium. The removal of the prelabeled phospholipid acyls also occurred with phospholipid acyls which had been synthesized from [1-(14C)]acetate 3 days earlier. The results demonstrate the fact that the apparent permanently retained phospholipid acyl groups found in L-cells could in fact be displaced through experimental manipulations.


The importance of the lysophosphatidylcholine and choline moiety of bile phosphatidylcholine in lymphatic transport of fat. A luminal supply of biliary phosphatidylcholine is important in the translocation of absorbed fat into lymph and in the amount and composition of phosphatidylcholine concurrently synthesized. This study was undertaken to determine whether the effect was due to absorbed lysophosphatidylcholine, to a specific (1-palmitoyl) biliary lysophosphatidylcholine or to extra choline supplied by lysophosphatidylcholine. Rats with bile fistulae and thoracic duct lymph fistulae were given test meals of oleic acid and monoolein (molar ratio 2 : 1) infused duodenally for 8 h. Addition of choline chloride to the test meal increased lymphatic output of triglyceride and phospholipid but not to values found previously in rats with supplements of bile phosphatidylcholine or with bile ducts intact. Addition of dioleoyl phosphatidylcholine increased triglyceride and phospholipid output to values found in rats with intact bile ducts. Since dioleoyl phosphatidylcholine was as efficient as biliary phosphatidylcholine it was concluded that a luminal supply of 1-palmitoyl lysophosphatidylcholine was not essential. It seemed likely from the smaller effect of supplemented choline and from the fatty acid composition of lymph phosphatidylcholine that the essential requirement was a supply of absorbed lysophosphatidylcholine for rapid reacylation to phosphatidylcholine.


Changes in the structural and metabolic heterogeneity of phosphatidylcholines in the developing rat lung. In the present study the developmental profiles of the structural and metabolic heterogeneity of rat lung phosphatidylcholine are presented. The individual molecular species of phosphatidylcholine at different stages of the developing rat lung were analyzed as diacylglycerol derivatives. The metabolic heterogeneity of rat lung phosphatidylcholine was also studied by incubation using lung slices with radioactive precursors. The results obtained were as follows: 1. A significant increase of lung phosphatidylcholine during perinatal development (up to 1 day after birth) was found to be largely due to the increase of dipalmitoyl species. The percentage of palmitoyl-palmitoleoyl species also increased from --4 to --1 day of gestation, while palmitoyl-oleoyl species were found to decrease during development. Other molecular species showed no significant changes. 2. The incorporation of [1-14C]palmitoyl lysophosphatidylcholine into saturated phosphatidylcholine was relatively low at the earlier stage of fetal development, but it increased significantly in the last stage and reached its maximum at one day prior to birth and one day prior to the marked accumulation of dipalmitoylphosphatidylcholine occurring in the lung. In contrast, the incorporation of [3H]glycerol into saturated phosphatidylcholine remained, without showing a marked alteration during development. These results suggest that the lysophosphatidylcholine pathway which is mainly attributed to transacylation mechanism between 2 molecules of lysophosphatidylcholine may contribute to the marked production of dipalmitoylphosphatidylcholine in the lung in the last stage of gestation.


Comparison of molecular structure of glycerolipids in rat lung. The major individual molecular species of glycerolipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and diacylglycerol of rat lung were determined quantitatively as diacylglycerol acetates. 1. In three glycerolipids such as phosphatidylcholine, phosphatidylglycerol and diacylglycerol, the main molecular species was dipalmitoyl. The species was not detectable in phosphatidylethanolamine and was not of quantitative importance in phosphatidylinositol. 2. The oligoenoic molecular species in phosphatidylethanolamine and phosphatidylinositol contained palmitoyl and stearoyl species in almost equal amounts, while those of phosphatidylcholine, phosphatidylglycerol and diacylglycerol were principally of the palmitoyl types. 3. Tetraenoic species of phosphatidylinositol and diacyl types of phosphatidylethanolamine were predominantly of stearoyl types. Tetraenoic diacylglycerol also contained significant amounts of stearoyl-arachidonyl species. The value was about 3-fold that of palmitoyl-arachidonyl species. However, palmitoyl types were rather higher in phosphatidylcholine and phosphatidylglycerol. 4. The alkenyl-acyl types of lung phosphatidylethanolamine consisted predominantly of polyenoic species, namely, tetraenoic, pentaenoic and hexaenoic species. The pentaenoic and hexaenoic species of all glycerolipids examined consisted mainly of palmitoyl types.


Structure of neutral glycolipids in bovine thyroid tissue. Four asialo glycolipid fractions have been isolated from bovine thyroid glands. The structures were elucidated by partial hydrolysis, periodate oxidation, permethylation analysis and sequential enzymatic degradation studies. The following structures were identified: GL-1a glucosyl-beta-(1 leads to 1)ceramide; GL-1b galactosyl-beta-(1 leads to 1)ceramide: GL-2 galactosyl-beta-(1 leads to 4)glucosyl-beta-(1 leads to 1)ceramide: GL-3 galactosyl-alpha-(1 leads to 4)galactosyl-beta-(1 leads to 4)glucosyl-beta-(1 leads to 1)-ceramide; GL-4 N-acetylgalactosaminyl-beta-(1 leads to 3)galactosyl-alpha-(1 leads to 4)galactosyl-beta-(1 leads to 4)glucosyl-geta-(1 leads to 1)ceramide.


Liquid crystals and cholesterol nucleation during equilibration in supersaturated bile analogs. In recent work, apparent liquid crystal agglomeration to form typical solid cholesterol microcrystals was frequently observed photomicrographically in bile samples from prairie dogs fed a cholesterol-enriched diet, prior to solid crystal formation. We therefore have conducted a systematic study of time-course lipid compositional changes in the mesophase and micellar phase constituents of bile analog solutions while undergoing cholesterol nucleation during equilibration. On the basis of these studies, we conclude that the nucleation process for microcrystal formation most likely occurs within the mesophase component which is only the first of a two-step transition in a sequential series of physical ordering processes. We deduce that mesophase formation must have a lower kinetic energy requirement and that the second step (microcrystal formation) must be rate limiting. In keeping with theoretical considerations, structural evidence for increased hydration is demonstrable near the point of complete equilibration when the mesophase is dissolving.


Lipoprotein lipase of cultured mesenchymal rat heart cells. I. Synthesis, secretion and releasability by heparin. Cell suspensions prepared from rat hearts were separated by replating into F1, F2 and M cultures, and cultured for 3--11 days. Lipoprotein lipase activity was highest in the F1 cultures which consisted mainly of non-beating, mesenchymal cells. The enzyme activity was released into the medium only after addition of heparin. The release occurred by an initial rapid phase and a continuous slow phase. Both the rapid and the slow release of enzyme activity by heparin were inhibited by about 70% after a 4 h pretreatment with colchicine. Thus, it seems that the vesicular transport is responsible for the translocation of lipoprotein lipase to the cell surface also during the slow process of release. The residual activity in the colchicine treated cultures was higher than in the controls indicating that no inhibition of enzyme synthesis occurred. The slow phase of enzyme release continued also after removal of heparin from the medium but was reduced markedly when protein synthesis was inhibited by cycloheximide. Thus the increase in total enzyme activity encountered after exposure to heparin resulted from stimulation of new enzyme synthesis. The half-time of lipoprotein lipase in the F1 cultures was 35 min and full restoration of enzyme activity was found 60 min after complete removal of cycloheximide from the system. These data indicate that the culture system can be used to study regulation of new enzyme synthesis and its turnover.


Distribution of radioactivity in myelin lipids following subcutaneous injection of [14C]stearate. Blood fatty acids are an important parameter for the synthesis of brain myelin as exogenous stearic acid is needed: after subcutaneous injection to 18-day-old mice this labelled stearic acid is transported into brain myelin and incorporated into its lipids. However the acid is partly metabolized in the brain by elongation (thus providing very long chain fatty acids, mainly lignoceric acid) or by degradation to acetate units (utilized for synthesis of medium chain fatty acids as palmitic acid, and cholesterol). These metabolites are further incorporated into myelin lipids. The myelin lipid radioactivity increases up to 3 days; most of the activity is found in phospholipids; their fatty acids are labelled in saturated as well as in polyunsaturated homologues but sphingolipids, especially cerebrosides, contain also large amounts of radioactivity (which is mainly found in very long chain fatty acids, almost all in lignoceric acid). The occurrence of unesterified fatty acids must be pointed out, these molecules unlike other lipids, are found in constant amount (expressed in radioactivity per mg myelin lipid).


