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Biomedical subjects

A Jonas

Publications and source records attributed to A Jonas.

At least 73 records · Page 4Linked to original sources
PubMed1993

Structural and functional properties of natural and chemical variants of apolipoprotein A-I.

Four isoforms of human apolipoprotein A-I (apo A-I): the normal allele product and the corresponding Lys-107 deletion mutant, and apo A-I with sulfoxidized Met-112 and Met-148 residues and the corresponding reduced form, were investigated in their lipid binding properties, structures, and abilities to activate lecithin-cholesterol acyltransferase. All apo A-I isoforms reacted completely with palmitoyloleoylphosphatidylcholine to give reconstituted high density lipoprotein (rHDL) particles with diameters of 96 A. These particles reacted with low density lipoprotein (LDL) and lecithin-cholesterol acyltransferase (LCAT) equally well, except that the Lys-107 deletion mutant was resistant to structural rearrangements in the presence of LDL. The spectral measurements revealed only minor structural differences among the free apo A-I forms or among their rHDL products, but showed a decreased stability of the Lys-107 deletion mutant and the isoform with reduced Met towards denaturation by guanidine hydrochloride. The results demonstrate that these specific alterations of the apo A-I sequence, which change the helix orientation and hydrophobic moment in one or two putative lipid binding regions, are not sufficient to disrupt the overall properties of the apo A-I complexes with lipid nor to impair significantly their ability to activate LCAT.

Amino Acid Sequence↗
PubMed1993

Reaction of lecithin: cholesterol acyltransferase with a water soluble substrate: effects of surfactants.

The reaction of fatty acid esters of p-nitrophenol, including the butyrate ester (PNPB) with lecithin: cholesterol acyltransferase (LCAT) has already been described (Bonelli, F.S. and Jonas, A. (1989) J. Biol. Chem. 264, 14723-14728) as a means to investigate the events at the active site of LCAT in the absence of interfacial interactions. Since various surfactants at low concentrations are known to affect the reaction of LCAT with particulate substrates, we set out to analyze their effects on the enzyme in solution using the PNPB substrate to monitor enzyme activity. All the surfactants studied by us (sodium dodecyl sulfate (SDS), dodecyltrimethyl ammonium bromide (DTAB), sodium laurate, sodium cholate, Triton X-100, and BIGCHAP) were able to interact with LCAT below their critical micellar concentrations. The ionic detergents caused inhibition of LCAT at concentrations ranging from 10(-4) to 10(-3) M, whereas the two nonionic detergents actually activated the enzyme in a similar concentration range. From the kinetic constants, the patterns of inhibition, and the well documented effects of the detergents on other proteins, we propose that SDS binds cooperatively to LCAT and elicits inhibitory structural changes; laurate and cholate bind to specific sites either in the active site cavity or in negative effector sites elsewhere; and the nonionic detergents may produce a slight interfacial activation of the phospholipase reaction near their critical micellar concentrations.

Binding Sites↗
PubMed1993

The number of amphipathic alpha-helical segments of apolipoproteins A-I, E, and A-IV determines the size and functional properties of their reconstituted lipoprotein particles.

The objective of this work was to determine the role of the amphipathic alpha-helical structural units of human apolipoproteins A-I, E, and A-IV in defining the sizes and reactivities with lecithin:cholesterol acyltransferase (LCAT) of their reconstituted lipoprotein particles. We prepared reconstituted high density lipoprotein (rHDL) particles with each of the three apolipoproteins in two weight ratios with lipid: 2.7/0.07/1 and 1.35/0.04/1, palmitoyloleoylphosphatidylcholine/cholesterol/apolipoprotein, by the sodium cholate dialysis procedure; and examined the rHDL product sizes and distributions by nondenaturing gradient gel electrophoresis. The rHDL particles were also incubated with low density lipoprotein (LDL), and with LDL plus LCAT, to observe any structural modifications due to phospholipid transfers to LDL and to cholesterol esterification by LCAT. In addition, we examined the average structural properties of the original rHDL by several fluorescence methods and circular dichroism spectroscopy, and determined their reaction kinetics with LCAT. The results indicate that the diameters of the largest rHDL particles, containing two apolipoproteins per particle, correlate with the maximum number of putative amphipathic alpha-helical segments in their sequences, and that smaller particles of this class may arise from the removal of one or more alpha-helical segments from contact with lipid. Furthermore, the larger particles may be converted into the smaller ones upon loss of phospholipid to LDL, and may form one or two well defined products when reacted with LCAT. In general, the subclasses of particles have distinct spectroscopic properties, consistent with a different apolipoprotein folding in particles containing different proportions of phospholipid to apolipoprotein. Furthermore, the different apolipoprotein structures lead to significant differences in reactivity with LCAT.

Apolipoprotein A-I↗
PubMed1993

D-lactic acidosis: a treatable encephalopathy in pediatric patients.

