Reconstitution of glucose-transporting vesicles from erythrocyte membranes disaggregated in detergent.

With the eventual aim of purifying a membrane transport system by using reconstitution of transport activity as an assay, I showed that if, after the erythrocyte membrane is solubilized in deoxycholate, the detergent is removed, membrane vesicles re-form which retain glucose-transport activity. They take up and release D-glucose in preference to L-glucose and the uptake and release are sensitive to Hg2+ and phloretin. Release of tracer D-glucose is competitively inhibited by transported sugars inside the vesicles and increased by unlabelling D-glucose in the outside medium. Uptake of tracer is increased so much by preloading vesicles with unlabelled transported sugars that the tracer is probably concentrated against a gradient. When the membrane is solubilized, two proteins that span the membrane can be separated, suggesting that it will be possible to fractionate the membrane before reconstitution.


Incorporation of [14C]shikimate into plenazines and their further metabolism by Pseudomonas phenazinium.

1. During growth of Pseudomonas phenazinium on l-threonine medium, phenazine pigment formation commenced early and 1,6-dihydroxyphenazine 5,10-dioxide (iodinin) was the major component. Growth on l-[U-(14)C]threonine showed that when growth was complete about 25% of the label had been incorporated into phenazines and 30% into cell substance. 2. The addition of d-[2,3,4,5(n)-(14)C]shikimate to cultures at different phases of growth showed that the greatest efficiency of incorporation (about 70%) occurred in the mid- to late-exponential phase. Phenazines accounting for most of the (14)C supplied were iodinin and 9-hydroxyphenazine-1-carboxylate plus 2,9-dihydroxyphenazine-1-carboxylate. Radioactivity incorporated into cell substance was about one-third of the amount found in phenazines. 3. Kinetic studies showed that radioactivity from a pulse of [(14)C]-shikimate was incorporated into phenazines immediately, without a discernible lag, and into all detectable phenazines simultaneously rather than sequentially. 4. Radioactive phenazines isolated from culture media were fed to growing cultures and their metabolism was studied. The results supported a scheme for the biosynthesis of iodinin and 1,8-dihydroxyphenazine 10-monoxide by a branched pathway. 5. It is proposed that phenazine-1,6-dicarboxylate is the common precursor of all naturally occurring phenazines.


Control of isocitrate lyase synthesis in Chlorella fusca var. vacuolata. The basal activity of the enzyme and the kinetics of induction.

1. Isocitrate lyase activity was measured in non-induced Chlorella fusca var. vacuolata cells. 2. During exponential autotrophic growth about 1-2 molecules of the enzyme per cell were present. 3. In light-limited cultures the amount of the enzyme increased to 10-20 molecules/cell. 4. When autotrophic cultures were placed in the dark, the basal activity of isocitrate lyase increased after a 2h lag so that after 8h in the dark there was a 500-fold increase in activity. 5. When isocitrate lyase was induced (by addition of acetate and removal of illumination) in autotrophic cultures which had been growing exponentially, the full induced rate of enzyme synthesis was obtained after 70-80min. 6. When light-limited autotrophic cultures were induced, the rate of isocitrate lyase synthesis was maximal after only 40-50min. 7. These data are consistent with a catabolite-repression control co-ordinated with photosynthetic activity,which may be independent of the specific inducing effect of acetate.


Heterogeneity of proteoglycans in developing chick limb cartilage.

Proteoglycan heterogeneity was studied during the maturation of embryonic-chick limb cartilage in vivo. The results suggest that during the differentiation of limb-bud cartilage the aggregated forms of proteoglycans increase between stages 24 and 35, whereas the non-aggregated or monomeric forms decrease. Only one link protein is found in stage-24 limb buds, whereas two are present at stage 35. Evidence suggests that the synthesis of link proteins may be a regulatory factor in limb chondrogenesis.


Comparison of the biliary excretion of the four isomers of bilirubin-IX in Wistar and homozygous Gunn rats.

The biliary excretion of the four isomers of bilirubin-IX was studied in Wistar rats (JJ) and homozygous Gunn rats (jj). Synthetic preparations of 14C-labelled pigments were used. 1. After intravenous administration, the alpha-isomer was rapidly excreted in conjugated form in bile of Wistar rats. In Gunn rats excretion was insignificant. In contrast, both rat species promptly excreted the non-alpha-isomers at rates that were comparable with that found for bilirubin-IXalpha in Wistar rats. 2. In normal rats about 16% of the beta- and delta-isomers and at least 50% of the gamma-isomer were excreted as ester conjugates of the injected parent bile pigments. Conjugation of the beta- and delta-isomers had occurred exclusively at the carboxyl groups of pyrrole ring D and C respectively. For bilirubin-IXgamma no preference for any carboxyl group could be established. 3. In homozygous Gunn rats the non-alpha-isomers were apparently excreted chemically unaltered. This suggests that, as for bilirubin-IXalpha, conjugation of the non-alpha-isomers is also deficient in Gunn rats.


Characterization of the major diazo-positive pigments in bile of homozygous Gunn rats.

Bilinoid pigments in bile of homozygous Gunn rats (jj) were analysed either after formation of dipyrrolic ethyl anthranilate azo derivatives or as the unmodified parent tetrapyrroles. 1. T.l.c. of the azo derivatives revealed seven major unconjugated components which were structurally characterized by chemical tests, spectrophotometry and mass spectrometry. In addition, two minor components were identified as azodipyrrole (A+B)-glucoside and azodipyrrole (A+B)-beta-d-glucuronide. 2. Extraction and t.l.c. of the tetrapyrrolic pigments showed 13 major yellow diazo-positive bands. Four of them, accounting for 59% of total diazo-positive material, were identified as unconjugated bilirubin-IXalpha, -IXbeta, -IXgamma and -IXdelta. A fifth band (16%) was characterized as a mixture of two isomeric monohydroxyl derivatives and another band (8%) as a dihydroxyl derivative of bilirubin-IXalpha. 3. Although unconjugated bilirubin-IXalpha constitutes one-third of total diazo-positive material in bile of our strain of Gunn rats, the daily amount excreted represented only about 3-4% of daily bilirubin production. 4. Phototherapy caused a 2.2-fold increase in the biliary output of diazo-positive bilinoids, but did not affect markedly their composition. However, an additional diazo-negative pigment, accounting for one-third of total yellow colour, was observed but was not identified. Mass-spectral data on two dipyrrolic azopigments have been deposited as Supplementary Publication SUP 50076 (3 pages) with the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1977) 161, 1.


The effect of insulin on the washout of [45Ca]calcium from adipocytes and soleus muscle of the rat.

Insulin stimulates the washout of 45Ca from preloaded isolated fat-cells, whole epididymal fat-pads and isolated soleus muscles of the rat. This effect occurs within 10 min after the addition of the hormone, and it can be detected at concentrations down to those measured in rat plasma. When K+ is omitted from the washout medium, the effect on soleus muscles is more pronounced and increases with the time of exposure.


The UDP-N-acetylglucosamine-asparagine-sequon N-acetyl-beta-D-glucosaminyltransferase activity in preparations of rough endoplasmic reticulum from regenerating rat liver.

The UDP-N-acetylglucosamine-asparagine-sequon N-acetylglucosaminyltransferase activity in preparations of rough endoplasmic reticulum from rat liver is less than 1% of that in preparations from rabbit liver. The activity of the enzyme is increased about 20-fold in preparations from regenerating rat liver within 48h after partial hepatectomy. A smaller, but still marked, increase (8-10-fold) occurs in preparations of rough endoplasmic reticulum from sham-operated rats.


Synthesis of cartilage-specific proteoglycan by suspension cultures of adult chondrocytes.

Chondrocytes isolated from larynges of adult pigs were cultured as cell suspensions for at least 4 days before use. During continuous-labelling experiments in nutrient medium for 18h with 35SO42- and [3H]glucosamine as precursors, some macromolecular polyanionic material was synthesized which behaved on gel chromatography as proteoglycan. Gel chromatography on Sepharose 2B showed that a proportion of the proteoglycans in the medium appeared to be aggregated, and was dissociated in 4M-guanidinium chloride. Moreover the dissociated proteoglycan interacted with hyaluronic acid. Newly synthesized proteoglycan was larger than the average total cetylpyridinium chloride-precipitable material assayed as uronic acid alone.


Effects of dolichol monophosphate on galactose incorporation into glycoconjugates of cell cultures.

Cultured-cell homogenates catalysed the incorporation of galactose from UDP-galactose into protein and sphingolipid acceptors. Dolichol monophosphate stimulated the incorporation of galactose into glycoproteins, but it did not affect the rate of glycosylation of either exogenous or endogenous glycosphingolipids. It is proposed that, under certain conditions, galactose may be incorporated into glycoproteins via polyisoprenol intermediates, as is the case with N-acetylglucosamine and mannose.


Cellular transport of lysosomal enzymes: an alternative hypothesis.

Hickman & Neufeld [(1972) Biochem, Biophys. Res. Commun. 49, 992-999] have proposed that lysosomal enzymes reach the lysosomes by means of exocytosis and subsequent pinocytic reincorporation. The results leading to this conclusion are re-assessed and an alternative explanation is advanced that relates to the necessity for membrane recycling in endocytic cells.


Permeability of the liver cell membrane to quinolinate.

Quinolinate was taken up by both rat and guinea-pig liver cells. Equilibrium was reached after approx. 20 min with rat cells, but guinea-pig cells had not achieved a steady state after 60 min. There was no evidence to suggest that quinolinate is rapidly metabolized by either species. The concentrations of quinolinate attained in rat and guinea-pig cells after short periods of incubation with 0.5 mM-quinolinate did not inhibit gluconeogenesis. These results raise further doubts as to the mechanism of quinolinate action in liver.


Isolation and characterization of membrane proteins responsible for attachment of polyribosomes to rough microsomal fraction of rat liver.

A protein fraction which has a high affinity for polyribosomes was isolated from rough microsomal membranes of rat liver. The mode of polyribosome binding to this fraction (R-fraction) was studied by using CsCl equilibrium centrifugation and compared with that for stripped rough microsomal membranes. The following were found. (1) The polyribosome-binding cpacity of the R-fraction was heat-labile and sensitive to trypsin, and was suppressed by increasing KCl concentration and addition of 0.1 mM-aurintricarboxylic acid. (2) Of the four subfractions obtained by gel filtration of the R-fraction on a Sephadex G-200, only the R1-fraction, eluted at the void volume, showed a high affinity for polyribosomes. The polyribosome-binding capacity of the R1-fraction decreased with time on storage at 4 degrees C. (3) The R1-fraction contained three major proteins with mol. wts. 108,000, 99,000 and 65,000.