Studies on the properties of the singlet oxygen-like factor produced during lipid peroxidation. The singlet oxygen reaction product of various trapping agents is observed during enzymic and nonenzymic peroxidation of microsomes as well as during the peroxidation of pure lipids extracted from microsomes. We now wish to report that purified fatty acid hydroperoxide alone, as well as peroxidized microsomal lipid and cumene hydroperoxide also form the singlet oxygen reaction product with 2,5-diphenylfuran. The reaction product (cis-1,2-dibenzoylethylene) was observed to be formed in an anaerobic system, with or without EDTA. The data indicate that a reaction of hydroxyl radicals with 2,5-diphenylfuran cannot account for the formation of dibenzoylethylene in these systems. These results are consistent with a hypothesis that the singlet oxygen-like factor was formed from the lipid peroxides per se and, in addition, supports the possibility that either the peroxides can react directly with diphenylfuran to produce dibenzoylethylene or that the self-reaction of organic peroxides may form an intermediate product which can react directly with singlet oxygen-trapping agents to produce substances which are identical to a reaction of the trapping agents with singlets oxygen.


Transformation of arachidonic acid into thromboxane B2 by the homogenates of activated macrophages. The homogenates of activated macrophages obtained from liquid paraffin-injected guinea pig peritoneum were incubated with [14C]arachidonic acid or with radioactive prostaglandin endoperoside [14C]prostaglandin H2. The major radioactive metabolite in both cases was thromboxane B2, which was identified by NaBH4 reduction, rechromatography and autoradiography.


Distribution of prostaglandin biosynthetic pathways in organs and tissues of the fetal lamb. Five prostaglandins, i.e. prostaglandins E2, F2alpha and D2, 6-keto-prostaglandin F1alpha and thromboxane B2, were measured by mass spectrometry. Homogenates of fetal lamb brain, lung, liver, spleen and kidney and the ductus arteriosus, aorta and pulmonary artery formed different amounts of each product. Although the main prostaglandin in the fetal organs was prostaglandin E2, arterial tissue formed mostly 6-keto-prostaglandin F1alpha. These results demonstrate significant differences between organs and tissues in the relative direction of the 'prostaglandin synthetase' enzyme complex.


A simple and novel method for tritium labeling of gangliosides and other sphingolipids. A very simple method for introducing tritium specifically into the ceramide portion of gangliosides, neutral glycosphingolipids and sphingomyelin has been developed using potassium boro [3H]hydride and palladium as catalyst. In this way six different gangliosides, five different neutral glycosphingolipids and sphinomyelin with high specific radioactivity were prepared for the first time. This simple procedure which does not require sophisticated apparatuses is suitable for large scale preparation of tritium-labeled sphingolipids as well as for nanogram quantities of individual sphingolipids so as to serve for analytical purposes. During the course of the labeling procedure no degradation of even the most labile trisialosyl-gangliotetraosylceramide has been observed. The yield of tritiated compounds is almost quantitative. The specific radioactivity depends on the unsaturation of the ceramide moiety and the specific activity of the boro[3H]hydride employed.


Steroid-protein interactions. 40. The effect of fatty acids on progesterone binding to human serum albumin. Human serum albumin was delipidated by solvent extraction or by treatment with charcoal. Progesterone complexes formed with these albumin preparations had higher association constants than those formed with the untreated samples. The charcoal method of delipidation resulted in somewhat higher affinity constants than extraction with chloroform/methanol. Addition of 5 mol lauric acid per mol albumin reduced the association constant of the progesterone complex by approx. 50%. Studies with lauric, myristic, and palmitic acid showed that the decrease of binding affinity for progesterone was proportional to the amount of fatty acid added to albumin, and to its chain length. These results confirm and extend our previous findings of inhibition of progesterone binding to human albumin by long-chain fatty acids.


Transfer of phosphatidylcholine facilitated by a component of human plasma. A constituent of lipoprotein-free (p greater than 1.21) human plasma from normolipemic donors facilitates the transfer of diacyl phosphatidylcholine from unilamellar egg yolk phosphatidylcholine liposomes to liver mitochondria. The active component is heat labile, has a hydrated density greater than 1.25 and an apparent molecular weight of more than 100 000. The presence of this protein in plasma may facilitate movement of diacylphospholipids between the surfaces of lipid-containing particles such as lipoproteins and erythrocytes. Knowledge of the properties and behavior of this protein are important in designing methods of drug therapy based on encapsulation in biodegradable lipid vesicles.


Stereoconfiguration of phosphatidylglycerol in the membrane of bacteriophage PM2 and in its host, Pseudomonas BAL-31. Turnover of the acylated and unacylated glycerol moieties of phosphatidylglycerol was examined during infection of Pseudomonas BAL-31 by bacteriophage PM2. No turnover of either glycerol moiety was observed in infected or inunfected cells. The stereochemical configuration of phosphatidylglycerol from both virus and host was determined and proved to be 3-sn-phosphatidyl-1'-sn-glycerol. These results exclude a mechanism of mobilizing lipids for the virus by acylation and deacylation of 3-sn-phosphatidyl-1'-sn-glycerol in the host membrane to form 1-sn-phosphatidyl-3'-sn-glycerol in the membrane of PM2.


Serine ethanolamine phosphodiester: a major component in chicken semen. The phosphodiester, serine ethanolamine phosphodiester, has been found by 31P NMR to be present in chicken semen and the structure was confirmed by chromatographic and spectroscopic techniques. The presence of serine ethanolamine phosphodiester in chicken semen completes the analogy between the distribution of this compound in the tissues of the chicken and that of glycerol 3-phosphorylcholine in mammals.


Studies on a phospholipase B from Penicillium notatum. Substrate specificity. 1. The action of a highly purified phospholipase B from Penicillium notatum on 1-O-alk-1'-enyl-2-acyl-, 1-O-alkyl-2-acyl-, 1,2-diacyl-, 1-acyl- and 2-acyl-sn-glycero-3-phosphocholine, monoacyl-, diacyl- and triacylglycerols, cholesteryl oleate and p-nitrophenyl acetate was studied. 2. The hydrolysis products of the monoethermonoacylglycerophospholipids were identified as fatty acids, 1-O-alk-1'-enyl-sn-glycero-3-phosphocholine and 1-O-alkyl-sn-glycero-3-phosphocholine. The hydrolysis rates were in the following order: 1,2-diacyl-sn-glycero-3-phosphocholine greater than 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphocholine greater than 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine. 3. 1-Acyl-sn-glycero-3-phosphocholine was hydrolyzed about 15 times faster than 2-acyl-sn-glycero-3-phosphocholine. 4. Monoacylglycerols were hydrolyzed at the optimal pH 4.0, but diacyl- and triacylglycerols were not hydrolyzed at various pH values between 4.0 and 9.0. 5. Cholesteryl oleate and p-nitrophenyl acetate were not hydrolyzed.


Galactolipid formation in chloroplast envelopes. I. Evidence for two mechanisms in galactosylation. Two different enzymes for galactosylation occur in isolated chloroplast envelopes of spinach leaves, UDPgalactose-diglyceride galactosyltransferase and galactolipid-galactolipid galactosyltransferase. The first enzyme is responsible for the biosynthesis of monogalactosyldiglyceride, UDPgalactose being donor of the galactosyl moiety. The second enzyme is responsible for the biosynthesis of digalactosyldiglyceride and higher homologues. The optimum pH for monogalactosyldiglyceride synthesis was found to be 7.5, for digalactosyldiglyceride synthesis 6.5. After incubation at pH 7.4 a Lineweaver-Burk plot indicated two binding sites for UDPgalactose on the UDPgalactose-diglyceride galactosyltransferase with different affinities for UDPgalactose and different activities. Indirectly the galactolipid-galactolipid galactosyltransferase was shown to respond similarly to various UDPgalactose concentrations. However, the second reaction also proceeds in absence of UDPgalactose. It was concluded that the second enzyme does not require the presence of UDPgalactose, but that galactosyl transfer proceeds by direct exchange of galactosyl groups between molecules of galactolipids, or via unknown lipid intermediates, not detected in our system. Results of other investigations will be discussed in the light of the present data.


Lipase-colipase interactions during gel filtration. High and low affinity binding situations. The interaction of porcine pancreatic lipase and colipase was studied during gel filtration in columns eluted with a variety of buffers. High and low affinity binding situations were observed under different conditions. Low affinity binding could only be detected at the high lipase-colipase concentrations encountered during batch purification (10(-3)-10(-4) M). Even in this situation the rapid dissociation of the weak complex during filtration resulted in considerable separation of the two proteins. High affinity binding of lipase to colipase was observed at protein eluant concentrations as low as 10(-8) M on columns equilibrated with oleic acid-taurodeoxycholate mixed micelles. This binding did not take place on columns equilibrated with simple bile salt and mixed phosphatidylcholine-cholesterol-bile salt micelles. Colipase alone exhibited strong binding to phosphatidylcholine and fatty acid mixed bile salt micelles when applied together in a sample on columns eluted with pure bile salt micelles, lipase did not. The relevance of the high affinity complex to the lipase . colipase . substrate complex is discussed.