A 20-month old infant, who had short bowel syndrome following extensive surgery for a mid gut volvulus, developed hyperchloremic acidosis, with a large anion gap after enteral feeding was instituted. The organic acidosis was at least partly due to an increased concentration of D-lactic acid. This patient, as did five other pediatric patients, presented with a typical encephalopathy syndrome. Early recognition of this syndrome and treatment with an intestinal antibiotic and discontinuation of enteral feeding enabled prompt correction of the hyperchloremic acidosis and a rapid clinical recovery.

Acidosis, Lactic↗
PubMed1992

High-resolution NMR study of the pressure-induced unfolding of lysozyme.

The pressure-induced reversible unfolding of lysozyme was investigated by high-resolution proton magnetic resonance spectroscopy by following the proton spectra of the following residues: His-15 epsilon 1, Trp-28 epsilon 3, Leu-17 delta 2, Cys-64 alpha, and Trp-108 epsilon 3. The experiments were performed at pH 3.9 and 68.5 degrees C in the pressure range from 1 bar to 5 kbar both in the absence and presence of tri-N-acetylglucosamine (tri-NAG). From the pressure-induced changes of the equilibrium between the native and denatured forms of lysozyme, the reaction volumes (delta V) were calculated for each residue. Small but statistically significant differences in delta V were found for residues located in different regions of the protein. For example, delta V for the disulfide bonded Cys-64 alpha is smaller than the delta V's found for the other residues. In particular, the effect of tri-NAG binding to lysozyme was a change of delta V from -10.3 +/- 0.6 cm3/mol to -18.1 +/- 1.7 cm3/mol for the Trp-108 epsilon 3 residue which is located close to the active site. It is important to note that the Cys-64 alpha residue also senses the binding of the substrate analog. The ability to detect statistically significant differences for delta V of individual residues located in different regions of lysozyme represents the main result of these experiments.

Hydrogen-Ion Concentration↗
PubMed1992

Basal and meal-stimulated pepsinogen secretion in preterm infants: a longitudinal study.

In order to establish longitudinal normal values for basal and meal-stimulated pepsinogen secretory function in preterm infants, we studied 44 preterm infants with gestational ages of 28-36 weeks during the period of nasogastric tube feeding. Three age groups were evaluated: gestational ages of 28-30, 31-33, and 34-36 weeks. Basal pepsinogen did not change with postnatal age in any of the groups. Significant meal-stimulated pepsinogen secretion appeared during the third postnatal week in infants of 28-30 weeks of gestation. In the groups of infants of 31-33 and 34-36 weeks of gestation, significant meal-stimulated pepsinogen secretion was apparent in the first postnatal week. This study suggests that maturation of pepsinogen secretion appears at 31 weeks of gestation, independent of early feeding.

Gastric Mucosa↗
PubMed1992

Mucosal peptic activity during Helicobacter pylori infection in pediatric patients.

Intramucosal peptic activity may participate in the genesis of acute and chronic superficial gastritis. The proteolytic activity of homogenates of gastric mucosa (antrum and body) and duodenum were measured at pH 2.0 (total peptic activity) after exposure to pH 8.0 (pepsinogen) and the activated pepsinogen (pepsin) was calculated in pediatric patients investigated for the presence of Helicobacter pylori (H pylori), 122 antral, 77 stomach body, and 74 duodenal biopsies were examined in 43 H pylori positive patients, 51 controls, and 28 H pylori negative gastritis patients. Activated pepsinogen was significantly reduced in the stomach of H pylori positive patients only. Pepsinogen values were similar in all the anatomical areas tested in all patients. In 13 H pylori positive patients reinvestigated three months after antibiotic therapy, antral mucosal activated pepsinogen activity increased significantly (mean pretreatment 1.56 (1.0) U/mg protein versus mean post-treatment 2.72 (1.7) U/mg protein) and reached values comparable with controls. The decreased activated pepsinogen activity in association with normal pepsinogen content observed in the antrum of H pylori positive gastritis patients indicate local pepsin inactivation or alternately enhanced removal into the gastric lumen or backflow into the circulation.

Adolescent↗
PubMed1992

Continuous fluorescence assay for lecithin:cholesterol acyltransferase using a water-soluble phosphatidylcholine.

A water-soluble fluorescent phosphatidylcholine, 1,2-bis[4-(1-pyreno-butanoyl]-sn-glycero-3-phosphocholine (DPybPC) has been used to develop a sensitive, continuous assay for pure lecithin:cholesterol acyltransferase (LCAT) in solution. The monomeric substrate allowed us to examine the reaction of LCAT in the absence of a lipid/water interface in terms of the sensitivity of the enzymatic reaction to anions, ionic strength, apolipoproteins A-I and A-II, and a series of lysophosphatidylcholines and fatty acids. In contrast to the reaction of LCAT with aggregated phosphatidylcholines, the reaction of DPybPC with LCAT was not significantly affected by anions, ionic strength, nor apolipoproteins, indicating that these are only effectors of the interfacial reaction. Lysophosphatidylcholines and fatty acids inhibited LCAT in a chain-length-dependent manner below the critical micellar concentrations of these amphiphiles, indicating that the products of the LCAT reaction can bind to the enzyme and affect its kinetics even in the absence of an interface.