Iron-transport characteristics of vesicles of brush-border and basolateral plasma membrane from the rat enterocyte.

Vesicles of brush-border and basolateral plasma membrane were prepared from enterocytes of the rat small intestine. The separateness of these two varieties of plasma membrane was confirmed by appropriate enzyme assays. The uptake of Fe2+ by these membrane vesicles was studied, and the results suggest differences between the two types of membrane in both the amount of Fe2+ taken up and in the rate of uptake. At low (up to 3 micrometer) concentrations of Fe2+, uptake by both membrane types showed evidence of saturation and could be blocked with the thiol inactivator N-ethylmaleimide. The studies suggest that Fe2+ is taken into an osmotically active space by a process of facilitated diffusion at low concentrations, but that at higher concentrations the process appeared to obey first-order kinetics. The data provide further evidence for the existence of functional polarity in the epithelial cell of the small intestine.


Role of haem in the synthesis and assembly of cytochrome P-450.

By using 3-amino-1,2,4-triazole, an inhibitor of haem synthesis, and 2-allyl-2-isopropylacetamide, a drug that degrades the haem moiety of cytochrome P-450, the involvement of haem in cytochrome P-450 synthesis and assembly was investigated. Phenobarbital was used to stimulate apo-(cytochrome P-450) synthesis. Degradation of preformed cytochrome P-450 haem does not result in a concomitant release of the apoprotein from the endoplasmic reticulum. The availability of haem for cytochrome P-450 synthesis in the normal animal is not rate-limiting. Prolonged inhibition of haem synthesis in vivo decreases the rate of apo-(cytochrome P-450) synthesis, although this effect is not discernible under conditions of short-term inhibition of haem synthesis. Under the former conditions exogenous haemin is able to counteract the decrease in the rate of apoprotein synthesis. In animals receiving successive injections of phenobarbital plus 3-amino-1,2,4-triazole, compared with those receiving phenobarbital only, the holo-(cytochrome P-450) content measured spectrally shows a greater decrease than could be accounted for by the decrease in the content of the total apoprotein. In addition to less haem being available under these conditions, the free apoprotein appears to have undergone some modification, such that its haem-binding capacity is considerably decreased. This particular effect could be due to a direct interaction of 3-amino-1,2,4-triazole or its metabolites with cytochrome P-450 rather than a consequence of haem deficiency. Apo-(cytochrome P-450) is capable of binding to the endoplasmic reticulum in a form and at a site, which can be reconstituted with haemin to yield the functional protein.


Turnover of myelin proteins in mouse brain in vivo.

The incorporation of tyrosine into proteins was measured after the subcutaneous implantation of a pellet of [14C]tyrosine in mice. This method keeps the specific radioactivity of free tyrosine fairly constant and makes it possible to follow incorporation up to a 10-day period. At the end of 10 days most of the protein-bound tyrosine was replaced (i.e. most protein turned over) in lung, liver, heart, kidney and spleen; about half was replaced in brain, one-quarter in muscle. The rate of protein turnover in myelin was approx. 40% of that of whole brain proteins; at 10 days one-fifth of the myelin proteins were replaced. All protein components of myelin measured were in a dynamic state; incorporation decreased in the following order, Wolfgram greater than DM-20 greater than basic greater than proteolipid proteins. The incorporation of tyrosine into each protein fraction was greater in the 0-5-day than in the 5-10-day period, indicating heterogeneity of metabolic rates. The results show that after myelination at least a portion of each protein component of myelin is undergoing significant metabolic turnover. In the adult, myelin components are not stable, but turnover is heterogeneous, and each protein may be compartmentalized. Turnover can be influenced by a variety of factors.


Development of mitochondrial energy metabolism in rat brain.

1. The development of pyruvate dehydrogenase and citrate synthase activity in rat brain mitochondria was studied. Whereas the citrate synthase activity starts to increase at about 8 days after birth, that of pyruvate dehydrogenase starts to increase at about 15 days. Measurements of the active proportion of pyruvate dehydrogenase during development were also made. 2. The ability of rat brain mitochondria to oxidize pyruvate follows a similar developmental pattern to that of the pyruvate dehydrogenase. However, the ability to oxidize 3-hydroxybutyrate shows a different developmental pattern (maximal at 20 days and declining by half in the adult), which is compatible with the developmental pattern of the ketone-body-utilizing enzymes. 3. The developmental pattern of both the soluble and the mitochondrially bound hexokinase of rat brain was studied. The total brain hexokinase activity increases markedly at about 15 days, which is mainly due to an increase in activity of the mitochondrially bound form, and reaches the adult situation (approx. 70% being mitochondrial) at about 30 days after birth. 4. The release of the mitochondrially bound hexokinase under different conditions by glucose 6-phosphate was studied. There was insignificant release of the bound hexokinase in media containing high KCl concentrations by glucose 6-phosphate, but in sucrose media half-maximal release of hexokinase was achieved by 70mum-glucose 6-phosphate 5. The production of glucose 6-phosphate by brain mitochondria in the presence of Mg(2+)+glucose was demonstrated, together with the inhibition of this by atractyloside. 6. The results are discussed with respect to the possible biological significance of the similar developmental patterns of pyruvate dehydrogenase and the mitochondrially bound kinases, particularly hexokinase, in the brain. It is suggested that this association may be a mechanism for maintaining an efficient and active aerobic glycolysis which is necessary for full neural expression.


The activities of 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase in hearts and mammary glands from ruminants and non-ruminants.

1. The activities of 2-oxoglutarate dehydrogenase (EC 1.2.4.2) were measured in hearts and mammary glands of rats, mice, rabbits, guinea pigs, cows, sheep, goats and in the flight muscles of several Hymenoptera. 2. The activity of 2-oxoglutarate dehydrogenase was similar to the maximum flux through the tricarboxylic acid cycle in vivo. Therefore measuring the activity of this enzyme may provide a simple method for estimating the maximum flux through the cycle for comparative investigations. 3. The activities of pyruvate dehydrogenase (EC 1.2.4.1) in mammalian hearts were similar to those of 2-oxoglutarate dehydrogenase, suggesting that in these tissues the tricarboxylic acid cycle can be supplied (under some conditions) by acetyl-CoA derived from pyruvate alone. 4. In the lactating mammary glands of the rat and mouse, the activities of pyruvate dehydrogenase exceeded those of 2-oxoglutarate dehydrogenase, reflecting a flux of pyruvate to acetyl-CoA for fatty acid synthesis in addition to that of oxidation via the tricarboxylic acid cycle. In ruminant mammary glands the activities of pyruvate dehydrogenase were similar to those of 2-oxoglutarate dehydrogenase, reflecting the absence of a significant flux of pyruvate to fatty acids in these tissues.


Effect of a nutritional shift on the degradation of abnormal proteins in the mouse liver. Decreased degradation during rapid liver growth.

1. The intravenous injection of puromycin to mice 0.5 min after administration of radioactive leucine resulted in the release of labelled ribosome-bound nascent protein chains with the next 0.5 min. 2. During the subsequent 13 min, 40% of the liver protein radioactivity disappeared. The rate of this process was already maximal 0.5 min after the injection of puromycin, with no apparent lag. 3. Evidence is presented that this phenomenon represents the selective degradation of puromycinyl-peptides: (a) the magnitude of this fraction corresponded to the calculated proportion of protein radioactivity in nascent chains at the time of the puromycin effect; (b) the size distribution of the proteins disappearing between 2 and 14 min was smaller than that of those retained at 14 min; and (c) when the injection of puromycin was delayed for 5 min, or when the leucine pulse was interrupted by the injection of cycloheximide (rather than puromycin), the fraction disappearing within 14 min was much smaller. 4. The degradation of puromycinyl-peptides was much slower in the rapidly growing livers of animals recovering from a protein depletion than in the protein-depleted controls. It is concluded that the large decrease in the overall rates of total liver protein degradation previously described during liver growth is a general phenomenon, also affecting the rate of scavenging of abnormal proteins.


Regional differences in ribonuclease content of rat and mouse kidney.

Kidney cortex, red medulla and white medulla were separated into nuclei, mitochondria, microsomal and 105000g supernatant fractions. Assay of RNAase (ribonuclease) activity at pH7.8 revealed that, for each subcellular fraction, activity was much greater in cortex than in red or white medulla; this was true for both free RNAase and total (free plus latent) RNAase. For example, the free RNAase activity in the 105000g supernatant of cortex was 5 and 8 times higher than in red and white medulla respectively. No latent RNAase activity was found in any particulate fraction. Latent supernatant RNAase activities (suggesting presence of bound RNAase inhibitor) were similar in cortex and medulla. The cortex supernatant contained minimal free RNAase inhibitor, whereas that of the red and white medulla showed about one-third and one-tenth respectively of the inhibitor activity measured in liver. Adrenalectomy did not change RNAase activity in any fraction nor the content of free RNAase inhibitor in the kidney supernatant, but did decrease the liver RNAase inhibitor content by 40%. In supernatants from mouse kidney, both free and total RNAase activities of both cortex and red medulla were similar to those of rat red medulla. Mouse cortex contained appreciably higher amounts of free RNAase inhibitor than rat cortex. The difference between the rat and mouse cortical RNAase activity and inhibitor content may help explain the relative ease with which satisfactory renal polyribosome profiles were obtained from mouse kidneys. Our results, as well as those of Kline & Liberti [(1973) Biochem. Biophys. Res. Commun.52, 1271-1277], showing that renal red and white medulla are more active than cortex in protein synthesis, are consistent with the hypothesis that the RNAase-RNAase-inhibitor system may participate in the regulation of protein synthesis.


Inhibition of protein degradation in isolated rat hepatocytes.