Conformation of a stable intermediate on the folding pathway of Staphylococcus aureus penicillinase. The partly unfolded intermediate (state H) of penicillinase from Staphylococcus aureus PC1 is stable in 0.8 M guanidinium chloride at pH 7.0. It has been characterized by measurements of intrinsic viscosity, sedimentation and diffusion coefficients, leading to an equivalent hydrodynamic volume of five times that of the native penicillinase molecule. Values of alpha-helix content calculated from circular dichroism were 27% for penicillinase and 16% for state H. A multi-domain model is proposed for penicillinase in which the domains can separate without appreciable change in secondary structure. This model is important in understanding the means by which the enzyme activity can be controlled, and corresponds to a kinetic pathway of folding.


Studies on the reduction and reoxidation of the disulfide bonds of the alpha and beta subunits of human choriogonadotropin. Reoxidation of the disulfide bonds of the alpha-subunit of human choriogonadotropin after their complete reduction yields a product which is indistinguishable from the native subunit in its electrophoretic pattern in polyacrylamide gel and in its ability to recombine with the beta subunit of bovine lutropin. The circular dichroism of reoxidized human choriogonadotropin-alpha is essentially identical to that of the native alpha-subunit, except for slightly more negative ellipticity in the region of 240 mm. Hybrid hormone preparations obtained by recombination of reoxidized or native human choriogonadotropin-alpha with native lutropin-beta exhibit identical electrophoretic patterns in polyacrylamide gels, elution profiles in gel filtration, receptor binding activities, and CD spectra. However, reoxidation of human choriogonadotropin-beta under the same conditions does not yield a product which resembles the native beta subunit in its electrophoretic pattern on gels, its CD spectrum or its ability to recombine with the alpha subunit.


Fluorescence energy transfer between epsilon-ATP at the nucleotide binding site and N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide at Cys-373 of G-actin. The method of fluorescence energy transfer is used to measure the distance from the nucleotide binding site to Cys-373 of G-actin. The fluorescent ATP analogue 1-N6-ethenoadenosine 5'-triphosphate was used as donor and N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide was used as acceptor. From the measurements of the efficiency of fluorescence energy transfer by both static and time resolved fluorometries, the distance between nucleotide binding site and Cys-373 residue of G-actin was calculated to be about 30 A.


Similarity of antigelatin factor and cold insoluble globulin. Antigelatin factor, a protein capable of complexing denatured collagen, was separated from human serum by adsorption onto immobilized collagen. Antiserum raised against the material binding to denatured collagen permitted the development of a radioassay for the determination of antigelatin factor in which the complex of antigelatin factor and denatured 125I-labeled collagen is precipitated with this antiserum. Further purification of antigelatin factor was achieved by chromatography on DEAE-cellulose yielding an electrophoretically homogeneous protein. Its migration rate in dodecyl sulfate-polyacrylamide gel electrophoresis was identical with that of cold insoluble globulin (molecular weight approx. 440 000) prepared from human plasma by a published procedure amended by DEAE-cellulose chromatography. Reduction of disulfide bonds yielded subunits of molecular weight approx. 220 000, indistinguishable from those of cold insoluble globulin. The amino acid composition of both proteins was very similar. Immunological identity of both proteins was demonstrated by gel diffusion against monospecific anti-cold insoluble globulin antiserum. Closely related binding curves were obtained if denatured 125I-labeled collagen was reacted with increasing amounts of either cold insoluble globulin or antigelatin factor and the complexes formed were precipitated with anti-cold insoluble globulin antiserum. In addition, antigelatin factor and cold insoluble globulin mediated the fixation of denatured 125I-labeled collagen to trypsinized macrophages in the same way. Therefore, it is concluded that antigelatin factor and cold insoluble globulin are identical or very closely related proteins.


The effect of limited fragmentation of porcine 19 S IgM on agglutination and complement fixation. Evidence that at least two intact subunits are required for complement fixation by IgM antibody.antigen complexes. Limited fragmentation of pig IgM anti-Salmonella with pepsin gave a 12 S product which agglutinated Salmonella almost as efficiently as the original IgM but the complement fixing ability of the complexes was substantially lowered. The 12 S product was shown to consist of molecular entities having intact Fc regions with an average of 3 or 4 intact Fab arms still attached. Our results appeared to show that removal of Fab arms by pepsin was a random process and on this basis a mathematical model was set up. From this model it was possible to calculate the frequencies of molecular entities which would give rise to the observed average of 3 or 4 Fab arms. The most likely explanation of our results is that complexes need to be formed by molecules having at least two intact subunits before efficient complement fixation takes place.


Isolation of a granulocyte colony inhibitory factor derived from human polymorphonuclear neutrophils. An inhibitory factor obtained from mature human granulocytes which suppresses granulocyte and monocyte-macrophage colony formation by an action on the endogenous colony stimulating factor-producing cells has been partially purified and characterized. The methods for purification consisted of a combination of ultracentrifugation, DEAE-Sephadex chromatography, SDS polyacrylamide gel electrophoresis and isoelectric focusing. The material had a molecular weight range of 102--128 000 and an isoelectric point between pH 6.2 and 6.4. The inhibitory factor was found to be heat stable and glycoprotein in nature.


Isolation and characterization of the 165 000 dalton protein component of the M-line of rabbit skeletal muscle and its interaction with creatine kinase. The M-line protein component of molecular weight 165 000 was isolated and purified from rabbit skeletal muscle using ion exchange chromatography. Gel electrophoresis, in the presence and absence of sodium dodecyl sulfate, revealed the protein to be homogeneous. Sodium dodecyl sulfate gel electrophoresis and low speed sedimentation equilibrium studies in 0.5 M KCl, 50 mM potassium phosphate gave a molecular weight of 165 000 suggesting the protein to be made up of a single polypeptide chain. Circular dichroism spectra revealed the presence of two negative dichroic bands located at 216 and 208 nm, indicative of the presence of some beta-structure. Ellipticity values at these two wavelengths were --6500 +/- 400 and --7500 +/- 400 deg . cm2 . dmol-1, respectively. Addition of 165 000 component lowered the enzymatic activity of creatine kinase M-line protein and the nature of the inhibition was found to be a competitive one. When the protein was mixed with creatine kinase in a 1 : 1 mole ratio in a medium consisting of 0.2 M KCl, 25 mM Tris, 1 mM dithiothreitol (pH 8.0), low speed sedimentation equilibrium studies gave a molecular weight of 260 000 +/- 10 000 for the complex, indicative of an interaction of the two components of the M-line.


Laser Raman investigations of intact single muscle fibers. Protein conformations. Raman spectra, in the frequency region of the protein vibrations, of intact single muscle fibers of the giant barnacle are presented. Strong bands at 1521 and 1156 cm-1 in the spectra are attributed to resonance-enhanced Raman bands of membrane-bound beta-carotene. Many bands of the myofibrillar proteins are also observed, and at least three spectral features confirm that these proteins adopt a predominantly alpha-helical structure: (1) the amide I band at 1648 cm-1, (2) the weak scattering in the amide III region, and (3) a strong skeletal C-C stretching band at 939 cm-1. Deuterated fibers have also been examined in order to find the exact shape of the amide III band. The presence in the fibers of paramyosin, which is only found in catch muscles, is also apparent from the spectra.


Histone H2B variants from the erythrocytes of an amphibian, a reptile and a bird. Histones H2B have been isolated from the terminally differentiated diploid erythrocytes of three different classes, amphibia (Xenopus laevis), reptilia (Crocodilus niloticus) and aves (Gallus domesticus). Partial amino acid sequences revealed three regions of sequence variation, each variant involving a single amino acid substitution.


Isolation and characterization of the predominant protein in nuclear ribonucleoprotein particles from rat liver. The predominant protein of the nuclear ribonucleoprotein particles of rat liver was isolated by polyacrylamide gel electrophoresis. The polypeptide represented 35% to 40% of the total mass of the protein moiety. Its molecular weight was estimated to be 38 000 and its NH2-terminal residue was found to be threonine. The amino acid composition is unique in having a high content of glycyl residues (20%) and NG-dimethylarginine (14% of total arginyl residues).


Primary structure of rat liver apoferritin. The amino end. Rat liver apoferritin is known to have a blocked amino end. From a pronase digest of rat liver apoferritin we have isolated and purified by ion-exchange chromatography the blocked N-terminal tripeptide. Its sequence and the nature of the blocking group were shown to be Ser-Ser-Gln and an acetyl moiety, respectively. The N-terminal sequence of rat liver apoferritin is thus N-acetyl-Ser-Ser-Gln, which coincides with the N-terminal sequence of horse-spleen apoferritin, the only other apoferritin studied structurally at present.


