Is automatic disinfection between each endoscopy mandatory? Société Royale Belge de Gastro-entérologie

PURPOSE: The effects of chronic chloroquine administration on serum proteins and free amino acids of rabbits and the accompanying ultrastructural changes in the retina were investigated. METHODS: Thirty pigmented rabbits were injected intramuscularly with chloroquine diphosphate (14 mg/Kg bw). Fifteen rabbits served as control. Blood samples drawn after 1, 2, 3, 4, 5, 6 and 12 months were assayed for serum proteins by Bio-analytic kits and horizontal electrophoretic technique. Free amino acids were determined by Beckman analyzer. Central retinal tissue was fixed in 4% glutaraldehyde, embedded in araldite CY212 and sectioned for ultrastructural examination. RESULTS: Histopathologic changes were apparent within three months, initially affecting the pigment epithelium and photoreceptors and later involving the remaining neuroretinal layers. Hypoproteinemia gradually developed mainly due to a sharp drop in albumin and alpha 1 and 2 globulin fractions, inspite of an increase in beta and gamma globulin fractions. The concentration of non-essential amino acids varied considerably from a depletion of taurine, aspartine, glutamine, tryptophan and arginine to an increase of serine, alanine, GABA and ornithine. The most outstanding effect was the disappearance of tyrosine and its return to normal value at the end of the experiment. CONCLUSION: The disturbances in protein pattern and free amino acid levels are an indication of chloroquine toxicity and may be implicated in the retinopathy.     

Protein hydrolysis by immobilized enzymes

The enzymic hydrolysis of some proteins (insulin-B-chain-S-sulfonate, S-aminoethylated lysozyme, bovine serum albumin) by immobilized peptidolytic enzymes is reported. Sepharose-bound pronase, trypsin and a protease from Thermoactinomyces sp. (MP), the latter both cross linked by glutaric dialdehyde and an exopeptidase mixture containing Sepharose-bound leucine aminopeptidase, carboxypeptidase A and a crude preparation of prolidase were used. After enzymic hydrolysis nearly all amino acids, except proline, were recovered in a 100% yield compared to the value of an acid reference hydrolysate. Tryptophan and methionine, which are partially destroyed by acid hydrolysis in the presence of oxygen could be recovered completely.     

Effect of amino acids on purified rat intestinal brush-border membrane aminooligopeptidase

When a hexapeptide, Leu-Trp-Met-Arg-Phe-Ala, or a pentoapeptide, Leu-Trp-Met-Arg-Phe, was incubated in vitro with a purified aminooligopeptidase from rat small intestinal mucosa, the respective C-terminal dipeptides, Phe-Ala and Arg-Phe, were observed to be resistant to hydrolysis. The resistance of these C-terminal dipeptides to hydrolysis was found to be due mainly to the accumulation of inhibitory hydrophobic amino acids liberated in the incubation mixture. The hydrolysis of various peptides by the brush-border membrane peptidase is inhibited to a varying extent by the hydrophobic amino acids L-tryptophan, L-methionine, L-isoleucine, L-leucine, L-tyrosine, and L-phenylalanine, but not the D-form of these amino acids. The inhibition of the hydrolysis of three dipeptides by hydrophobic amino acids showed these amino acids to be competitive inhibitors (same Vmax, the maximal velocity of the enzyme reaction; different Km, the substrate concentration at which the enzyme reaction is half maximal) of one of the dipeptides while exhibiting a mode of inhibition that was not competitive (different Vmax, different Km) with either of the other two dipeptides. These data indicate that the effect of amino acids on the hydrolytic rate of the brush-border membrane aminooligopeptidases must be considered in studies of intestinal hydrolysis and absorption of peptides.     