1. Isolated parenchymal cells were prepared by collagenase perfusion of livers from fed rats that had been previously injected with [(3)H]leucine to label liver proteins. When these cells were incubated in a salts medium containing glucose, gelatin and EDTA, cellular integrity was maintained over a period of 6h. 2. Cells incubated in the presence of 2mm-leucine to minimize radioactive isotope reincorporation released [(3)H]leucine into the medium at a rate accounting for the degradation of 4.5% of the labelled cell protein per h. 3. Degradation of [(3)H]protein in these cells was inhibited by insulin and by certain amino acids, of which tryptophan and phenylalanine were the most effective. 4. Protein degradation was decreased by several proteinase inhibitors, particularly those that are known to inhibit lysosomal cathepsin B, and by inhibitors of cell-energy production. 5. Ammonia inhibited degradation, but only at concentrations above 1.8mm. Aliphatic analogues of ammonia were effective at lower concentrations than was ammonia. 6. High concentrations of ammonia inhibited degradation by 50%. The extent of this inhibition could not be increased further by the addition of the cathepsin B inhibitor leupeptin, which by itself inhibited degradation by approx. 30%. 7. The sensitivity of proteolysis in isolated hepatocytes to these various inhibitory agents is discussed in relation to their possible modes of action.


Preparation of plasma-membrane subfractions from isolated rat hepatocytes.

1. Rat livers were dissociated into their constituent cells by perfusion through the portal vein with a medium containing collagenase, and hepatocytes separated from non-parenchymal cells. 2. It is shown that the procedure described by Wisher & Evans [(1975) Biochem. J. 146, 375-388] for preparation of plasma membranes from liver tissue when applied to isolated hepatocytes also yielded subfractions of similar morphology and marker-enzyme distribution. 3. Thus the distribution of alkaline phosphodiesterase, 5'-nucleotidase and the basal and glucagon-stimulated adenylate cyclase among two 'light' vesicular and one 'heavy' junction-containing plasma-membrane subfractions paralleled that reported for tissue-derived plasma-membrane subfractions. 4. Increased recoveries and specific activities of plasma-membrane marker enzymes were obtained when soya-bean trypsin inhibitor was included in the collagenase-containing perfusion media used to dissociate the liver. 5. Polyacrylamide-gel-electrophoretic analysis of the corresponding plasma-membrane subfractions prepared from liver tissue and isolated hepatocytes were generally similar. 6. The results indicate that the functional polarity of the hepatocyte's plasma membrane is retained after tissue dissociation. The damage occurring to plasma-membrane ectoenzymes by the collagenase-perfusion procedure is discussed.


Metabolic fates of diethylstilboestrol sulphates in the rat.

The metabolic fates and modes of excretion of diethylstilboestrol mono[35S]sulphate and diethylstilboestrol di[35S]sulphate were studied in the rat. Both of the esters were desulphated to some extent in vivo. In addition, significant amounts of radioactivity appeared in the bile as diethylstilboestrol mono[35S]sulphate monoglucuronide. The percentage of the dose appearing in bile as the diconjugate was substantially greater in experiments with diethylstilboestrol mono[35S]sulphate than with diethylstilboestrol di[35S]sulphate. Whole-body radioautography and studies with isolated perfused liver confirmed the liver as the major metabolic organ for both esters. When the metabolite diethylstilboestrol mono[35S]sulphate monoglucuronide isolated from the bile was reinjected, it was excreted in the bile unchanged. Studies in vitro demonstrated that both esters were substrates for arylsulphatase C with Km values in the range 52-76 micrometer. The metabolic fates and modes of excretion of the esters are discussed in relation to the enzyme complement of rat liver.


The effects of acetate, metal cations, phenobarbitone, porphyrogens and substrates of glycine acyltransferase on the utilization of haem by rat liver apo-(tryptophan pyrrolase).

1. The utilization of haem by rat liver apo-(tryptophan pyrrolase) under basal conditions and after enhancement of the enzyme activity by various mechanisms was studied under the influence of treatments affecting various aspects of liver haem metabolism. 2. These treatments were: benzoate and p-aminobenzoate as substrates of glycine acyltransferase, acetate as an inhibitor of 5-aminolaevulinate synthase activity, enhancement of 5-aminolaevulinate dehydratase by aluminium, destruction of haem and inhibition of ferrochelatase by porphyrogens, increased haem utilization by phenobarbitone and enhancement of haem oxygenase activity by metal cations. 3. The results show that the haem saturation of the apoenzyme is sensitive to all these treatments. 4. The possible usefulness of tryptophan pyrrolase in studying the regulation of liver haem is suggested.


Rapid partial purification of placental glucocerebroside beta-glucosidase and its entrapment in liposomes.

1. A glucocerebroside beta-glucosidase-rich detergent-free preparation was obtained from human placentas by a rapid method combining affinity chromatography on concanavalin A-Sepharose and organic-solvent precipitation. In a typical preparation about 11000 units of the enzyme purified 1500-fold were obtained from five placentas in 2 days. 2. The enzyme preparation also contained other hydrolases, but the extent of their purification was much smaller. 3. Studies on entrapment in liposomes showed that all glucocerebroside beta-glucosidase activity used could be incorporated in neutral egg phosphatidylcholine-cholesterol liposomes. Association with liposomes appeared to discriminate against other proteins, including some of the hydrolases, thus contributing to further purification of the enzyme. More than 95% of the liposome-associated enzyme activity was latent.


Stimulation of calcium accumulation in cardiac sarcolemma by phosphorylase kinase.

After incubation with phosphorylase kinase, calcium accumulation in cardiac sarcolemma was increased from 65+/-2 to 95+/-3nmol/10min per mg of protein. Under these conditions, phosphorylase kinase catalysed phosphorylation of membranes. This phosphorylation was hydroxylamine-insensitive, was stimulated by Ca2+ ions and was unaffected by 3':5'-cyclic AMP.


Enhanced plasma persistence of therapeutic enzymes by coupling to soluble dextran.

Conjugation of carboxypeptidase G and arginase, two enzymes of therapeutic interest, to a soluble dextran significantly enhanced plasma persistence in normal and tumour-bearing mice. A prolonged decrease in arginine concentrations in plasma of tumour-bearing mice was demonstrated by using the dextran-linked arginase. Gel filtration of dextran-enzyme conjugate showed that enzyme activity co-chromatographed as a single peak with carbohydrate, and enzyme was shown to be covalently linked to the dextran.


Some observations on the choice of detergent for solubilization of the human erythrocyte membrane.

Solubilization of the human erythrocyte membrane by seven detergents is described. Components released into the supernatant or retained in the residue were identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Two non-ionic detergents exhibiting little u.v. absorption were more efficient than u.v.-absorbing Triton X-100. Evidence is presented of an interchange between protein PAS 1 and protein PAS 2.


Self-digestion of human erythrocyte membranes. Role of adenosine triphosphate and glutathione.

Intact human erythrocytes incubated at 37 degrees C, pH7.4, release a sialoglycopeptide similar in its chemical composition, immunological and aggregation properties to the glycopeptide released by isolated 'ghost' membranes. The presence of ATP or reduced glutathione at physiological concentrations in the incubation medium of 'ghost' membranes inhibits this self-digestion process.


Synthesis, purification, and chemical characterization of the amino-terminal 1-34 fragment of bovine parathyroid hormone synthesized by the solid-phase procedure.

Peptides prepared by solid-phase synthesis during a systematic study of structure-activity relations in parathyroid hormone have been subjected, after sequential purifications, to rigorous analysis of purity by a variety of analytical techniques including sequence analysis by the Edman procedure. The present paper undertakes a critical appraisal of the utility of different tests of peptide purity and the use of the procedures in guiding and monitoring optimal synthesis strategies. Sequence analysis of a peptide representing the amino-terminal 34 residues of bovine parathyroid hormone (bPTH-(1-34)) revealed the presence of at least 30% of contaminating error peptides which were undetected by other analytical procedures. The major contaminant was identified as a peptid in which glutamine at position 29 was deleted. A repeat synthesis using fluorescamine rather than ninhydrin to monitor the coupling reaction resulted in a preparation that lacked the contaminant resulting from deletion. These findings demonstrate the particular value of sequence analysis in the evaluation of purity of peptides synthesized by the solid-phase technique.


Dual-mode cooperative binding of adenosine 5'-triphosphate to poly(L-lysine).

Equilibrium dialysis of adenosine triphosphate interacting with polylysine at low polymer concentration yield Scatchard exhibiting an apparent non-cooperative binding mode superimposed on a positively cooperative one. The same behavior has been reported in the literature for various mononucleotides and basic poly(amino acids). It is pointed out here that both modes must not be  considered as independent but to be mutually exclusive. Applying a pertinent theoretical approach yields higher degrees of cooperativity than obtained for mutual independence. Another necessary modification in the analysis of data takes into account that one ligand interacts with more than one equivalent binding contact (i.e., an elementary charge here). This leads to a further enhancement of the actual cooperativity. Increasing the polymer concentration in the binding experiments reveals a cooperative effect also in the first binding mode. This is physically interpreted and theoretically analyzed in terms of bound dimers stabilized by base stacking.


Chromatin replication in vitro. Properties of a HeLa nuclear system.

An in vitro HeLa chromatin replication system was developed and characterized. Purified nuclei synthesized DNA from replication forks which were undergoing chain elongation at the time of cell fractionation. DNA replication in isolated nuclei was deficient in DNA maturation functions; the Okazaki fragments synthesized in vitro accumulated and were ligated to high-molecular-weight DNA with low efficiency. The chromatin protein components of nuclei incubated in the DNA replication assay conditions were investigated for displacement, degradation, and exchange. Displacement of total nuclear protein during in vitro incubation occurred to the extent of 3%, and involved only the nonhistone nuclear proteins. Degradation was not detectable, assayed both by loss of acid-soluble radioactivity and by protein electrophoretic patterns in polyacrylamide gels. No detectable protein was exchanged from chromatin to exogenous DNA.


Purification and properties of the NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi.

The NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi have been purified to homogeneity as judged by single bands on sodium dodecyl sulfate gel electrophoresis. The overall purification for the NADH specific enzyme is 3000-fold and 4000-fold for the NADPH specific enzyme from a crude extract. The final step in the purification procedure is chromatography on a 5'-AMP-Sepharose 4B affinity column which results in approximately a 50-fold purification to a final specific activity of 31 mumol of NADH oxidized min-1 (mg of protein)-1 for the NADH specific FMN reductase. The NADPH specific reductase has been purified to a final specific activity of 51 mumol of NADPH oxidized min-1 (mg of protein)-1 using a NADP agarose affinity column, which results in a 70-fold purification. Molecular weights of 30 000 and 40 000 and Km's of 4.75 X 10(-5) M NADH and 4.0 X 10(-5) M NADPH have been determined for the pure NADH and NADPH specific FMN reductases, respectively. The NADPH specific FMN reductase does not utilize NADH, while the NADH specific enzyme does dehydrogenate NADPH with a maximal velocity one-tenth of that for NADH. Separate NADH and NADPH specific FMN reductases from Photobacterium fischeri could not be demonstrated.