Influence of fatty acid and time of focusing on the isoelectric focusing of human plasma albumin. In the isoelectric focusing of human plasma albumin, two major peaks of pI 4.8 and 5.6 are observed. As fatty acids are removed from the albumin either by defatting before the focusing experiment or gradually during the focusing experiment, the pI 4.8 peak diminishes and the pI 5.6 peak increases. The interpretation of this effect is confirmed by fatty acid analysis before and after focusing. Alkylation of the free sulfhydryl group changes the focusing position of the defatted peak to pI 5.7. Otherwise the isoelectric focusing pattern of human albumin is unaffected by the status of the free sulfhydryl group or by the ampholine concentration.


Four states of tyrosine residues in the fibrinogen molecule. The ionization of tyrosine residues in fibrinogen was studied by a spectrophotometric method. The total of 100 tyrosine residues in the fibrinogen molecule was classified into four states: (1) 28 tyrosine residues with pK 10.1 (m = 1.0). (2) tyrosine residues with pK 11.5 (m = 1.0), (3) 20 tyrosine residues with pK 12.2 (m = 3.0) and (4) 10 tyrosine residues non-ionizable. When fibrinogen was treated with 4 M guanidine . HCl, all of the tyrosine residues became ionizable with the ionization characteristics of pK 10.1 (m = 1.0). The ionization characteristics of tyrosine residues in plasmin-digested fibrinogen were similar to those of fibrinogen, while in CNBr-treated fibrinogen they were fairly different. The value, m, stands for the number of hydroxyl ions involved in the ionization of a tyrosine residue.


Binding of 4-methylumbelliferyl-beta-D-ribopyranoside to beta-D-xylosidase from Bacillus pumilus. The determination of the binding parameters of 4-methylumbelliferyl-beta-D-ribopyranoside, a fluorescent ligand of beta-D-xylosidase (exo-1,4-beta-xylosidase, EC 3.2.1.37) from Bacillus pumilus, is described. A single binding site per polypeptide chain (60 000 daltons) was found and the homogeneity of the binding sites in the dimeric or tetrameric forms of the enzyme were shown. The association constants, as a function of temperature and ionic strength, were obtained from equilibrium binding experiments and compared to the kinetically determined inhibition constants. The apparent discrepancies are attributed to a temperature, ionic strength and concentration dependent shift in the dimer-tetramer equilibrium of the enzyme and different affinities of the ligand for both oligomeric forms. Sedimentation velocity experiments seem to corroborate this hypothesis.


Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides. I. Mannosylated units. In order to evaluate the possibility in a pig thyroid rough microsomal system of a transfer of pre-assembled sugar cores from sugar-lipids to protein, we have examined after incubation with GDP-[14C]Man the compounds bearing labeled saccharides and have determined some properties of their released saccharide moieties. The [14C]Man material specifically soluble in CHCl3/CH3OH/H2O, 10:10:3, behaved on DEAE-cellulose and when treated with hot alkali and alkaline phosphatase as a lipid pyrophosphate (sometimes accompanied by some dolichol-P-[14C]Man). Its saccharide moiety, released by mild acid, exhibited properties (molecular size, sensitivity to alpha-mannosidase, affinity for concanavalin A and charge modification introduced by a strong reductive alkaline treatment) pointing to a polymannosylated N,N'-diacetylchitobiose containing an average of nine monosaccharide units (from six to twelve). The [14C]mannosylated glycoproteins have represented all the polymeric label remaining after lipid extraction. From the susceptibility of their pronase glycopeptides to a differential reductive alkaline hydrolysis, it was concluded that their label belonged mainly to N-glycosidically linked units. Released saccharides exhibited the same properties as those from lipids, a result substantiating the possibility raised from previous studies of a transfer of pre-assembled moieties.


Studies on lectins. XL. O-glycosyl derivatives of Spheron in affinity chromatography of lectins. Free monosaccharides can be used for direct glycosylation of Spheron, a spherical macroporous hydroxyalkyl methacrylate-ethylene dimethacrylate copolymer, in a reaction that proceeds at room temperature in dioxane medium under catalysis of dry HCl or BF3. Derivatives of L-fucose, D-galactose, D-glucose, D-mannose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine thus prepared from Spheron beads have been shown to be efficient affinity carriers in isolation of lectins from seeds of Canavalia ensiformis D.C. (concanavalin A), Dolichos biflorus L., Glycine soja (L.) Sieb. et Zucc., Lens esculenta Moench, Ricinus communis L., Ulex europaeus L. and from albumin glands of the garden snail Helix pomatia L.


Effect of blood group determinants on binding of human salivary mucous glycoproteins to influenza virus. We have demonstrated that the inhibitor of influenza B virus hemagglutination in human saliva is inactivated by neuraminidase and is associated with the mucous glycoprotein fraction (blood group substance) of this secretion. Inhibitory activity of saliva was found to be roughly proportional to its sialic acid content (r = 0.456). However, the minimal quantity of salivary sialic acid, neutral sugar, or blood group antigen required to inhibit virus hemagglutination was greater for secretors of A and B than for secretors of H and Lea blood group substances. Removal of terminal galactose from blood group B substance with alpha-galactosidase markedly decreased blood group B activity but increased blood group H and virus hemagglutination inhibitory activities of this glycoprotein. These data suggest that terminal alpha-linked galactose and, probably, N-acetyl-galactosamine interfere with access of influenza virus to binding sites on oligosaccharide chains of the mucous glycoprotein.


Evidence for a membrane-bound fraction of chick intestinal calcium-binding protein. After homogenization of intestinal mucosa from vitamin D-replete chicks and high speed centrifugation, the major proportion of the vitamin D-induced calcium-binding protein is present in the supernatant fraction. However, the centrifugate, after repeated washing, contains significant amounts of bound calcium-binding protein that can be solubilized by Triton X-100. The bound calcium-binding protein is identical to soluble calcium-binding protein by the criteria of immunological identity, electrophoretic mobility, and molecular size, as determined by gel filtration chromatography. The bound calcium-binding protein is only partially released by sonication, osmotic shock or by ribonuclease treatment. Bound and soluble calcium-binding protein are not present in rachitic chick intestine. The addition of calcium-binding protein to rachitic mucosa prior to homogenization does not yield a Triton X-100 solubilizable form, indicating that bound calcium-binding protein in vitamin D-replete intestine is not due to adsorption or vesicular entrapment of soluble calcium-binding protein. The overall evidence suggests that part of the intestinal calcium-binding protein is membrane-bound.


Isolation and structure determination of a new amino acid, alpha-amino-gamma, delta-dihydroxyadipic acid, from the hydrolysate of normal human urine. A new acidic amino acid has been isolated from the hydrolysate of normal human urine. The chemical structure of the amino acid was determined to be alpha-amino-gamma, delta-dihydroxyadipic acid, based on its physical properties involving nuclear magnetic resonance, infrared and mass spectrometry as well as chemical degradation and chemical synthesis.


Selenium proteins in ovine tissues: III. Distribution of selenium and glutathione peroxidases in tissue cytosols. Three 6 week-old lambs were injected with carrier-free selenium-75 as sodium selenite initially and again after 6 days. One lamb received no further injections whereas the other two received injections of either vitamin E or unlabeled Na2SeO3 when the first selenium-75 injection was given. Selected tissues were removed at autopsy 10 days after the first injection. The cytosol from homogenates of these tissues was subjected to gel chromatography, and the elution profiles determined for radioactivity, protein content, and glutathione peroxidase activity using either hydrogen peroxide or cumene hydroperoxide as substrates. The selenium-75 was found to be distributed mainly between 2 different MW peaks. The larger MW seleno-peak (90,000) possessed both glutathione:hydrogen peroxide oxidoreductase, and glutathione:cumene hydroperoxide oxidoreductase activities, but the smaller MW seleno-peak (about 10,000) possessed no glutathione peroxidase activity. A peak of about 60,000 daltons containing only glutathione:cumene hydroperoxide oxidoreductase activity and no selenium-75 was found primarily in the liver and kidney. Vitamin E had no effect on the elution profiles. Selenium status of the animal had only a minor effect on the selenium-75 distribution in the cytosol, but had a marked effect on the absolute amount of the label taken up by tissues.


The 1H-nmr spectra of mixed valence complexes of copper with derivatives of 1-amino-2,2-dimethyl-2-mercaptoethane. Paramagnetic cluster ions of the type Cu(II)6Cu(I)8L12ClZ, where Z Is charge and L a derivative of 1-amino-2,2-dimethyl-2-mercaptoethane, have been shown to produce detectable 1H-nmr absorptions. Linewidths are narrow enough to permit the use of high resolution instruments. Downfield shifting of all detectable resonances is indicative of S leads to Cu2+ charge transfer, which results in a positive spin density at sulfur. Exchange measurements with 67Cu indicate relatively static structures.


The origin of the intense absorption in azurin. The intense visible absorption band of native azurin and the corresponding transitions of the Ni(II) and the Co(II) derivatives were analyzed. The bands were shown to be analogous ligand-to-metal charge transfer transitions and the optical electronegativity of the ligand involved is estimated to be 2.6. Comparisons with small molecule systems strongly indicate that the donor involved is cysteine sulfur.