Distribution of radioactivity in the body and rate of incorporation of radioactivity into the tissue proteins of monogastric animals following intravenous injection of tracer amino acids

The studies were carried out with pigs and rats. The radioactive animo acids (14C leucine and 3H lysine) were administered to the pigs by way of a catheter tube into the jugular vein. Subsequently, the time pattern of the distribution of the specific amino acid radioactivity was followed in the TCE soluble and Tce precipitable fractions of the blood plasma (TCE= trichloro-acetic acid). The radioactive labelling in rats was carried out by injecting 14C leucine into the portal vein. The animals were killed after incorporation periods from 2 to 60 mins, and the levels of specific radioactivity were estimated in the TCE soluble and TCE precipitable fractions of the blood plasma, in the liver and in the skeletal muscles. The experimental results clearly indicated that the specific radioactivity of the tracer amino acids and the rate of incorporation of radioactivity into tissue proteins were greatly influenced by the size of the free amino acid pool within the range of distribution of the tracer. An estimation of the magnitude of the pool of free amino acids within the distribution range of the tracer can be obtained from the curve pattern for the decline of specific radioactivity of the corresponding free amino acid in the blood plasma. This pool exhibits a high rate of turnover. In all studies made to evaluate in vivo processes of protein synthesis by use of radioactive tracer amino acids it will be particularly important that consideration should be given to the specific radioactivity of the amino acid in the precursor pool for protein synthesis.     

Enteroplication for the prevention of intussusception recurrence in dogs: 31 cases (1978-1992)

Blood serum concentrations of protein, albumin, prealbumin, transferrin, amino acids and urea were measured in 31 healthy cows 0 to 6 weeks before and 3 to 8 weeks after parturition. In comparison to the precalving values the concentrations of albumin, prealbumin and transferrin were all lower after parturition. Alanine, glutamine, leucine, methionine, serine, and urea concentrations were also lower after calving. Multiple correlation analysis between plasma protein and amino acid concentrations reveals that the synthesis of export proteins in liver may be reduced due to limitation in amino acid availability.     

Compartmentation of albumin and ferritin synthesis in rat liver in vivo

Infusion of rats with [U-14C]glycine resulted in labelling of glycine and serine in plasma albumin and liver ferritin. The patterms of labelling in these two proteins were not similar, suggesting that each is synthesized from a different pool of free amino acids.     

Automatic dosage of total urinary proteins with centrifugal analyser

Automatic assay of urinary total proteins on a centrifugal analyser. The assay of urinary proteins is achieved opacimetrically after precipitation by trichloracetic acid. The addition of tensio-active agents lates the initiation of precipitation, helps the formation of a microprecipitate thus hold in suspension. The technique is linear from 0 to 2,5 g/l and for every albumin/globulins ratio between pure albumin and pure globulin.     

Molecular characterization of the GTPase-activating domain of ADP-ribosylation factor domain protein 1 (ARD1)

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins recognized as critical components in intracellular vesicular transport and phospholipase D activation. Both guanine nucleotide-exchange proteins and GTPase-activating proteins (GAPs) for ARFs have been cloned recently. A zinc finger motif near the amino terminus of the ARF1 GAP was required for stimulation of GTP hydrolysis. ARD1 is an ARF family member that differs from other ARFs by the presence of a 46-kDa amino-terminal extension. We had reported that the ARF domain of ARD1 binds specifically GDP and GTP and that the amino-terminal extension acts as a GAP for the ARF domain of ARD1 but not for ARF proteins. The GAP domain of ARD1, synthesized in Escherichia coli, stimulated hydrolysis of GTP bound to the ARF domain of ARD1. Using ARD1 truncations, it appears that amino acids 101-190 are critical for GAP activity, whereas residues 190-333 are involved in physical interaction between the two domains of ARD1 and are required for GTP hydrolysis. The GAP function of the amino-terminal extension of ARD1 required two arginines, an intact zinc finger motif, and a group of residues which resembles a sequence present in Rho/Rac GAPs. Interaction between the two domains of ARD1 required two negatively charged residues (Asp427 and Glu428) located in the effector region of the ARF domain and two basic amino acids (Arg249 and Lys250) found in the amino-terminal extension. The GAP domain of ARD1 thus is similar to ARF GAPs but differs from other GAPs in its covalent association with the GTP-binding domain.     