Effect of mixed solvents on the formation of horseradish peroxidase compound I. The importance of diffusion-controlled reactions.

In aqueous glycerol solutions, the rate constant for the formation of compound I of horseradish peroxidase from hydrogen peroxide is independent of the viscosity. However, whem m-chloroperbenzoic acid is used to form compound I, the rate is viscosity dependent, indicating that this reaction is diffusion controlled. The problems in obtaining accurate information about diffusion-controlled reactions are discussed as is the possible significance of diffusion control. Ethanol has been used in previous studies to decrease the dielectric constant of the medium but it was found that its dominant effect on horseradish peroxidase was to bind to the sixth coordination position of the heme ion. The stability of the native enzyme in different environments was tested by difference absorption and circular dichroic spectroscopy and new circular dichroic data for compound I are reported.


Resonance Raman studies of the purple membrane.

The individual resonance Raman spectra of the PM568 and M412 forms of light-adapted purple membrane from Halobacterium halobium have been measured using the newly developed flow technique. For comparison purposes, the Raman spectra of the model chromophores, all-trans- and 13-cis retinal n-butylamine, both as protonated and unprotonated Schiff bases, have also been obtained. In agreement with previous work, the Raman data indicate that the retinal chromophore is linked to the purple membrane protein via a protonated. Schiff base in the case of the PM568 and an unprotonated Schiff base for the M412 form. The basic mechanism for color regulation in both forms appears to be electron delocalization. The spectral features of the two forms are different from each other and different from the model compound spectra.


Purification and characterization of a peptide from the carboxy-terminal region of chick tendon procollagen type I.

A disulfide-bonded peptide with a molecular weight of about 100 000 was isolated from the medium of cultured chick embryo tendons. It was shown to be a trimer with two types of subunits in a 2:1 ratio, and tryptic fingerprinting and immunological evidence indicated that it was derived from the carboxy-terminal-precursor-specific region of procollagen. Amino acid analysis after reduction and alkylation indicated that the trimer contains about 30 residues of half-cystine involved in intrachain as well as interchain disulfide bonding. The interchain bonds could be reduced and alkylated under nondenaturing conditions. Carbohydrate analysis showed that each of the three peptide chains in the trimer contains about two residues of N-acetylglucosamine and about ten residues of mannose. This suggests the presence of one or two oligosaccharide units per chain.


Effect of linocmycin on the chlorophyll protein complex I content and photosystem I activity of greening leaves.

1. Greening barley and pea leaves treated with lincomycin have a reduced chlorophyll content. Lincomycin dose not alter the proportion of chlorophyll in chlorophyll-protein complex II (CPII) but greatly reduces that in chlorophyll-protein complex I (CPI). 2. Difference spectra show that chloroplasts from lincomycin-treated leaves are deficient in at least two long wavelength forms of chlorophyll ratio of chloroplasts is unaffected by lincomycin but the photochemical P-700/chlorophyll ratio is less than half of that of the control. It is less affected than the chlorophyll-protein complex I content. 4. Photosystem I activity expressed on a chlorophyll basis is unaffected by linocomycin but the light intensity for half saturation is increased 8-fold. 5. Chlorophyll-protein complex I apoprotein content is reduced by lincomycin. No evidence was found for an accumulation of its precursor(s). The relative abundance of major peptides of 18 000, 15 000 and 12 000 daltons in lincomycin-treated chloroplasts is attributed to a general inhibition of greening and associated membrane of formation.


Light-induced movement of magnesium ions in intact chloroplasts. Spectroscopic determination with Eriochrome Blue SE.

The metallochromic indicator Eriochrome Blue SE was used to measure light-induced internal movement of Mg2+ in intact chloroplasts. By dual-wave-length spectroscopy (measuring wavelength 554 nm, reference 592 nm) a light-induced, dark-reversible absorbance increase of Eriochrome Blue in samples of isolated intact chloroplasts was observed. The light/dark difference spectrum of Eriochrome Blue between 550 and 590 nm (reference wavelength 562 nm) indicated that this absorbance increase was caused by an increased concentration of free Mg2+ in a neutral or slightly alkaline chloroplast compartment. The signal was seen only with intact, but not with broken, envelope-free chloroplasts, which had lost most of their divalent cations. This is interpreted to show that the indicator responds to an increase of Mg2+ concentration in the chloroplast stroma, which represents an efflux of Mg2+ from the intra-thylakoid space caused by light-dependent proton pumping. As calculated from corrected values of the absorbance increase of Eriochrome Blue, the light-induced internal release of Mg2+ was close to 100 nequiv per mg chlorophyll at pH 7.6 and 250 nequiv at pH 7.1. This corresponds to a light-dependent increase in the concentration of free Mg2+ in the stroma of about 2 and 5 mM, respectively.


The binding of bicarbonate ions to washed chloroplast grana.

Radioactive labelling techniques show that isolated broken chloroplasts can take up HCO3-in the dark. There are two pools of binding sites for this ion on, or within, the thylakoid membranes. A smaller, high affinity pool exists at a concentration of one HCO3-bound per 380-400 chlorophyll molecules. Removal of HCO3-bound in this pool requires special conditions and results in greater than 90% inhibition of oxygen evolution. The inhibition is fully reversed when HCO3-is added back. HCO3-bound in the small pool does not necessarily exchange with free HCO3-in the dark or in light. Evidence presented suggests that this site is very near the site of action of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea. A second, much larger, pool of HCO3-binding sites also exists in a concentration approaching that of the bulk chlorophyll. These sites have a much lower affinity for HCO3-, and their function has not yet been determined.


Orientation and flexibility of the choline head group in phosphatidylcholine bilayers.

The average orientation and flexibility of the phosphorylcholine group are deduced from deuterium and phosphorus-31 nuclear magnetic resonance measurements of unsonicated phosphatidylcholine bilayers in the liquid crystalline state. The experimental data are consistent with a model in which the polar head group exhibits a restricted flexibility characterized by rapid transitions between two enantiomeric conformations. A completely flexible or a completely rigid head group structure can be excluded. The phosphorylcholine residue is found to be bent at the position of the phosphate group, due to a gauche-gauche conformation of the phosphodiester linkage. The choline dipole is aligned parallel to the plane of the membrane, which is in agreement with X-ray and neutron diffraction studies. The average orientation of the phosphorylcholine group is therfore the same as that of the phosphorylethanolamine head group.


Changes in structural organization of surface membrane during erythrocyte maturation.

The effect of concanavalin A and its succinylated derivative on cell agglutination and potassium compartmentation of mature and immature erythrocytes was observed. The binding of tetravalent concanavalin A to the surface glycoproteins of rabbit erythrocytes leads to a change in the properties of the surface membrane, which results in an induction of cell agglutination and concomitant release of potassium from the cells. Both of the phenomena induced by concanavalin A are temperature dependent, and observed at above 15 degrees C. Divalent succinylated concanavalin A, lacking the inducing activity of surface glycoprotein cross-linking into patches and caps, caused neither cell agglutination nor change in the potassium compartmentation of erythrocytes and reticulocytes. In the case of immature reticulocytes, however, remarkable agglutination of the cells was induced without a change in the potassium compartmentation after treatment with tetravalent concanavalin A. It is suggested that changes in the molecular organization of the surface membrane occur in which potassium compartmentation of the reticulocytes becomes more susceptible to surface glycoprotein cross-linking during cellular maturation.


Variations in microviscosity values induced by different rotational behaviour of fluorescent probes in some aliphatic environments.

Recently there have been large developments in the indirect estimation of phospholipid bilayer and membrane microviscosities through the use of fluorescent probes with the help of paraffin oils as standard solvents. Before applying this semi-empirical method to membrane systems, it seemed necessary to test: first, a large variety of probes (this has been done by many authors) and secondly, a large variety of aliphatic oils (there is little literature on these tests). The present paper shows the variations of the rotational relaxation rates of three probes in relation to the viscosities of some aliphatic oils. When changing the oil but keeping constant the macroscopic viscosity, large differences appear in the relaxation rates of a given fluorphore (a ratio of 30/1 is observed in the extreme cases). The microviscosities of membranes deduced from the probe motion will consequently exhibit large uncertainty, as is shown with dipalmitoyl phosphatidylcholine. The cause of these different behaviours must be looked for in the properties of the oil. Particularly, the anisotropy of the solute-solvent interaction in the site where the probe is located depends in part on the internal order of the solvent which is used as a reference.


Enzymatic synthesis of adenosine 5'-O-(3-[35S]-thiotriphosphate).

A method for the enzymatic synthesis of adenosine 5'-O-(3-[35S]thiotriphosphate) is described. The method involves the thiophosphate exchange of adenosine 5'-O-(3-thiotriphosphate) with [35S]thiophosphate with the aid of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase in the presence of 3-phosphoglycerate. The method is also applicable for the synthesis of guanosine 5'-O-(3-[35S]thiotriphosphate).


Studies on the glycosidases of semen. Further purification and characterization of two hexosaminidases from bull seminal plasma.

Two isozymes of beta-N-acetylglucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxy glucohydrolase, EC 3.2.1.30) (A and B) from bull seminal plasma were purified to homogeneity by isoelectric focusing having pI values of 5.31 and 6.78. The two proteins were glycoproteins with very similar amino acid composition but isozyme A contained more sialic acid than isozyme B. The molecular weights of isozyme A and B were estimated at 200 000 and 190 000 by gel filtration. Two identical subunits corresponding to molecular weights of 53 000 and 13 400 were obtained from hexosaminidase A and B when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Similar results were obtained when dissociation of the isozymes was effected with mercaptoethanol, guanidine hydrochloride and urea in presence of sodium dodecyl sulphate and the subunits separated by acrylamide gel electrophoresis. The two isozymes were more stable in frozen conditions than at the refrigerated temperature. Of the divalent ion tested, glucosaminidase and galactosaminidase activities of isozymes A and B were strongly inhibited by Hg2+ and Ag+ thus suggesting the presence of thiol groups in the two proteins. The two isozymes were active on natural substrates; isozyme B being more active than isozyme A.