Postinductive actinomycin D effects on the concentrations of cadmium thionein, zinc thionein, and copper chelatin in rat liver. The time courses of induction in rat liver of copper chelatin by copper, cadmium thionein by cadmium, and zinc thionein by copper, cadmium, and zinc were monitorg metal were used in order to avoid toxic effects, being 5 mg zinc, 0.5 mg copper, and 0.25 mg cadmium per kg body weight. Peak times of induction and half times of decay observed were: copper chelatin (9 h, 8.6 h), cadmium thionein (18 h, 6.80 days), and zinc thionein (zinc rats, 18 h, 10.1 h; copper rats, 9 h, 18.2 h; cadmium rats, 24 h, 4.53 days). Administration of actinomycin D (1 mg per kg body weight) at the peak times of induction of the various proteins had no effect on the concentrations of chelatin or cadmium thionein observed up to 24 hours later, but in the case of zinc thionein, induced by zinc, copper, or cadmium, elevated concentrations were observed up to 23 h after administration of the drug. Such behavior is reminiscent of superinduction previously seen with other proteins and enzymes. We postulate that the intracellular concentration of free zinc in liver is of fundamental importance in the induction of zinc thionein, and this can be distributed by exogenous copper or cadmium resulting in the induction of synthesis of zinc thionein.


A quantitative comparison of the time-course of sensitivity changes produced by calcium injection and light adaptation in Limulus ventral photoreceptors. The time-course of light and dark adaptation was quantitatively compared with the time-course of the onset of and recovery from desensitization produced by intracellular calcium injection in Limulus ventral photoreceptors. The onset of light adaptation tended to be faster (by 60-90 s) than the onset of desensitization produced by intracellular Ca++ injection. The initial portion of the time-course of dark adaptation was faster (about 10-20 s) than the time-course of recovery from desensitization  produced by  intracellular Ca++ injection. The final portion of recovery from Ca++ injection had the same time-course as a comparable dark adaptation.


Frequency domain analysis of asymmetry current in squid axon membrane. The change in capacity of squid axon membrane during hyper- and depolarizations was investigated in the absence of ionic currents after the membrane was treated with pronase. In the presence of the inactivation process (h parameter), failure to observe the gating current in the frequency domain was attributed to the rapid attenuation of the possible capacity change during depolarizations, which is likely to be due to the sodium activation process. Elimination of the h process would therefore enable us to observe the gating current in the frequency domain as the change in the capacitance component of membrane admittance. However, even after the inactivation process was abolished by pronase, the capacity of the axon membrane remained constant when ionic currents were blocked by external tetrodotoxin and internal Cs+ ion. Actually capacity was observed to decrease slightly with depolarization, contrary to the prediction based on the magnitude of gating currents.


Kinetics of the 580-nm ultrafast bacteriorhodopsin transient. We have observed, by low-temperature picosecond spectroscopy, a photo-induced transient at 580 nm in light-adapted bacteriorhodopsin. The transient was characterized by bleaching in the 550-585-nm regions within 6 ps and recovery in approximately 20 ps. The spectral intensity of the transient is found to be enhanced at lower temperatures, and the lifetime slightly elongated.


Activation of electron transfer reactions of the blue proteins. Thermal activation of electron transfer reactions of the blue proteins is considered in terms of vibronic coupling. Use of the electron paramagnetic resonance spectral distribution to obtain an estimate of the force constant for the relevant protein mode is proposed and demonstrated. This analysis leads to a model in which the configurations of the cupric and cuprous sites are displaced only a few degrees from each other, both being close to the configuration midway between planar and tetrahedral.


Exponentiated exponential model (Gompertz kinetics) of Na+ and K+ conductance changes in squid giant axon. The conductance changes, gK(t) and gNa(t), of squid giant axon under voltage clamp (Hodgkin and Huxley, 1952) may be modeled by exponentiated exponential functions (Gompertz kinetics) from any holding potential VO to any membrane clamp potential V. The equation constants are set by the membrane potential V, and include, for any voltage step in the case of gK, the initial conductance, gO, the asymptote conductance g, and rate constant k: gK = g exp(-be-kt) where b = 1n g/gO. Equations of similar form relate g and k to the voltage V, and govern the corresponding parameters of the gNa system. For the gNa, the fast phase y = y exp (-be-kt) is cut down in proportion to a slow process p = (1 - p)e-k't + p, and thus gNa = py. The expo-exponential functions involve fewer constants than the Hodgkin-Huxley model. In particular, the role of the n, m, h parameters appears to be filled largely by 1n (g/gO) in the case of gK and by 1n (y/yO) in the case of gNa. Membrane action potentials during current clamp may be computed from the conductances generated by use of the appropriate differential forms of the equations; diverse other membrane behaviors may be predicted.


Light-scatter analysis of microalgae. Correlation of scatter patterns from pure and mixed asynchronous cultures. A method is described for the first time for rapid and accurate discrimination among several algal types by their light-scattering properties alone. Using a multiangle light-scattering flow system, we obtained light-scatter patterns for individual cells in asynchronous cultures of Chlorella, Chlamydomonas, and Anacystis. The patterns are consistent and distinct for each species. By these signatures, each algal type can be recognized within mixtures.


Phase transitions in phosphatidylcholine dispersion observed with an interference refractometer. An interferometer is used to measure the refractive index change accompanying the crystal-to-liquid-crystal phase transition in the dispersion of phosphatidylcholines. Two separate methods of obtaining the refractive index change are employed: the first method analyzes the intensity transmitted through a spatial filter and the second method utilizes a piezoeletric crystal-based electronic compensator. The results of the two methods agree well. The accuracy of the apparatus (6 X 10(-6)) permitted us to use a very dilute sample to detect the phase change. Only a fraction of a milligram of dry lecithin is needed to observe the change. The result confirms conclusively that the major reason for the turbidity change at the transition temperature is the alteration in the refractive index of the lipid membranes. The fractional change in the refractive index does not agree well with the fractional change in the density of lipid molecules in vesicles.


Energy transfer in the purple membrane of Halobacterium halobium. The absorption spectrum of the primary photoproduct (the bathoproduct, or K) of the purple membrane protein (PM) at-196 degrees C has a maximum at 628 nm and an extinction coefficient of 87,000. Knowing the absorption spectrum allowed us to calculate the quantum efficiencies for PM to K and K to PM conversion at -196 degrees C. Direct measurements of these quantum yeilds at -196 degrees C gave 0.33 +/- 0.05 and 0.67 +/- 0.04, respectively. Determination of relative quantum efficiencies for PM to K and K to PM conversion by analysis of the absorption spectra of several photostationary-state mixtures of PM and K at -196 degrees C, however, gave wavelength-dependent quantum efficiencies that appear to be greater than 1. These anomolous results can be readily explained in terms of energy transfer from PM to K within the trimer clusters of pigment molecules which exist in the purple membrane. A model for such a transfer predicts an efficiency of energy transfer from PM to K of about 43%.


The fluorescence from the chromophore of the purple membrane protein. The fluorescence from the purple membrane protein (PM) of Halobacterium halobium and its relation to the primary photochemical events have been studied. The emission spectrum at 77 degrees K has structure, with peaks at 680, 710-715, and 730-735 nm. The excitation spectrum shows a single peak centered at 580 nm. This and a comparison of the fluorescence intensity at 77 degrees K under a variety of conditions with the amounts of the bathoproduct (or K, the only photoproduct seen at this temperature) formed suggest that the source of the fluorescence is the purple membrane itself, not the photoproduct. From the difference in several of their properties, we suggest that the fluorescing state of the pigment is different from the excited state which leads to photoconversion.


Head rotation or dissociation? A study of exponential rate processes in chemically skinned rabbit muscle fibers when MgATP concentration is changed. The mechanical response of fully activated muscle bundles (one to five fibers) to sinusoidal length perturbation ( approximately 0.4% L(0)) was studied as a function of MgATP concentration. The frequency response (0.25-167 Hz; corresponding to 1 ms time resolution) of chemically skinned rabbit muscle fibers was resolved into three exponential rate processes, (A), (B), and (C). At 20 degrees C, the apparent rate constants associated with the fast exponential lead (2pic = 388-588 s(-1)) and the oscillatory work (2pib = 59-116 s(-1)) both increase with increment of the MgATP concentration from 1 to 5 mM, and they both saturate for further increase. Over the whole range of MgATP concentrations the slow exponential lead (2pia = 9-7 s(-1)) remains constant. The effect of MgATP on processes (B) and (C) can be interpreted in the context of the biochemical evidence, in which MgATP enters the cross-bridge cycle after the desorption of the product, and the binding of MgATP to rigorlike cross-bridges promotes a rapid dissociation of actomyosin (Lymn and Taylor, 1971. Biochemistry.10:4617-4624.). The effect is not predicted by a model for force generation in which head rotation dominates the fast component ("stage 2" of Huxley and Simmons, 1971. Nature (Lond.).233:533-538. and 1973. Cold Spring Harbor Symp. Quant. Biol.37:669-680.), and head dissociation dominates the slow component ("phase 4" of Huxley, 1974. J. Physiol. (Lond.).243:1-43; Julian et al., 1974. Biophys. J.14: 546-562.).