Physico-chemical properties and partial N-terminal amino acid sequence of chicken serum albumin

Studies have been made on the molecular weight, solubility, electrophoretic mobility, isoelectric point and N-terminal fragments containing 4 amino acids of the serum albumin in two strains of hens and their hybrids. In all the animals studied, the albumin had Asp as the N-terminal amino acid. Amino acid sequence in the 4-acid fragments was also identical: NH2--Asp--Ala--His--Lys. With respect to all the physico-chemical parameters investigated (except isoelectric point), proteins of the parental strains and of their hybrids did not exhibit significant differences.     

Amino acid kinetics during hemodialysis

The kinetic of essential amino acids as well as of histidine and alanine in bilateral nephrectomized rabbits was investigated during a 3 hours hemodialysis. Dialysis, elimination and incorporation rates into plasma proteins were determined for all applied amino acids. Total elimination rates of all the investigated amino acids varied. From 5.31% (leucine) to 75.51% (alanine) of the injected labelled amino acids were eliminated in the dialysate. There was an exponential decrease in the dialysis of essential amino acids and histidine during the period of investigation due to the high incorporation rate into plasma proteins. The kinetic of alanine was different due to a slow incorporation rate leading to a higher elimination rate. The fast incorporation of intravenously applied essential amino acids and histidine during dialysis demonstrates that the existing protein deficiency in renal failure can be influenced positively by infusion of amino acids.     

Statistical analysis of predicted transmembrane alpha-helices

Statistical analyses were undertaken for putative transmembrane alpha-helices obtained from a database representing the subset of membrane proteins available in Swiss-Prot. The average length of a transmembrane alpha-helix was found to be 22-21 amino acids with a large variation around the mean. The transfer free energy from water to oil of a transmembrane alpha-helix in bitopic proteins, -48 kcal/mol, is higher than that in polytopic proteins, -39 kcal/mol, and is nearly identical to that obtained by assuming a random distribution of solely hydrophobic amino acids in the alpha-helix. The amino acid composition of hydrophobic residues is similar in bitopic and polytopic proteins. In contrast, the more polar the amino acids are, the less likely they are to be found in bitopic proteins compared to polytopic ones. This most likely reflects the ability of alpha-helical bundles to shield the polarity of residues from the hydrophobic bilayer. One half of all amino acids were distributed nonrandomly in both bitopic and polytopic proteins. A preference was found for tyrosine and tryptophan residues to be at the ends of transmembrane alpha-helices. Correlated distribution analysis of amino acid pairs indicated that most amino acids are independently distributed in each helix. Exceptions are cysteine, tyrosine, and tryptophan which appear to cluster closely to one another and glycines which are preferentially found on the same side of alpha-helices.     

Detection of phosphotyrosine in glutaraldehyde-crosslinked and alkali-treated phosphoproteins following their partial acid hydrolysis in gels

Soluble fractions and particulate extracts from human prostate, and extracts from rat-liver membranes were used as a source of kinases to phosphorylate endogenous proteins in the presence of gamma- 32P-labeled ATP. Histone was also added as a substrate in order to compare the direct partial acid hydrolysis of phosphoproteins in gels to an indirect procedure involving partial acid hydrolysis after extraction in sodium dodecyl sulfate followed by precipitation with acetone. These procedures led to recoveries of 32P-labeled material of 90% and 40%, respectively, with a similar proportion of radiolabeled phosphoamino acids. Several 32P-labeled phosphoproteins separated in gels were therefore directly HCl-hydrolyzed and their phosphoamino acids were quantitated either prior to, or after glutaraldehyde crosslinking, with and without alkali treatment. By preventing protein losses occurring in hot alkali, glutaraldehyde crosslinking increased by an average factor of 6.5 the 32P-labeled material available for phosphoamino-acid analyses. For eight phosphoproteins analyzed, the overall effect of combined glutaraldehyde and alkali treatments was a relative decrease in phosphoserine (up to 8-fold), with concomitant relative increases in phosphotyrosine and phosphothreonine (up to 62- and 6-fold, respectively). This method will especially be useful for the detection of pTyr, a less abundant phosphoamino acid, in proteins which suffer from poor transfer efficiency in Western blot, are weakly antigenic towards anti-phosphotyrosine antibodies, can hardly be extracted from a gel and for identification of protein tyrosine kinases renatured in gels.     