A two-step purification of mouse liver glyoxalase I and evidence of its dimeric constitution.

Glyoxalase I (S-lactoyl-glutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) was purified from Swiss mouse liver to homogeneity by a rapid, two-step procedure involving hydrophobic and affinity chromatography. Homogeneity was established by multiple electrophoretic determinations and by sedimentation equilibrium centrifugation. The purified enzyme exhibited a specific activity of 944 I.U./mg protein an has a molecular weight of 43 000. The enzyme was shown to be a dimer by sodium dodecyl sulfate disc gel electrophoresis and is apparently composed of identical subunits of molecular weights approximating 21 500.


Evidence for the presence of a trypsin inhibitor within rabbit and mouse tumour cells.

Post-granule supernatant fractions obtained from induced tumour cells in rabbits and from Ehrlich ascites tumour cells in mice have been shown to contain a protease inhibitor, inhibiting trypsin (EC 3.4.21.4) and neutral proteases located in the cytoplasm of the cells. This inhibition was found to be irreversible over the time period studied, independent of the time of enzyme incubation and independent of the extent of trypsin digestion within an insoluble substrate (within the limits of linear enzyme kinetics).


Studies of the thermodynamics and nature of interaction between serum albumin and penicillins.

The possibility of a calorimetric determination of the number of homogeneous independent binding sites on the protein molecule surface is presented, together with the possibility of the determination of interaction heat and association constants for the binding of small molecules to one such site. Thermodynamic characteristics of interaction of 10 penicillins and methyl orange with primary and secondary binding sites of bovine serum albumin have been obtained using this technique. The data show that hydrogen bonds between the component parts of the complexes studied are absent. The main contribution to free energy change at forming these complexes is made by hydrophobic interactions. Electrostatic forces are also of some importance, their contribution into the complex formation is more significant in the case of quinacillin and carbenicillin which have an additional charged carboxyl group.


A photoacoustic study of newborn rat stratum corneum.

We have applied photoacoustic spectroscopy, a new spectroscopic technique, to the study of intact stratum corneum, a translucent membrane with high ultraviolet light scattering, and have obtained clear spectra in the 220-450 nm region. The photoacoustic technique has been used to study the effects of hydration and chemical alteration of the stratum corneum. Newborn rat stratum corneum shows marked spectral changes during the initial 60 h maturation period. These changes are attributed partly to the environmental and conformational changes occurring within the stratum corneum matrix during this time period. We also postulate the possibility of a novel molecular change within the keratin protein to account for our results.


The low temperature magnetic circular dichroism spectra of iron-sulphur proteins. I. Oxidised rubredoxin.

Variable temperature magnetic circular dichroism spectra have been measured on oxidised Clostridium pasteurianum rubredoxin. Evidence has been obtained for the presence of two one-electron charge-transfer transitions, sulphur to ferric ion, in the region 15 000 to 28 000 cm-1. The first moment of the lower energy band is consistent with it being the orbital transition t1 non-bonding sulphur orbital, to the 2 e ferric d-orbital. The magnitude of the spin-orbit coupling constant in the lower excited state has been determined and shown to be small compared with the axial distortion. The splitting of the low energy band observed in the absorption spectrum can therefore be equated directly with the axial distortion of the lowest excited charge-transfer state. Finally, the potential utility of making saturation experiments at very low temperatures has been examined.


The low temperature magnetic circular dichroism spectra of iron-sulphur proteins. II. Two-iron ferredoxins.

Variable temperature magnetic circular dichroism (MCD) spectra of a number of two-iron ferredoxins have been measured. The spectra of fully oxidised spinach and Spirulina maxima ferredoxin are independent of temperature between room temperature and 18 K, showing that no contribution to the room temperature MCD spectrum arises from the small population of low-lying excited states originating from the exchange coupling. However, the low temperature MCD spectra of the half-reduced proteins spinach and Spirulina maxima ferredoxin and adrenodoxin are all reasonably intense and temperature dependent. An interpretation of the spectrum of the charge-transfer region is suggested by starting with the assignments previously obtained from rubredoxin.


Natural fluorescence properties of brome mosaic virus protein.

Some of the fluorescence properties of brome mosaic virus protein in different states of aggregation (dimer, capsid) have been studied, in particular the emission spectra, fluorescence quantum yields and lifetimes, as well as the effect of external quenchers and of temperature on the fluorescence. Brome mosaic virus protein, which contains two tryptophan and five tyrosine residues per monomer, displayed an unusual fluorescence spectrum maximum of 308 nm at pH 7.4 when excited at 280 nm. The emission maximum was shifted to 327 nm when excited at 295 nm. Analysis of the results showed that the tyrosine emission is characterized by a high value for the quantum yield (phi = 0.07), which is consistent with a location of most of these residues in helical regions of  the protein, while the tryptophan emission is strongly quenched (phi=0.035). The effects of external quenchers suggested that two kinds of tryptophan residues might exist, one buried (phi=0.056) and one exposed, the quantum yield of the latter being particularly low (phi=0.014). The tryptophan fluorescence quenching is partially removed at pH 8.4 and totally eliminated after chemical (guanidinium chloride) or thermal denaturation of the protein. Formation of capsid induced an additional quenching of the exposed tryptophan residue while interaction with the RNA in the virus did not modify the emission parameters of the protein.


The influence of temperature and urea on intrinsic fluorescence of Streptomyces subtilisin inhibitor. A study by fluorescence polarization and quenching.

Intrinsic fluorescence of new microbial protease inhibitor, Streptomyces subtilisin inhibitor was studied by observing fluorescence polarization degree and lifetime in the temperature range 25-81 degrees C. Striking thermal changes in these fluorescence properties of tryptophan residues were observed. The apparent molecular volumes for tryptophan and tyrosine residues in the native form were determined to be 89 and 75 A3, respectively. The fluorescence quenching by Br- or Cs+ was investigated to obtain a microenvironmental information around tryptophan residues both in the native and denatured form. Cs+ quenches the fluorescence slightly stronger than Br-, implying that there is not any distinctive electrostatic interaction between tryptophan residues and their neighborhood.


Naja mossambica mossambica venom. Purification, some properties and the amino acid sequences of three phospholipases A (CM-I, CM-II and CM-III).

Three phospholipases A, CM-I, CM-II and CM-III, were purified from Naja mossambica mossambica venom by gel filtration on Sephadex G-50 followed by ion-exchange chromatography on CM-cellulose. They comprise each 118 amino acid residues and are close-linked by seven intrachain disulphide bridges. The complete primary structure of the three phospholipases A have been elucidated. The sequences and the invariant amino acid residues of CM-I, CM-II and CM-III resemble those of phospholipases A from other snake venoms and also from porcine pancreas. However, the letality (LD50 values) of the three phospholipases A from Naja mossambica mossambica venom, differ among themselves, and are also much higher than the LD100 value encountered for notexin from Notechis scutatus scutatus venom.


Structural properties of the glial fibrillary acidic protein. Evidence for intermolecular disulfide bonds.

Glial fibrillary acidic protein was purified from 4 M urea extracts of bovine brain by DEAE Bio-Gel A chromatography, 30% ammonium sulfate precipitation and hydroxylapatite chromatography. Subunits of about 54 000 daltons are present in solution as polydisperse distributions of polymers largely constrained by the presence of interchain disulfide linkages. Circular dichroism measurements indicate a native conformation containing some alpha-helical structure. The relevance of these findings to the cytoskeletal function of intermediate (80-100 A) filaments is discussed.


Electrophysiologic behavior modification of frontal EMG in cerebral-palsied children.

Four cerebral-palsied children participated in the following ABAB design: speech and motor pre-electrophysiologic behavior modification (EMB) evaluation; frontal EMG EMB, six weeks; speech and motor post-EBM evaluation; six weeks no training; speech and motor reevaluation; EMG EBM, four weeks; speech and motor evaluation. Auditory and visual feedback of frontal EMG was monitored by cumulative integration of frontal EMG voltage. The children were shaped by setting a cumulative voltage threshold (CVT). If the child's cumulative integrated frontal EMG voltage fell below the CVT at the end of each 60-sec epoch, a reward was automatically dispensed from a Universal Feeder. Frontal EMG decreased significantly over the initial twelve trials. Correspondingly, improvement was noted for the children in speech and motor skills. Follow-up six weeks later showed increased frontal EMG voltage and deterioration of speech and motor function. Reinstitution of frontal EMG EBM produced reacquisition of low frontal EMG and some recovery of speech and motor function. Collectively, these results indicate that frontal EMG EBM shows promise as an additional treatment modality in the habilitation of cerebral palsy children with spasticity.


The use of feedback training and a stimulus control procedure to obtain large magnitude increases in heart rate outside of the laboratory.

A series of eight single-subject experiments were performed to test the feasibility of using a stimulus control procedure to teach subjects to control heart rate in the absence of feedback and outside of the laboratory. Four of the six subjects who completed the experiments were able to demonstrate increases in heart rate of from 15 to 35 beats per minute without the assistance of feedback and outside of the laboratory. These increases were consistently produced over six daily sesssions. Through the use of a systematic replication series of experiments, it was possible tentatively to rule out age, sex, and basal heart rate of subject, as well as type of previous feedback training and number of prior training sessions, as accounting for the differential success.


Affinity chromatography systems for artificial liver support.

The physical, adsorptive, and flow properties of two albumin conjugated agarose materials have been investigated in vitro to determine whether they could be used in artificial liver support systems to enhance the removal of toxic protein-bound substances. The results of binding of chenodeoxycholic acid and unconjugated bilirubin and flow experiments with blood and saline suggested that there could be advantages in using the SepharoseR rather than Bio--GelR albumin agarose in future haemoperfusion systems. However, further modifications are needed before the former can be successfully scaled up for human use.


Electric field effects in bacteriorhodopsin.

Exposure of aqueous suspensions of fragments of the purple membrane of Halobacterium halobium to electric field pulses leads to transient linear dichroism phenomena. The effects are interpreted in terms of field-induced alignments of the bacteriorhodopsin chromophore. Two observed relaxation times (tau) are attributed to rotation of the whole membrane fragments (tau s approximately 100 ms), and to a much faster reorientation of the chromophore within membrane (tau f approximately 260 microns).


Interpretation of the small-angle X-ray diffraction of collagen in view of the primary structure of the alpha1 chain.