Profiles in altered metabolism I--the organic acids accumulating in acute non-diabetic ketoacidosis associated with alcoholism. Several organic acids, among them the acidic catabolites of the branched chain amino acids and tyrosine, have been found to be elevated in the sera of non-diabetic patients presenting acute ketoacidosis associated with alcohol abuse. These findings are interpreted in terms of insufficiency of dietary co-factors required for their further catabolism. Butane-2,3-diol is also found frequently elevated in the urine of these patients and suggests interception of hydroxyethyl thiamine pyrophosphate by circulating high levels of acetaldehyde.


Pharmacokinetics of nabilone, a psychotropically active 9-ketocannabinoid, in the dog. Utilization of quantitative selected ion monitoring and deuterium labeling. Quantitative selected ion monitoring methods for the determination of plasma concentrations of nabilone, a psychotropically active 9-ketocannabinoid, and two carbinol metabolites using deuterium labeled internal standards are described. These specific methods have a lower limit of sensitivity of about 2 pmol ml-1 with a coefficient of variation of less than 4%. The utility of these methods was demonstrated by in vivo studies of single dose and steady state pharmacokinetics of nabilone and its carbinol metabolites in the dog.


Mass spectrometry of dimethylthiophosphinates of aromatic hydroxy compounds. The mass spectra of 20 differently substituted dimethylthiophosphinic esters of aromatic hydroxy compounds are presented. Fragmentation routes were investigated using high resolution mass measurements, decoupled metastable determinations and deuterium labelling. All compounds exhibited abundant molecular ions and typical phosphorus-containing ions. Characteristic elimination processes strongly dependent upon the respective type of substitution were observed. Due to their high stability, their great ease of formation and their good gas chromatographic properties these new types of derivatives are of special interest for establishing gas chromatography mass spectrometry profiles of acidic catecholamine metabolites.


Comparison of gas chromatography mass spectrometry methods for the determination of delta9-tetrahydrocannabinol in plasma. A method for the identification of delta9-tetrahydrocannabinol by gas chromatography mass spectrometry has been developed, and this method has been compared with other techniques, such as detection via thin-layer chromatography using tritium labeled delta9-tetrahydrocannibinol and a dual gas chromatographic method. The gas chromatographic mass spectrometric method was found to be equal or superior to other techniques and has the added advantage of being highly specific for the compound analyzed. An alternate approach using chemical ionization is also described; however, this procedure does not show significant advantages over the electron impact method. These methods show a practical lower detection limit of 500 pg ml-1 of plasma in clinical practice.


A selective ion monitoring assay for probenecid. A selective ion monitoring assay for probenecid (p-(dipropylsulfamyl)benzoic acid) using m-(di-isobutylsulfamyl)benzoic acid as an internal standard is described. The method has been applied to the determination of probenecid concentrations in human cerebrospinal fluid and lends itself to incorporation into the selective ion monitoring procedures used to quantitate other acidic and neutral compounds in such samples.


Neutrophil transfusion: effect of storage and of collection method of neutrophil blood kinetics. The kinetics in blood of autologous neutrophils collected by phlebotomy, filtration leukapheresis (FL), or intermittent-flow centrifugation (IFC), labeled with 32P-diisopropylfluorophosphate, and stored at 4 degrees C for up to 2 days were measured in 41 normal subjects. Mean initial recovery for unstored IFC cells was 34.0%, compared to 7.9% for unstored FL cells. Blood half-lives were 4.1 and 2.7 hr for unstored IFC and FL cells, respectively. With neutrophils collected by phlebotomy and stored in whole blood for 1-2 days, posttransfusion recoveries and blood half-times were significantly decreased. Storage of both IFC and FL preparations resulted in only moderate kinetic abnormalities in comparison to the unstored cells. These studies indicate that the ability of unstored IFC cells to circulate is basically normal, whereas that of unstored FL cells is significantly impaired. The data further suggest that these neutrophil concentrates might be stored for 1-2 days prior to transfusion.


"Concentration x time" methotrexate via a subcutaneous reservoir: a less toxic regimen for intraventricular chemotherapy of central nervous system neoplasms. Neurotoxicity associated with intrathecal methotrexate therapy has been shown to correlate with elevated concentrations of the drug in the cerebrospinal fluid as well as with the total cumulative dosage. In our study 19 patients with meningeal leukemia were randomized to receive courses of intraventricular methotrexate via an Ommaya reservoir consisting of either single injections of 12 mg/sq m/dose or a low-dose "concentration x time" (C x T) schedule of 1 mg every 12 hr for 3 days. There were no significant differences between the two treatment groups in the rate of remission induction, the number of relapses, or the durations of remission. The mean (+/- 1 SD) cumulative methotrexate dose was 66 +/- 41 mg/sq m in the C x T group and 173 +/- 64 mg/sq m in the 12 mg/sq m/dose group (p less than 0.005). Neurologic toxicity occurred in one of the eight patients in the C x T group and in seven of ten patients in the 12 mg/sq m/dose group (p less than 0.05). These observations suggest that the C x T dosage schedule is less neurotoxic and equally effective in the treatment of central nervous system leukemia.


Granulocyte modulation of endotoxin-stimulated colony-stimulating activity (CSA) production. Incubation of human macrophages with endotoxin resulted in significantly enhanced colony-stimulating activity (CSA) production by these cells. Preincubation of endotoxin with mature granulocytes abolished this stimulatory effect. The stimulatory effect of endotoxin on macrophage CSA production was not abolished, however, by preincubation with NaF-treated granulocytes, granulocyte membranes, or nonphagocytic cells (lymphocytes or erythrocytes). These data suggest that mature granulocytes may play a role in the modulation of CSA production and granulopoiesis by inactivation of stimulatory materials such as endotoxin.


Inhibition in vivo of mouse granulopoiesis by cell-free activity derived from human polymorphonuclear neutrophils. Cell-free extracts from human polymorphonuclear neutrophils (PMN) inhibited rebound granulopoiesis in the bone marrow and spleen of mice pretreated with cyclophosphamide to remove endogenous PMN. Absolute numbers of granulocytemonocyte progenitor cells (CFU-C) and net endogenous colony-stimulating activity (CSA) production were found to be increased 3 days after cyclophosphamide in the bone marrow and 6 days after in the spleen. Administration of PMN extract to the drug-treated mice prior to rebound granulopoiesis substantially decreased CSA production and CFU-C but not spleen B lymphocyte colony-forming cells. In addition, mice treated with PMN extract had decreased levels of CSA in serum and in conditioned medium of marrow-free bone, heart, and lung cultures. Inhibition was reversed by injection of bacterial lipopolysaccharide. Extracts from PMN of patients with chronic myelogenous leukemia, inactive in vitro, had no effect in vivo. These results demonstrate that inhibitory activity derived from PMN can control granulopoiesis in vivo.


Hereditary nonspherocytic hemolytic anemia and hexokinase deficiency. An 11-yr-old child with mild chronic hemolytic anemia was found to have decreased red cell hexokinase activity in spite of the reduced mean age of her red cell population. Similar decreases in red cell hexokinase activity were documented in the patient's parents and in one sib. The red cells were morphologically normal. Red cell 2,3-DPG levels were normal and ATP and glucose-6-phosphate levels were diminished. The kinetic properties, electrophoretic mobility, and thermal stability of the residual red cell hexokinase were normal or nearly so. Glucose consumption of the hexokinase-deficient cells was not appreciably decreased, probably because less of the potent inhibitor glucose-6-phosphate was present in the erythrocytes. It is likely, although not certain, that in this patient nonspherocytic hemolytic anemia resulted from hexokinase deficiency.


Effects of actinomycin D in vivo on murine erythroid stem cells. Low-dose actinomycin D (Acto) selectively suppresses murine erythropoiesis without decreasing erythropoietin (Ep) production. We used the plasma clot system to determine the stage of erythroid differentiation at which this inhibition occurs. Late erythroid precursors, CFU-E, and less differentiated committed erythroid stem cells, BFU-E, were assayed in CF1 mice given Acto 75-82 microgram/kg/day or saline subcutaneously for 5 days. We also assayed pluripotent (CFU-S) and committed granulocyte-monocyte (CFU-C) stem cells. Reticulocytes and marrow and spleen nucleated erythroid precursors were decreased by 99% in the Acto-treated mice; tibial marrow CFU-E were decreased by 97% and splenic CFU-E by 99%. Tibial BFU-E were not decreased by Acto, although there was a 66% diminution in splenic BFU-E. Acto increased tibial CFU-S, but splenic CFU-S and tibial and splenic CFU-C were unchanged. Thus Acto inhibits erythropoiesis by suppressing the ability of immediate committed erythroid precursors of CFU-E or CFU-E themselves to differentiate further in response to Ep. Acto does not affect survival or proliferation of the less differentiated cells--CFU-C, CFU-S, and marrow BFU-E. The suppression of splenic BFU-E in Acto-treated mice may indicate that marrow and splenic BFU-E are basically different stem cells. Alternatively, Acto treatment may impair migration of BFU-E from marrow to spleen.