Quantitative microanalysis of cell walls of Saprolegnia diclina Humphrey and Tremella mesenterica Fries

Cell walls of the fungi Saprolegnia declina Humphrey and Tremella mesenterica Fries were analyzed quantitatively. Particular attention was paid to the hydrolysis and analysis of neutral sugars, amino sugars and amino acids. These components, together with total lipids, total uronic acids and the ashed residue, accounted for more than 90% by weight of the original dry cell wall preparation. There were substantial losses of amino acids during hydrolysis; however, analytical recovery approached 100% when total protein was calculated from the total nitrogen analysis. The analytical procedures were reproducible (+/- 3% for amino acids and amino sugars, and +/- 5-10% for other components) when applied to individual cell wall preparations. However, even under carefully standardized conditions, different cell wall preparations from the same species showed variable composition. Glucose was the predominant neutral sugar in the cell wall polymers of both species. The amino acid compositions were remarkable in that neither species contained detectable levels of cyst(e)ine. Hydroxyproline was detected in both species. The report from Tremella mesenterica is the first for this amino acid from the cell wall of a Basidiomycete.     

Pathway for uptake and degradation of X-prolyl tripeptides in Streptococcus mutans VA-29R and Streptococcus sanguis ATCC 10556

The growth of Streptococcus mutans and Streptococcus sanguis in the oral environment requires that these micro-organisms be able to degrade salivary proteins and to assimilate the resulting peptides as an amino nitrogen source. Our research is aimed at the definition of the proteolytic enzyme systems in these oral streptococci which allow them to utilize such substrates. In the present work, the nature of the hydrolytic activity expressed by S. mutans VA-29R and S. sanguis ATCC 10556 against X-Pro4-nitroanilide and X-Pro-Y tripeptide substrates was investigated. This activity was predominantly associated with a cytoplasmic dipeptidyl peptidase which preferentially catalyzes the release of an N-terminal dipeptide from substrates in which proline is the penultimate residue. These streptococci also possess a second cytoplasmic peptidase, pepD, which catalyzes the hydrolysis of X-Pro dipeptides. We found that Gly-Pro-Ala or Ala-Pro-Gly were transported into the bacterial cells only when an energy source such as glucose was present. Peptide uptake was time-dependent, and selective exodus of peptide-derived amino acids from the bacterial cells occurred during peptide uptake. Results from these studies provide evidence that S. mutans VA-29R and S. sanguis ATCC 10556 possess a pathway for the complete degradation of X-Pro tripeptides. Transport of the peptides into cells prior to hydrolysis provides an efficient way by which all amino acids of a peptide may be obtained at an energy expense equivalent to that associated with the transport of just one amino acid. In light of the abundance of proline in salivary polypeptides, this degradative pathway could be an important component in the proteolytic pathway for salivary polypeptide utilization in these oral streptococci.     

Oncotic pressure and edema formation in hypoalbuminemic HIV-infected patients with proteinuria