The small-angle X-ray diffraction pattern of collagen has been calculated using the sequence of the alpha 1 chain and a Hodge-Petruska scheme for the packing of the collagen molecules. The molecular stagger giving the best fit of calculated-to observed structure factors has been found to be 236 or 237 amino acid residues for three tendon collagens. But this result depends on the appoximation that the  molecular conformation is uniform throughout the molecule. A comparison of the observed and calculated electron density profiles in axial projection leads to a corrected model, in which the COOH-terminal telopeptide is contracted in a way suggesting a saddle-shaped electron density distribution near the collagenase site.


Characteristics of sodium tail currents in Myxicola axons. Comparison with membrane asymmetry currents.

Sodium currents after repolarization to more negative potentials after initial activation were digitally recorded in voltage-clamped Myxicola axons compensated for series resistance. The results are inconsistent with a Hodgkin-Huxley-type kinetic scheme. At potentials more negative than -50 mV, the Na+ tails show two distinct time constants, while at more positive potentials only a single exponential process can be resolved. The time-course of the tail currents was totally unaffected when tetrodotoxin (TTX) was added to reduce gNa to low values, demonstrating the absence of any artifact dependent on membrane current. Tail currents were altered by [Ca++] in a manner consistent with a simple alteration in surface potential. Asymmetry current "off" responses are well described by a single exponential. The time constant for this response averaged 2.3 times larger than that for the rapid component of the Na+ repolarization current and was not sensitive to pulse amplitude or duration, although it did vary with holding potential. Other asymmetry current observations confirm previous reports on Myxicola.


The noneffect of a large linear hydrocarbon, squalene, on the phosphatidylcholine packing structure.

The interaction of squalene with liposomes and monolayers of dipalmitoyl phosphatidylcholine (DPL) has been studied by differential scanning calorimetry, Raman spectroscopy, and surface potential measurements. Mole ratios of squalene to DPL up to 9 to 1 were studied. In contrast to small, nonpolar molecules, which profoundly influence the structure of lipid bilayers as detected by changes in both their thermodynamic phase transition parameters and membrane fluidity, this large, nonpolar, linear hydrocarbon is devoid of such influences. It is clear from our data that a large nonpolar molecule such as squalene, having no polar group that might anchor it to the aqueous interface, cannot intercalate between the acyl chains either below or above the phase transition of DPL. This behavior is not compatible with models that treat the bilayer interior as a bulk hydrocarbon, and suggests that great caution should be exercised in extrapolating partition coefficients based on bulk hydrocarbon measurements to lipid bilayers.


The organization of repeated nucleotide sequences in the replicons of mammalian DNA.

Chinese hamster ovary cells were irradiated with 100-5,000 rads of X-rays and inhibition of the initiation of replicons after irradiation was demonstrated by analyzing nascent DNA sedimented in alkaline sucrose gradients. The renaturation kinetics of DNA synthesized during 60 min of incubation after irradiation was compared with that of DNA synthesized during the 60 min after sham irradiation and with that of parental DNA. Nascent DNA from cells whose replicon initiation was inhibited renatured faster than nascent DNA from control cells in the COt range of repeated nucleotide sequences, suggesting that regions of the replicon not close to origins are enriched in repeated sequences and that regions close to origins are enriched in unique sequences. A class of repeated nucleotide sequences may be involved in the regulation of replicon initiation.


A single-file model for potassium transport in squid giant axon. Simulation of potassium currents at normal ionic concentrations.

A physical model for potassium transport in squid giant axon is proposed. The model is designed to explain the empirical data given by the Hodgkin-Huxley model and related experiments. It is assumed that K(+) moves across the axon membrane by single-file diffusion through narrow pores. In the model a pore has three negatively charged sites that can be occupied alternatively by K(+) or by a gating particle, GP(++), coming from the external surface. GP(++) is considered to be part of the membrane rather than a diffusible component of the surrounding solutions. A high activation barrier for GP(++) is supposed at the inner membrane border so that it cannot change over to the internal surface. Therefore potassium diffusion can be blocked by GP(++) penetrating into the pores. This mechanism controls the dynamic behaviour of the model. The time-dependent probabilities of the pore states are described by a system of differential equations. The rate constants in these equations depend on the ionic concentrations, the membrane voltage, and the electrostatic interaction between ions in a single pore. Detailed computational tests for normal composition of external and internal solutions show that the model agrees remarkably well with the stationary and dynamic behaviour of the Hodgkin-Huxley model. However, the hyperpolarization delay is not reproduced. A structural modification, concerning this delay and the way in which GP(++) is attached to the membrane, is proposed, and the qualitative behavior of the model at varied external and internal concentrations is discussed.


Asymmetry currents and admittance in squid axons.

The complex admittance of squid (Loligo pealei) axon was measured rapidly (within 1 s) with pseudo-random small signals and discrete Fourier transform techniques under guarded, "space-clamp" conditions and during suppression of ion conduction. Asymmetry currents were measured by paired step clam pulses of +/-70 mV from a holding potential of -97 mV and gave an apparent capacitance of 0.36 muF/cm2. However, the admittance data showed no change in capacitance at holding potentials from -97 to -67 mV and gave a decrease of 0.07 of 0.15 muF/cm2 at -37 mV. The failure to observe a capacitance increase at low membrane potentials suggests the following possibilities: (a) the asymmetry current is a displacement current that inactivates completely with time, and (b) the asymmetry current is not a displacement current and arises from large signal effects (i.e., delayed nonlinearity in ionic current) on the membrane.


On the photocycle and light adaptation of dark-adapted bacteriorhodopsin.

Pulsed Nd laser (25 ns, 530 nm) photolysis experiments were carried out at room temperature in aqueous suspensions of dark- and light-adapted fragments of the purple membrane of Halobacterium halobium. It is shown that the (50%) 13-cis isomeric component (BR13-cis) of dark-adapted bacteriorhodopsin (BRDA) undergoes a photocycle involving a characteristic transient absorbing in the neighborhood of 610 nm. At relatively high excitation intensities BR13-cis is converted to the same 410 nm (M) transient that characterized the photocycle of the all-trans isomer (BRtrans) of light-adapted bacteriorhodopsin (BRLA). This process, which competes with the generation of the "610" species, is attributed to the photo-induced conversion, during the pulse, of BR13-cis (or of its primary photoproduct "X") to a species in the BRtrans photocyte. The relationship between these observations and the mechanism of BRDA hv leads to BRLA adaptation at low excitation intensities (for which a quantum yield limit, 0 less than or equal to (3.5 +/- 0.7) X 10(-2) , is established) is discussed.


Synthetic pigment analogues of the purple membrane protein.

Nonphysiological analogues of retinal have been shown to form pigments in reactions with the apoprotein of the purple membrane of Halobacterium halobium. Both the all-trans and 13-cis isomers of a retinal analogue, having an elongated chain with an extra double bond, formed pigments. Unlike the native all-trans and 13-cis retinal1-based pigments, the new pigments were not interconvertible with each other and were unstable against hydroxylamine. When incorporated into phospholipid vesicles, they showed no proton pumping activity upon illumination. The ability of the extended-length retinal to form pigments contrasts with its nonreactivity with opsin (apoprotein of rhodopsin), suggesting a less stringent binding site for the purple membrane chromophore. All-trans retinal2 also combined with bleached purple membrane to form a blue pigment absorbing at ca. 590 nm. Like the native purple membrane, the blu membrane showed proton pumping activity upon illumination in phospholipid vesicles.


Interaction of calcium with negative lipids in planar bilayer membranes. Influence of the solvent.

The interaction of Ca++ with acidic phospholipids in black lipid films and lipid bilayers formed from two monolayers was studied by measuring their physical stability and conductance. It was found that the addition of CaCl2 to only one side of lipid bilayers formed from phosphatidylserine or cardiolipin does not appreciably change these parameters. In contrast, black films are unstable to the asymmetric addition of CaCl2. Therefore, the destabilizing effect of Ca++ cannot be attributed to a surface charge difference. The only variation in composition between both bilayer membranes, namely the solvent content of the bilayer, seems to be responsible for the distinctive effect of Ca++. A tentative explanation is presented.


Molecular dynamics through 2H spin-lattice relaxation time measurements with an enzyme-inhibitor complex: lysozyme and methyl N-acetyl glucosaminides.

Deuteron spin-lattice relaxation times of specifically labelled methyl N-acetyl-D-glucosaminides associated to lysozyme were measured from 1H and 2H NMR spectra through bandshape analysis and FT inversion-recovery technique, respectively. Model calculations were carried out in order to assess the limits of the extreme narrowing approximation for the systems studied. Rotational correlation times of the acetamido methyl groups were analyzed in terms of anisotropic overall reorientation combined with internal rotation. The acetamido methyl group undergoes fast internal rotation in the alpha-glycoside complex about an axis nearly parallel with the major ellipsodial axis of lysozyme. More rotational freedom is likely to occur in the beta-glycoside complex.


Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method. I. Autoassociation of 2-aminopurine.

A general equation was derived, describing fluorescence quantum yield and lifetime of an autoassociating compound in liquid solutions. The autoassociation of 2-aminopurine in aqueous solution was examined within the range from 0 to 90 degrees C. The compound seemed to associate cooperatively. The thermodynamic parameters of polymerization change with temperature, so that its free enthalypy deltaG = 0.0797 T2 + 45.4  T - 7893. The dimerization enthalpy and entropy are approximately temperature-independent (deltaH2 = -4.17 kcal/mol deltas2 = -10.9 e.u.), although the function: delta g2 = -0.0308T2 +30.3T -  7213 fits experimental points better. The observed dependences can be explained by the increasing role of the hydrophobic effect with temperature and size of the aggregates. The association rate constants were determined, and a two-step reaction mechanism was demonstrated. The first step is diffusion-controlled. The second is characterized by an activation energy of approximately 2  kcal/mol and an encounter distance of approximately 8.3 angstroms.


Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method. II. Heteroassociation between 2-aminopurine and thymidine.

Heteroassociation between A and B compounds in liquid solution was considered. Provided that concentration of A molecules is low, a general equation describing fluorescence quantum yield and lifetime of compound A as a function of B molecules concentration was derived. The heteroassociation between 2-aminopurine and thymidine in aqueous solutions was examined within the range of temperatures 0 to 90 degrees C. The equilibrium constants of the first step of association, namely heterodimer formation, were determined and its thermodynamic parameters (deltaH equals - 2.76 kcal/mol, deltaS equals - 5.9 e.u.) were calculated. The observed changes of the stacking rate constants with temperature confirm the two-step mechanism of the reaction. The activation energy (approximately 10.7 angstroms) are only slightly larger than in the case of 2-aminopurine autoassociation, most probably because of a stronger solvation of thymidine molecules.


Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method. III. Intramolecular association of 9,9'-[1,3-propylene]-bis-2-aminopurine.

General equations relating fluorescence quantum yield and lifetime of a compound with its intramolecular stacking equilibrium and kinetics were derived. Intramolecular stacking association of 9,9'-[1,3-propylene]-bis-2-aminopurine in aqueous solution was examined within the range of temperatures from 0 to 90 degrees C. A two-state thermodynamic model of the association was verified. The stacking enthalpy and entropy can be taken, with a good approximation, as temperature-independent (deltaH equals - 2.0 kcal/mol, deltaS=-3.25 e.u.) although the function deltaG=-0.00886T2 + 8.847 T - 2876 describes more precisely the observed changes of stacking free enthalpy with temperature. The association rate constants were detemined. Activation energy of the reaction (2 kcal/mol) is the same as in the case of association between free 2-aminopurine molecules. It confirms a two-step mechanism of the process. The advantages and shortcomings of the fluorescence quenching method are discussed.


Thermoosmosis through charged membranes.

Thermoosmosis through oxidized collodion and collodion-sulfonated polystyrene interpolyrene interpolymer membranes has been observed in KCl solutions of various concentrations. The effective temperature difference acting for thermoosmosis was determined by measuring the thermal membrane potential appearing on both sides of membrane. It was found that the velocity of thermoosmosis is proportional to the effective temperature difference and the proprtionality constant (themoosmotic coefficient) is a function of electrolyte concentration. The dependence of the thermoosmotic coefficient of charged membranes on the electrolyte concentration is found to have a characteristic feature.


Fluorescence decay and quantum yield characteristics of acridine orange and proflavine bound to DNA.

Fluorescence properties (quantum yield, decay curve, lifetime and polarization) of acridine orange and proflavine bound to DNA were examined as a function of nucleotide to dye (P/D) ratio. First, mean fluorescence lifetimes were determined by the phase-shift measurements. The lifetime and quantum yield of acridine orange increased in a parallel fashion with increasing P/D ratio. There was no parallel relation between the lifetime and quantum yield for proflavine; the lifetime showed a minimum around  P/D equals 10...


Equilibrium gel permeation: measurement of solute partitioning by direct fluorescence scanning.

The feasibility fo using fluorescence detection in quantitative gel permeation measurements has been explored. It is shown that the effect of scattering by the gel matrix can be evaluated in terms of pathlength-dependent turbidity functions for excitation and emission wavelengths. Experimental studies were carried out to evaluate these funcitons in cross-linked dextran gels (Sephadexes) and in agarose gels (Sepharoses). Empirical turbidity functions derived for these gels have a simple form, leading to accurate simplifying approximations for the scattering correction required in a fluorescence gel permeation measurement. Using this approach...


The problem of nuclease activity in nucleic acid hybridization reactions. Theoretical considerations.

The presence of nuclease during DNA-DNA or DNA-RNA hybridization reactions alters the kinetics of hybrid formation. Unfortunately, while the effect (even of small amounts of nucleas) on the Cot curve may be large, it may not be readily detectable. The effect of various types of nuclease is shown. As many nucleases cause a shift in the position rather than a change in shape of the curve, all studies involving nucleic acid hybridization should assay for the presence of nucleases and care must be taken to avoid their presence as contaminants.


Kinetics of monensin complexation with sodium ions by 23NaNMR spectroscopy.

The kinetics of the sodium binding to the ionophore monensin (Mon) in methanol has been studied by 23NaNMR spectroscopy. Fast quadrupole relaxation of the bound sodium affected the relaxation rate of the free sodium through an exchange process between these two species. The exchange was found to be dominated by the reaction: Na+ + Mon-in equilibrium MonNa. The dissociation rate constant at 25 degrees C is 63 s(-1), with an activation enthalpy of 10.3 kcal/mol and activation centropy of -15.8 cal/mol deg. These results indicate that the specificity of the binding of sodium ions to monensin is reflectied in the relatively slow dissociation process. The entropy changes indicate that the activated monensin-sodium complex undergoes a conformational change, but the existence of a conformational change in monensin anion prior to complexation is excluded.


Thermodynamics of the interaction of daunomycin with DNA.

The association constant for the interaction of daunomycin with DNA was determined as a function of temperature (using [3H] daunomycin in conventional equilibrium dialysis cells) and ionic strength (using a spectrophotometric titration method). The association constant varied between 3.1 X 10(-6) M(-1) (4 degrees C) and 3.9 X 10(5) M(-1) (65 degrees C). The free energy change was -8.2 to -8.8 kcal/mol, the enthalpy change -5.3 kcal/mol and the entropy change +10 to +11 eu, all values being consistent with that expected of an intercalation process. The apparent number of intercalation sites detected (0.15 to 0.16 per nucleotide) was independent of temperature. The large positive entropy change accompanying the interaction appears to be due to extensive release of water from the DNA and daunomycin. The apparent number of binding sites increased dramatically with decrease of ionic strength, although the apparent association constant remained largely unaffected by ionic strength.


Spectral properties of chlorophyll a in liquid crystal.

Solution of chlorophyll a in liquid crystals mixture (MBBA + EBBA) was investigated. Chl molecules are in LC in low electric field oriented. They can be divided into two groups: one strongly interacting with LC and subjected to reorientation by the electric field, and another weakly interacting with the solvent and insensitive to the voltage applied. The emission spectrum of the first type of chlorophyll is strongly perturbed. At higher voltages, the pigment molecules orientation in the plane of the electrode is another. Pigment absorption and emission anisotropy provides information about the reorientation of LC molecules. Even at high (10(-3)M) Chl concentration and regular pigment array, the interaction between the pigment and solvent exceeds pigment-pigment interaction because the solvent appears to have a stronger influence on the Chl spectrum.


[Automatic data processing for organisational aid to blood donor centers in emergency hospitals].

A test model of a computer-off-line system at the Community Hospital Blood Bank in Herford collecting approximately 3.000 units of blood annually is described. The computer system presented, now in operation for more than one year, has proven its value for general blood bank administration by its time-saving effect and the optimalized documentation of donor- and blood-related data. The computer system described is particularly recommended for smaller, hospital-based blood transfusion services. Its introduction is even justified in institutions preparing less than 5.000 units of blood per year. Prerequisite for an economical operation of the computer system is the participation of the hospital management in other computer activities, i.e. book-keeping, accounting etc.


[Erythropoiesis in iron deficiency].

The kinetics of erythroblast proliferation were studied by means of quantitative 14C-autoradiography in 5 patients showing anemia due to infection or malignancy, in 7 patients suffering from iron deficiency anemia, and in two individuals with bleeding enemia. Compared with a group of 5 healthy persons a markedly reduced turnover of erythroblasts was found in the anemia due to infection, malignancy, and iron deficiency, whereas this turnover was normal or increased in the case of bleeding anemia. The reduction is caused by a progressively decreasing rate of erythroblast proliferation and maturation with advancing development into mature cells. No indications of a change in the number of cell divisions were found in the anemia of infection, malignancy, and of iron deficiency, nor was there evidence of an intramedullary death of nucleated red cell percursors. The imbalance between production and loss of red cells causing anemia shows a different pattern in the 3 groups of disease: In bleeding anemia the insufficiency of supply is not yet apparent from the rate of erythroblast turnover giving weight to the factor of blood loss. In anemia due to infection, malignancy, and iron deficiency the but moderately increased rate of red cell destruction cannot be compensated because of several impairments: The rate of erythroblast turnover is reduced, and, in addition, a moderate portion of maturing cells is destroyed, probably at the reticulocyte stage. As the most significant factor, the bone marrow is unable to compensate the anemia by an effective erythroblast hyperplasia. In iron deficiency this hyperplasia is inadequately low, in infection and malignancies, however, it is more or less missing.


Cytochemical observations in preleukemic states and remission of akute leukemia.

In preleukemia and in remission of acute leukemia abnormal mononuclear cells ("string of bead" cells) have been observed and characterized with cytochemical methods. These cells may be resistant to cytostatic therapy and may be characterized as a source of a newly developing leukemic blast cells. Preleukemia and leukemic remissions seem to be identical states. Based on the morphological and cytochemical parallelity of cells in CFU-c-enriched fractions of the bone marrow and the so called string of bead cells we consider these cells to be closely related to the human commited stem cell.


[In-vitro studies with human transfer factor].

Human transfer factor was prepared from "buffy coat" of human donors by ultra filtration. These batches of transfer factor were tested for their ability to increase DNA-synthesis of antigen(mitogen) incubated lymphocytes. An increase of DNA-synthesis was measured by the ratio of appr. 2. Column chromatographic separation of transfer factor on sephadex G-10 or DEAE cellulose columns showed an elution pattern with many peaks. In the lymphoblastogenese assay some peaks were found to be stimulatory, others in contrast inhibitory. It seems likely from our results that transfer factor represents a mixture of different chemical compounds with activating or inhibitory action on lymphoblastogenesis.


Accumulation and elimination of dieldrin in muscle tissue of channel catfish.

Dieldrin accumulation and elimination in muscle tissue of channel catfish (Ictalurus punctatus) from water and food was determined in the laboratory. Twenty-eight-day exposure of fish 150-225 mm and 350-400 mm long to 75 parts per trillion (ng/liter) dieldrin resulted in the larger catfish consistently accumulating more dieldrin than the smaller fish. After 28 days of elimination, dieldrin levels in both size groups were nearly equal. Catfish exposed to 2 ppm (mg/kg) dieldrin through their diets accumulated significantly more dieldrin in muscle than did fish exposed to 75 pptr in water. When fish were exposed to dieldrin both in food and water, dieldrin from both sources contributed to the total dieldrin load. Large catfish accumulated more dieldrin from food and water than did smaller catfish. After 28 days of elimination, levels of dieldrin were not significantly different in muscles of 150-225 mm and 350-440 mm catfish exposed via both food and water.


Liver cadmium levels in North Carolina residents who died of heart disease.