Isoelectric focusing of human hemoglobin: its application to screening, to the characterization of 70 variants, and to the study of modified fractions of normal hemoglobins. Isoelectric focusing on slabs of acrylamide gel was adapted for the screening of abnormal hemoglobins, the characterization of 70 human variants, and the study of minor fractions of normal hemoglobin. The screening method was as fast and inexpensive as conventional techniques, allowed the simultaneous analysis of some 50 samples of whole blood, and yielded resolution superior to that obtained by other methods with hemolysates. Among the 70 variants, 31 mutants could not be separated from HbS by cellulose acetate electrophoresis. The characterization technique of electrofocusing allowed us to distinguish between most variants. Only one mutant, Hb Galveston, could be confused with HbS. Hb Köln, the most frequent unstable mutant, exhibited a special pattern. HbA1C was separated from HbA. Preliminary results indicate that quantitation of HbA1C by gel scanning is feasible.


Cation permeability alterations during sickling: relationship to cation composition and cellular hydration of irreversibly sickled cells. Sickle erythrocytes (RBC) incubated under 100% nitrogen for 4 hr manifested marked Na gain with an equivalent K loss. There were no changes in cell total cation or water content under these conditions, and no irreversible sickle cells (ISC) were formed. In contrast, sickle RBC incubated for 24 hr under 100% nitrogen in a glucose-free Na medium containing calcium manifested marked ISC formation. ISC formed under these conditions also had elevated Na content, although K content was much more reduced, and consequently ISC were cation depleted and dehydrated. When sickle RBC were incubated 24 hr under 100% nitrogen in a glucose-free K medium containing calcium no ISC formed and there were no major changes in cation or water content. These studies indicate that the Na+K content and dehydration of ISC was not directly related to the increased cation permeability associated with sickling. Rather, the ISC changes appear to reflect the well-known Gardos effect (K and water loss occurring in ATP-depleted RBC incubated with calcium). In addition, these studies suggest that ISC formation per se is related to K and water loss, since no ISC were formed when ATP-depleted sickle RBC were deoxygenated in calcium-containing high-K media that prevented K loss and dehydration.


Effect of cold storage on the sensitivity of and calcium influx into rat, rabbit and guinea pig portal veins. After cold storage treatment (5-7 days at 2 degrees C) the sensitivity to norepinephrine (NE) was increased in rabbit portal vein, decreased in rat portal vein and not altered in guinea pig portal vein. Cold storage decreased the sensitivity to methoxamine (ME), Ba++ and Ca++ in rat and guinea pig veins but not in rabbit veins and failed to alter the sensitivity to K+ in all veins tested. Cold storage increased 45Ca-influx while it reduced the NE content and 3H-NE uptake of all portal veins examined. NE and ME-induced 45Ca-influx was greater in cold-stored veins that in fresh veins from rabbit and guinea pig. K+ decreased 45Ca-influx in all cold-stored veins. These results indicate that cold storage induces a prejunctional supersensitivity in rabbit portal vein but not in guinea pig and rat portal veins. However, cold storage increased 45Ca-influx in all three veins.


Action of deoxycorticosterone acetate on the central artery of the rabbit ear. An inhibitor of extraneuronal uptake, deoxycorticosterone acetate (DOCA), increased the sensitivities of the rabbit ear artery to extraluminal and to intraluminal epinephrine, each approximately threefold. The effect on extraluminal epinephrine was increased in cocaine-treated or chronic sympathetic denervated arteries. That the sensitising actions of DOCA were due to inhibition of extraneuronal uptake was suggested (1) by the relative magnitudes of its sensitising effects on the three amines [in decreasing order, epinephrine greater than isoprenaline (beta-stimulation) greater than norepinephrine] and (2) by its ability to prevent the potentiation of epinephrine by normetanephrine. That the uptake system was readily saturated was suggested by the observation that the sensitising actions of DOCA were decreased by procedures which decreased the potencies of epinephrine and isoprenaline, namely, phentolamine, and doses of isoprenaline eliciting constriction via alpha-stimulation, respectively.


Effect of hormones on the glycosaminoglycan content of canine renal arteries. The effect of hypophysectomy and replacement therapy with individual hormones such as somatotropin, cortisone, estradiol, progesterone and testosterone on the glycosaminoglycan (GAG) composition of renal arteries was studied in young beagles. The renals from normal animals showed a uniformly low total GAG content of about two thirds of the value found for aorta and coronary arteries. Neither hypophysectomy nor hormone replacement altered the GAG content, with the exception of estradiol which showed a marked effect by raising the hyaluronic acid content to nearly double the normal value. These results by being at variance with those obtained for aorta and coronary arteries provide further evidence of the differential hormone sensitivity that exists in the various segments of the canine arterial tree with respect to GAG metabolism.


Successful prevention of coumarin-induced hemorrhagic skin necrosis by timely administration of vitamin k1. Successful prevention of the progression of incipient hemorrhagic skin necrosis by timely administration of vitamin K1 in a woman treated with phenprocoumon is presented. From a critical review of the literature strong evidence emerges that coumarin necrosis does only occur in cases with severe initial drug induced hypocoagulability. Non- recognition thusfar of its importance is due to insufficient knowledge of the biological activities of thromboplastin preparations presently used in the laboratory control of oral anticoagulation. All well documented cases with apparently adequate Quick values were monitored with Faktor VIII insensitive thromboplastin. Therefore, such preparations should no longer be used in anticoagulant control.


[Current aspects on evaluation of assays of factor-VIII activity, factor-VIII associated protein and factor-VIII neutralizing antibody (author's transl)]. In evaluating factor-VIII activity it should be noted that regarding the remaining activity of deficient plasma a linear reference curve is achieved. In the standard population factor-VIII activity and factor-VIII associated protein are distributed approximately lognormally. Due to this distribution certain results have been gained for the optimal choice of localisation and dispersion measures. It is assumed that the proportions of neutralized factor-VIII activity in plasma are distributed according to Poisson. The applicability of the Poisson distribution was also proved for the free factor-VIII activity portion. Due to the Poisson distribution the antibody unit is clearly defined, thus eliminating a further discussion on the establishment of an arbitrary standardized antibody unit.


Abnormal binding of spectrin to the membrane of erythrocytes in some cases of hereditary spherocytosis. In two cases of hereditary spherocytosis that we have examined, spectrin was bound abnormally tightly to the erythrocyte membrane, and could not be released by low ionic strength dialysis. This type of behaviour occurs in normal red cells only after heating above 50 degrees C. It appears that some cases of spherocytosis may be due to the presence of a protein which is abnormally temperature sensitive.


Platelet size distribution in mice following immunothrombopenia and vincristine administration. In the present study platelet size distribution was investigated after induction of immunothrombocytopenia by rabbit-anti-mouse-platelet-serum (RAMPS) and after vincristine-induced thrombocytopenia. The platelet size distribution after a single dose of RAMPS showed a shift to larger volumes at day 1 and 2, and a decrease to slightly smaller volumes than normal at day 8. These differences, however, were not statistically significant. After vincristine administration, a dose-dependent increase of the platelet size distribution was demonstrated, which lasted from day 1-7. It is suggested that in immune-induced thrombocytopenia the young platelets released from bone marrow megakaryocytes are not exclusively large platelets. On the other hand the early appearance of large platelets after vincristine administration points to a toxic or segregating effect of vincristine on circulating platelets. Therefore, in our experiments, the platelet size is not suitable for the differentiation of young and old platelets.


The influence of selenium on the level of mercury and metallothionein in rat kidneys in prolonged exposure to different mercury compounds. Mercuric chloride, phenylmercuric chloride, ethylmercuric chloride /0,23 mg Hg/kg/ and methylmercurycyan guanidine /0,46 mg Hg/kg/ were orally administered to rats every second day for 14 weeks. The same doses of the above mentioned mercury compounds were administered alternately with sodium selenite /0,18 mg Se/kg/ to parallel groups of rats at the same time. The level of total and inorganic mercury and of metallothionein was determined. All mercury compounds increased the level of metallothionein in rat kidneys. In rats which received only selenium the level of metallothionein was twice lower in the kidneys in relation to the physiological level of this protein. Selenium eliminated the stimulation of biosynthesis of metallothionein by mercury.