Human immunodeficiency virus nephropathy (HIVN) continues to challenge nephrologic consultative services at major urban institutions. Although noted in the literature, the decreased incidence of peripheral edema in HIVN has been unexplained to date. In HIV patients, total proteins frequently are found to be elevated due to an elevated globulin fraction. The impact that plasma proteins, specifically globulins, have on the total oncotic pressure has not been reported in HIVN, but may play a role in the paucity of edema noted in this proteinuric population. To evaluate the contributions of serum globulin to the total oncotic pressure and the presence or absence of edema in HIVN, we randomly selected 27 patients with proteinuria greater than 2.5 g/24 hr and serum albumin less than 3.1 g/dL from patients presenting to the nephrology outpatient clinic at the University of Miami/Jackson Memorial Hospital. Seventeen of the patients (63%) had a known diagnosis of HIV infection (group 1). These patients were subdivided into two subgroups: those presenting with clinically evident edema on physical examination (n = 7 [41%]; group 1A) and those who had an absence of edema (n = 10 [59%]; group 1B). Conversely, group 2 comprised 10 patients without known HIV infection, of whom six (60%) had edema (group 2A) and four (40%) did not (group 2B). Blood pressures were noted, and mean arterial pressure was calculated using standard formulas. Serum albumin, serum total proteins, and urine total proteins were measured using standard laboratory methods. Oncotic pressures for albumin (alpha), globulin (beta), and total protein (c) were calculated using the following formula: COPpl = alpha(2.8c + 0.18c2 + 0.012c3) + beta(0.9c + 0.12c2 + 0.004c3). We used Student's t-test to analyze the data. There is no significant difference between the albumin concentrations of HIV patients without edema (group 1B) and non-HIV patients with edema (group 2A), with mean concentrations of 2.3 +/- 0.1 g/dL versus 2.3 +/- 0.15 g/dL, respectively (P = NS). Group 1B, however, has a total oncotic pressure of 17.1 +/- 1.5 mm Hg, whereas both groups with edema (groups 1A and 2A) have statistically significant lower total oncotic pressures (12.1 +/- 2.3 mm Hg and 12.9 +/- 1.1 mm Hg, respectively; P < 0.05). The globulin oncotic pressures may account for some of the differences in total oncotic pressures, being significantly higher for those patients without edema in group 1B compared with group 2A (7.1 +/- 0.9 mm Hg v 3.9 +/- 0.4 mm Hg, respectively; P < 0.05). In patients with HIV, however, the presence or absence of edema is mandated by albumin concentration because both groups have similar globulin concentrations (group 1A 3.1 +/- 0.1 g/dL v group 1B 3.8 +/- 0.3 g/dL; P = NS). Mean arterial pressure does not play a role in edema formation in this study because the HIV patients without edema had the higher blood pressures (group 1B 97.8 +/- 4.7 mm Hg v group 2A 84.7 +/- 5.5 mm Hg; P < 0.05). We conclude that globulins play an important role in maintaining oncotic pressure in low albumin states. HIVN patients with increased serum immune globulin may benefit from higher globulin oncotic pressure, delaying the onset of clinical edema in the setting of proteinuria.     

Synthesis in vitro of intrinsic membrane proteins by free, membrane-bound, and Golgi apparatus-associated polyribosomes from rat liver

A fraction of intrinsic membrane proteins was prepared from the major membranous cell components of rat liver by extraction of the membranes with KCl and deoxycholate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the compositions of the intrinsic protein fractions from rough and endoplasmic reticulum, smooth endoplasmic reticulum. Golgi apparatus, plasma membrane, and nuclear envelope were similar to each other but distinct from that of mitochondria. Among endomembranes, differences were in the ratios of protein constituents plus a few protein bands of Golgi apparatus and plasma membranes not found in endoplasmic reticulum or nuclear envelope. The abilities of total rough endoplasmic reticulum, polysomes released from rough endoplasmic reticulum, and free polysomes to incorporate amino acids into the intrinsic protein fraction were tested in vitro. Polysomes bound to endoplasmic reticulum has the greatest capacity to synthesize proteins of this fraction as shown by co-purification of radioactive products and by immunoprecipitation. Although the majority of the radioactive products synthesized by bound polysomes were distinct from those synthesized by free polysomes, certain radioactive products synthesized by free polysomes also co-purified with intrinsic membrane proteins. The results show no absolute segregation between free and bound polysomes in the synthesis of intrinsic membrane proteins. However, the majority of these proteins appear to be synthesized by polysomes bound to the endoplasmic reticulum. Several intrinsic proteins found in plasma membranes do not appear in rough endoplasmic reticulum. To determine where these proteins were synthesized, the ability of other endomembrane components to support in vitro incorporation of [14C]leucine into protein was examined. In contrast to plasma membranes, isolated Golgi apparatus fractions did incorporate [14C]leucine to an extent greater than could be explained by contamination with rough endoplasmic reticulum. Golgi apparatus in situ and isolated from rat liver have polyribosomes associated with a zone of cytoplasm at the Golgi apparatus periphery occupied by tubules and vesicles. The polysomes are not directly attached to membranes as with rough endoplasmic reticulum and may represent a special class of "Golgi apparatus-associated" polysomes. The polysomes, when associated with Golgi apparatus membranes, incorporated amino acids in vitro. The products synthesized in vitro were analyzed by treatment with KCl and deoxycholate and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Certain proteins synthesized by the Golgi apparatus-associated polysomes remained insoluble after the treatment with KCl and deoxycholate. The proteins synthesized by the Golgi apparatus fraction had mobilities similar to proteins in plasma membranes which were absent from endoplasmic reticulum, and which were relatively minor components of Golgi apparatus...     