Using data from an autopsy series, we found a strong positive correlation between liver concentration of cadmium and death from heart disease. In view of recent experiments reported in the literature, the possibility that a low level of cadmium has a toxic effect on the cardiac conduction system is supported.


Further toxicologic studies with commercial and candidate flame retardant chemicals. Part II.

A number of commercial and candidate flame retardants were studied with regard to their toxicity to goldfish, inhibition of cholinesterase, inhibition of acetyl choline binding to its receptor and insecticidal properties. Several of the flame retardants were notably toxic to fish. Some of the compounds showed modest inhibition of cholinesterase and/or microsomal oxidases, but none inhibited acetyl choline receptor binding. Whereas several of the flame retardants showed little or no insecticidal properties when added alone to a housefly diet, piperonyl butoxide greatly synergised their toxicity to houseflies.


[Ventilatory and cardio-respiratory responses during exercise in chronic pulmonary restrictive disease (author's transl)].

Cardio-respiratory changes were studied during light exercise (20 or 40 W) in 45 patients with diminished vital total lung capacity. Diagnosis was : fibrosis (8 subjects, group I), lobectomy (7 subjects, group II), pachypleuritis (11 subjects, group III), thoracoplasty (7 subjects, group IV) and kyphoscoliosis (12 subjects, group V). Changes during exercise were characterized by : an increase in breathing rate much more marked than normally, with a lesser increase in tidal volume, so that ventilatory equivalent was steady, and below 30 except in group I; an increase in PaCO2, even in group I which was hypocapnic at rest, very marked in groups III and V, with worsening of hypoxemia in most cases; and enhancement of pulmonary artery hypertension, severe at rest already in group I.


Central haemodynamics during exercise in patients with restrictive pulmonary disease.

Pulmonary precapillary hypertension present only during exercise is the first stage of corpulmonale. Examination of the reaction of central haemodynamics to exercise is the only way to detect it. In a model situation like unilateral pulmonary artery occlusion (UPAO) in healthy subjects, an increase in pulmonary artery pressure (Ppa) during exercise is a linear function of restriction of the pulmonary vascular bed and blood flow increase. Central haemodynamics during exercise in younger subjects after pneumonectomy (PNE) resemble the situation with UPAO provided the remaining lung is intact. In older patients after PNE the same rise of blood flow is associated with a considerably greater rise of Ppa. PNE patients compared to healthy individuals increase their cardiac output during exercise more by increasing stroke volume. Patients with pulmonary fibrosis show a disproportionate rise in Ppa in relation to flow. This is due to reduced distensibility of the pulmonary system. Hypoxia also seems to be involved in pulmonary hypertension during exercise. Pulmonary wedge pressure rises at work but remains within the normal range, even in patients having marked hypoxaemia at rest and during exercise. In idiopathic intersitial pulmonary fibrosis (IIPF), pulmonary hypertension during exercise is greater than in other forms of pulmonary fibrosis. Inhalation of oxygen during exercise in patients IIPF leads to a reversal of hypoxaemia but the decrease of Ppa is small and due to a passive decrease of cardiac output. Finally right ventricular function in restrictive pneumopathies is discussed.


Peripheral limiting factors during exercise in chronic lung diseases.

In normal subjects the sensation of general fatigue subjectively limits the physical performance, whereas in patients with reduced respiratory function the sensation of breathlessness is the common limiting symptom. Patients with chronic obstructive lung disease may adopt an intermittent exercise pattern (e.g. when climibing stairs), in which the work periods are brief in duration but relatively high in intensity. The immediate ventilatory demand can in that way be reduced. The local metabolism of the working muscles can meet the increased demand, which demonstrates that peripheral factors are unlikely to limit the physical performance in these patients. For evaluating the importance of peripheral factors limiting the exercise tolerance of daily activities, such as stair-climbing or walking uphill, such activities should specifically be studied considerating also the exercise pattern shown by the patient. Even in bronchitic patients with reduced arterial oxygen tension there are no evidence that the skeletal muscles are working under more anaerobic conditions than in normal persons at the same relative work load. The blood lactate as related to the heart rate usually lies within normal limits. In patients with respiratory failure, however, lack of energy supply from the energy rich compounds may develop. Thus, only in extreme situations can the exercise performance of the respiratory patients be expected to be specifically limited by peripheral factors. Physical inactivity leads to changes in the muscle metabolism as in normal persons with reduced aerobic capacity in the muscles. The specific effects of steroid treatment on the muscle function in patients with chronic obstructive disease have not been studied, but may be an important factor limiting muscular performance at certain types of exercises.


[Adaptation of body plethysmography to the study of respiratory mechanics during muscular exercise (author's transl)].

A pressure-corrected integrated flow plethysmograph allowing the study of mechanical characteristics of the ventilatory system during muscular exercise is described. In order to limit the increase in temperature inside the body box during exercise, a forced convective flow passes through the box. Thermal exchanges induced by this flow have been studied. They are sufficient to allow muscular exercise during 15 minutes at a constant power of 100 W. It has been verified that this type of box allows measurement of the usual parameters of ventilatory mechanics: pulmonary volumes, compliance, resistance using the panting method. The measurement of resistance during spontaneous breathing inside the box is not possible.


Oxygen breathing during exercise in patients with diffuse interstitial fibrosis.

100% oxygen breathing during submaximal exercise in patients with diffuse interstitial lung disease diminishes cardiac output and pulmonary artery hypertension. The pressure drop in the pulmonary artery is most marked in patients with concomitant bronchial obstruction. At rest hyperoxia produced only minimal cardiopulmonary changes. The ventilatory and hemodynamic responses are discussed to find out if differences in these variables during oxygen breathing could be a reliable index for functional classification.


[Metabolic and respiratory parameters during muscular exercise in man (author's transl)].

Muscle can use ATP exclusively as the direct source of energy for contraction. The muscle ATP stores cannot provide more than 1 or 2 kcal of muscular work. tthe energy for resynthetizing ATP is supplied by three processes : the breakdown of creatine phosphate, anaerobic glycolysis and aerobic processes. These three mechanisms are characterized by different inertia, maximal output and capacity. Taking into account the part of aerobic processes in energy production, the physical fitness of an individual is usually expressed by its maximal oxygen uptake (VO2 max.). When the energy requirements cannot be met by aerobic reactions, the subject contracts an oxygen deficit which is compensated for during the recovery period by an oxygen uptake exceeding the rest requirements. During exercise tidal volume and ventilatory frequency are increased. The increase in ventilatory output is directly related to the workload until 75% of the maximal aerobic power is reached. For higher relative workloads the increase in ventilatory output is steeper. This increased ventilation allows the organism to limit the decreases in PaO2 and pH during exercises of high intensity.


The place of exercise testing in the investigation of patients with respiratory disorders.

Exercise testing may yield important information in the clinical assessment of patients with a wide variety of conditions. An objective measure of exercise capacity is obtained; limiting factors are identified; the adaptive capacity of the heart and lungs is measured; diagnoses are confirmed or excluded. This paper argues the case for a series of exercise testing procedures which vary from simple ("stages I and II") to complex ("stages III and IV"), which allows exercise testing to be widely applied to clinical investigation and which reserves complex and invasive procedures for the clinical situations in which the information is necessary. Examples are presented to illustrate the relative value of the procedures and the information obtained from each.


Value of simple parameters during exercise in chronic obstructive lung disease.

The value of simple physiological parameters as heart rate and peak expiratory flow rate, and bicycle ergometer, together with electrocardiogram is emphasized. Exercise capacity, exercise induced bronchoconstriction and/or electrocardiographic abnormalities are the conditions most important to look for. Recording tidal volume and respiratory air low might give additional information about disproportion between ventilatory "drive" and "performance".


[Therapeutic effects of physical training in patients with chronic respiratory insufficiency].

The therapeutic benefits of physical training for patients with chronic respiratory insufficiency must be assessed on the clinical, physiopathological and sociopsychological planes. As far as the clinical state is concerned, one observes a reduction of dyspnea and of the frequency of hospitalizations and an increase in the level of daily activity. On the physiopathological point of view, one observes an increase in the maximal capacity of exercise ranging from 1 to 33% resulting mostly from a better peripheral oxygen extraction. Pulmonary function is little modified on the spirographic level; however, one observes an improvement of blood gases at rest which seems to result from better alveolar ventilation. No systematic change in the circulatory variables is reported, except for a reduction of the PPA at rest. About the psychological factors, one observes a reduction of anxiety and an increase in motivation to perform muscular exercise. These results are difficult to obtain and often require a long rehabilitation. Socially, numerous obstacles are encountered to a social reinstatement : a relatively advanced age, a disadvantaged social environment, a low cultural level and an affectively weak surrounding. The solution to these different problems must be sought for each individual and the rehabilitation must be pursued continually; this often necessitates the hospitalization of the patient in a rehabilitation institute. On the basis of these studies, it seems certain that physical training is beneficial to patients with chronic respiratory insufficiency. However different programmes should be proposed for patients in an advanced or intial stage of their illness.


On the design and effectiveness of training regimes in chronic obstructive lung disease.

Most patients with chronic obstructive lung disease (COLD) show a favourable response to progressive endurance training. Symptomatology is reduced and effort tolerance is extended. However, there is little change of pulmonary function scores. Possible explanations of the clinical gains include 1) psychological encouragement, 2) improvements of mechanical efficiency, 3) restoration of cardiovascular fitness, thus breaking a vicous circle of dyspnoea, inactivity and worsening dyspnoea, 4) strengthening of the body musculature, thus reducing the proportion of anaerobic work, 5) biochemical adaptations reducing glycolysis in the active tissues, and 6) indirect responses to such factors as group support, with advice on smoking habits, breathing patterns and bronchial hygiene. tdespite immediate gains of performance, there is no reason to believe exercise can restore destroyed pulmonary tissue. Further study is thus needed to test whether training can influence long-term prognosis in COLD.


Neurosyphilis.

In the future it seems probable that cases of neurosyphilis will be seen sporadically by physicians and neurologists. As a result of the almost universal ingestion of antibiotics for trivial conditions, many cases of syphilis will be modified or arrested and some patients will be cured of the disease without ever knowing that they have acquired it. In others, developing neurosyphilis may be attenuated by inadequate chance treatment and the clinical and laboratory manifestations may become more subtle and difficult to recognize, leading to errors in diagnosis. Clinical experience of the varied manifestations of neurosyphilis has become less widespread among physicians and consequently greater care should be taken to ensure that the diagnosis is not missed.