Fetal effects of cadmium in pregnant rats on normal and zinc deficient diets. This investigation has shown that not only the extent of fetal resoprtion and malformation but also the types of malformation seen in rats depend upon the strain used and day of gestation. Furthermore, the effects of zinc deficiency and cadmium administration on the fetus can be at least additive, as was seen for malformations. For fetal resorption, zinc deficiency potentiated the action of cadmium.


A search for the presence of 2,3,7,8 tetrachlorodibenzo-p-dioxin in beef fat. Specimens of fat taken from steers which had grazed on rangeland previously treated with 2,4,5-T herbicides were analyzed for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A cleanup procedure resulting in a 500-fold concentration was followed by a gas chromatography-mass spectrometry detection technique. The limit of detection of TCDD (2.5 times peak to peak noise) was found to be in the 30-60 picogram range (3-6 ppt in beef fat using 10 gram samples). None of the sixteen samples comprising two of the three studies showed any response for TCDD. In the third study, in which the animals were confined to a fenced pasture sprayed in its entirety with a 2,4,5-T herbicide, samples from three of the seven animals gave a positive response at the extremely low level of 3 to 4 ppt TCDD, which is at the detection limit.


Acute toxicity of phosphate ester mixtures to invertebrates and fish. The acute toxicity of three commercial phosphate ester mixtures (hydraulic fluids) was tested against two invertebrates and four fishes. Pydraul 50E was more toxic than Houghtosafe 1120 in static and flow-through tests; Pydraul 115E was the least toxic. In flow-through tests these phosphate esters were less acutely toxic than PCB's. However, sublethal reactions to the phosphates suggest a need for chronic toxicity studies.


Resurfacing the sole: long-term follow-up and comparison of techniques. It has been hoped that this study would determine the ideal solution to resurfacing the sole of the foot. Instead it showed that the problem is greater than expected and previously described, that no solution is ideal and that weight-bearing on all resurfaced areas will be avoided by the patient wherever possible.


The two-stage face lift.

The two-stage face lift. Skin under tension with "creep", i.e. stretch. This probably accounts for the fact that by far the commonest postoperative complaint of face-lift patients is of inadequate lift. Creep occurs within a few hours of the operation but is at first masked by postoperative oedema. At the second stage half as much or more skin as was excised on the first occasion may be re-excised. The conservative face-lift incision which spares the sideburns is the most conventient for the 2-stage operation.


A subcutaneous pedicled cheek flap for reconstruction of alar defects. The anatomy of the blood supply around the alar region was studied and showed that a large flap of nasolabial skin would survive on a narrow subcutaneous pedicle containing sufficient arteries and veins to justify the term "axial patterned flap". The secondary defect is closed with another nasolabial flap. In many cases a definitive single stage repair may be achieved. In others, some secondary revision has been required.


Repeated exposure of rats to the convulsant agent flurothyl enhances 5-hydroxytryptamine- and dopamine-mediated behavioural responses. 1 Rats were convulsed once daily for 7 days by exposure to the inhalant convulsant agent, flurothyl (Indoklon, bis (2,2,2-trifluouroethyl)ether). Twenty four hours after the final convulsion the rats were injected with tranylcypromine (20 mg/kg) followed 30 min later by L-DOPA (50 mg/kg), a procedure which increases brain dopamine concentrations. The flurothyl-treated rats showed a greater locomotor activity response than rats that had not been convulsed.2 This enhanced response appears to be due to increased postsynaptic dopamine receptor sensitivity since flurothyl-treated rats also showed enhanced locomotor responses to methamphetamine (2 mg/kg) and apomorphine (2 mg/kg).3 Enhanced 5-hydroxytryptamine-induced activity responses following administration of tranylcypromine (20 mg/kg) and L-tryptophan (50 mg/kg) were also seen 24 h after the last of 10 daily flurothyl-induced convulsions.4 The increased 5-hydroxytryptamine response also appears to be due to increased postsynaptic sensitivity since the flurothyl-treated rats showed increased hyperactivity following administration of tranylcypromine (20 mg/kg) and the suggested 5-hydroxytryptamine agonist, 5-methoxy N,N-dimethyltryptamine (2 mg/kg).5 No change in the brain concentration of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, tryptophan, dopamine or noradrenaline was observed 24 h after the last of 10 daily flurothyl-induced convulsions, compared to untreated rats. The rate of 5-hydroxytryptamine accumulation after tranylcypromine/L-tryptophan treatment and of dopamine and noradrenaline accumulation after tranylcypromine/L-DOPA treatment was similar in both groups.6 Repeated flurothyl convulsion has the same effects on these behavioural tests as repeated electroconvulsive shock. Since both treatments have been used successfully to treat depression, it is suggested that the mechanism of action of electroconvulsive therapy may be by increasing postsynaptic responses to the monoamine neurotransmitters.


H1- and H2-receptors in the smooth muscle of the ruminant stomach. 1 The effects of histamine on the longitudinal and circular smooth muscle from the rumen and reticulum of the bovine stomach have been analyzed by the use of H1- and H2-receptor antagonists. 2 Histamine caused three different types of response of the smooth muscle preparations: a contraction, contraction followed by relaxation, and a relaxation. These responses were resistant to the effect of tetrodotoxin, atropine, combined treatment with alpha- and beta- adrenoceptor blocking agents, hexamethonium and also to guanethidine. 3 Mepyramine antagonized the contractile responses and metiamide antagonized the relaxation responses in a highly selective and competitive manner. 4 It is concluded that histamine-induced excitatory and inhibitory responses of the smooth muscles from the bovine forestomach are mediated by histamine H1- and H2-receptors, respectively.


Is acetylcholine involved in a dopamine receptor mediated hypothermia in mice and rats? 1 Apomorphine and oxotremorine caused a dose-related fall in core temperature in the mouse and a fall in core temperature preceded by an increase in tail-skin temperature in the rat.2 In both species the slope of the dose-response curve was greater for oxotremorine (8.2 +/- 0.6 mice, 0.9 +/- 0.1 rats) than it was for apomorphine (1.7 +/- 0.3 mice, 0.5 +/- 0.07 rats).3 The mouse was more sensitive than the rat to the effects of both agonists.4 Atropine (0.625 to 5 mg/kg) and hyoscine (0.5 and 1 mg/kg) caused a dose-related rightward shift of the dose-response curve to oxotremorine in mice, but pimozide (0.25 to 1 mg/kg) was ineffective. Similar results were obtained in the rat.5 Pimozide (0.125 to 1 mg/kg) caused a dose-related rightward shift of the dose-response curve to apomorphine in mice, but atropine (1.25 to 1 mg/kg) and hyoscine (0.5 and 1 mg/kg) were ineffective. Similar results were obtained in the rat.6 Intrahypothalamic injection of apomorphine (10 mug) and oxotremorine (1.25 mug) caused a fall in core temperature in rats. Pimozide (0.5 mg/kg i.p.) caused reversal of the effect of apomorphine but did not significantly change the response to oxotremorine. Atropine (2.5 mg/kg i.p.) blocked the effect of oxotremorine, but not that of apomorphine.7 These results suggest that there are both central dopamine and central muscarinic acetylcholine receptors which mediate a fall in core temperature in rodents, but do not support the hypothesis that any connection exists between these two receptor populations.


Pharmacological evidence for the involvement of Na+ channels in the release of catecholamines from perfused adrenal glands. Veratridine (0.1 mM) was found to be effective in producing an increase in the catecholamine output from perfused guinea-pig adrenal glands in the presence of high concentrations of hexamethonium (1.83 mM) and atropine (28.8 micrometer). The response to veratridine was abolished by removal of either Na+ or Ca2+ from perfusion media and by the addition of tetrodotoxin (0.1 micrometer). It is suggested that the response to veratridine may be due to an increase in the tetrodotoxin-sensitive Na+ permeability of chromaffin cell membranes.


Diminishing hypotensive effect of increasing doses of pindolol in DOCA/saline hypertensive rats. In DOCA-saline hypertensive rats, pindolol (4, 20 or 50 mg/kg orally) produced a hypotensive effect which was inversely related to dose. Following adrenal demedullation, a hypotensive response to the highest dose of pindolol was unmasked and the magnitude of the responses to lower doses was increased. The results suggest that adrenal catecholamines moderate the hypotensive effects of high doses of pindolol.


Release of vasopressin by enkephalin.

Release of vasopressin by enkephalin. Leu-enkephalin, its stable analogue [D-Ala2-D-Leu5]-enkephalin and the C-fragment of lipotropin (beta endorphin) injected intravenously in the rat produced antidiuretic responses which were inhibited reversibly by naloxone. It was shown for Leu-enkephalin that injection into the cerebral ventricles was at least ten times more effective than intravenous injection and for [D-Ala2-D-Leu5]-enkephalin that the antidiuretic response was associated with increased excretion of vaspressin in the urine.