Docking of nitrogenase iron- and molybdenum-iron proteins for electron transfer and MgATP hydrolysis: the role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein

Docking of the nitrogenase component proteins, the iron protein (FeP) and the molybdenum-iron protein (MoFeP), is required for MgATP hydrolysis, electron transfer between the component proteins, and substrate reductions catalyzed by nitrogenase. The present work examines the function of 3 charged amino acids, Arg 140, Glu 141, and Lys 143, of the Azotobacter vinelandii FeP in nitrogenase component protein docking. The function of these amino acids was probed by changing each to the neutral amino acid glutamine using site-directed mutagenesis. The altered FePs were expressed in A. vinelandii in place of the wild-type FeP. Changing Glu 141 to Gln (E141Q) had no adverse effects on the function of nitrogenase in whole cells, indicating that this charged residue is not essential to nitrogenase function. In contrast, changing Arg 140 or Lys 143 to Gln (R140Q and K143Q) resulted in a significant decrease in nitrogenase activity, suggesting that these charged amino acid residues play an important role in some function of the FeP. The function of each amino acid was deduced by analysis of the properties of the purified R140Q and K143Q FePs. Both altered proteins were found to support reduced substrate reduction rates when coupled to wild-type MoFeP. Detailed analysis revealed that changing these residues to Gln resulted in a dramatic reduction in the affinity of the altered FeP for binding to the MoFeP. This was deduced in FeP titration, NaCl inhibition, and MoFeP protection from Fe2+ chelation experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical composition of the capsule material of Staphylococcus aureus strain 1193/74

The capsule material of Staphylococcus aureus strain 1193/74 could be separated by precipitation with trichloroacetic acid and ethanol as well as by chromatography on DEAE-cellulose into 13 fractions. All fractions contained saccharides and uronic acids as well as amino acids and appeared in their qualitative composition rather similar. However, in quantitative composition and in chromatographic behaviour a rather high degree of heterogeneity could be observed. No clear cut separation of protein and polysaccharide material could be achieved in any fraction. It is supposed, therefore, that the capsule material does not represent merely an acidic polysaccharide, but contains certain amounts of amino acids or peptides varying in a rather wide range.     

Distribution of probon in various organs in acute poisoning]

The amino acids composition of summary proteins in unground buckwheat of four common and promising varieties grown in the Ukraine was investigated by using ion-exchange chromatography with an automatic analyzor Hd-1200 E. Between individual varieties of buckweheat no essential differences in the amino acids content were in evidence. The total proteins of the buckwheat grit contain high quantities of lysine, treonine, leucine, glutamic acid and arginine. The amino acids score was instrumental in determining the biological value and in eliciting amino acids limiting this value in different grits. These data may be made use of in the practice of public catering for estimating formulae of meals prepared with grits differently combined with other products securing an improved amino acids composition of ready-to-eat meals.