Evaluation Studies as Topic↗
PubMed1991

Transformations of reconstituted high-density lipoprotein subclasses as a function of temperature or LDL concentration.

The objectives of this study were to determine the structural changes in defined, reconstituted high density lipoproteins (rHDL) resulting from spontaneous phospholipid depletion in the presence or absence of low-density lipoproteins (LDL), to establish the precursor-product relationships among the rHDL particles and to assess the differences in behavior of rHDL particles containing apo A-I or apo A-II. The rHDL particles were prepared by the sodium cholate dialysis method, and were incubated in buffer at 50 degrees C, or in buffer containing different concentrations of LDL at 37 degrees C, for up to 24 h. The changes in the rHDL particle distributions with time were followed by non-denaturing gradient gel electrophoresis, and the rHDL were isolated at various time points for chemical analysis. We found that rHDL particles containing apo A-I or apo A-II lose phospholipid and gain cholesterol when incubated with LDL. Increasing LDL concentrations remove increasingly larger amounts of phospholipid. With phospholipid loss the apo A-I containing particles undergo major structural rearrangements that give rise to 78 A and 106 A particles from 86 A and 94 A precursors. The 78 A products appear to be the most stable, lipid-poor species. Reconstituted HDL particles prepared with apo A-II (94 and 101 A in diameter) are more resistant to structural rearrangements than the apo A-I counterparts under similar reaction conditions.

Apolipoprotein A-I↗
PubMed1991

Pressure effects on dipalmitoylphosphatidylcholine bilayers measured by 2H nuclear magnetic resonance.

The effects of pressure, up to 5 kbar, on multilamellar vesicles of 1,2-dipalmitoyl-sn-phosphatidylcholine perdeuterated in the acyl chains (DPPC-d62) were examined by using high-pressure NMR techniques. A deuterium probe was built, and the quadrupole splitting was measured against pressure at various temperatures. The experiments were performed on pure lipid bilayers in the liquid-crystalline state and on bilayers in the liquid-crystalline state containing the local anesthetic tetracaine. The results show that the order parameter of all segments of the acyl chains increases with pressure in the liquid-crystalline state. The more highly ordered regions of the chains are affected slightly more than the regions near the methyl ends. The addition of tetracaine increases the disorder of the chains, and pressure reverses the effect of anesthetic on the lipid as seen by the reversal of the changes in line shape and the measured order parameter.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Adaptive response of ileal mucosa to malnutrition in the rat: role of polyamines.

Mucosal disaccharidases and ornithine decarboxylase activities were measured in malnourished, preweaning (19 days), post weaning (24 days) and young adult (37 days) rats. Malnutrition resulted in decreased body weight, intestinal weight, DNA and protein content. Mucosal Prot/DNA ratios were elevated in the ileal segments of the 24 and 37 day rats. Preweaned malnourished rats had significantly enhanced lactase specific activity in both jejunal and ileal segments. Adult malnourished rats showed enhanced jejunal lactase and sucrase activities which were not accompanied by elevated ornithine decarboxylase values. Mucosal sucrase and ornithine decarboxylase specific activities were significantly elevated in the ileal segment of the 24 and 37 day old malnourished rats. Studies of adult rats showed that these increased specific activities were located in the mature enterocytes at the villus tip, and persisted during a 24 h diurnal cycle. DFMO administration for 4 days completely inhibited mucosal ornithine decarboxylase and abolished the rise of ileal sucrase activity. We concluded that the intestinal response to reduced food intake is age related and differs in the jejunum and ileum: ornithine decarboxylase and polyamines are involved in ileal adaptation to malnutrition in postweaned and adult rats.

Adaptation, Physiological↗
PubMed1991

Structural and functional properties of reconstituted high density lipoprotein discs prepared with six apolipoprotein A-I variants.

Six apolipoprotein A-I (apoA-I) variants containing the following amino acid changes: Pro3----Arg, Pro4----Arg, Lys107----0 (Lys deletion) Lys107----Met, Pro165----Arg, and Glu198----Lys, and the corresponding normal allele products, were isolated by preparative isoelectric focusing from heterozygous individuals. The apoA-I samples were reconstituted with palmitoyloleoyl phosphatidylcholine (POPC) or dipalmitoyl phosphatidylcholine (DPPC), and small amounts of cholesterol, into discoidal high density lipoprotein (HDL) complexes in order to examine their lipid binding and structural properties as well as their ability to activate lecithin:cholesterol acyltransferase (LCAT). Starting with initial molar ratios around 100:5:1 for phosphatidylcholine-cholesterol-apolipoprotein, all the normal and variant apoA-Is were completely incorporated into reconstituted HDL (rHDL). The rHDL particle sizes and their distributions were examined by nondenaturing gradient gel electrophoresis, before and after incubation with LDL, to assess the folding of apoA-I in the complexes. Intrinsic Trp fluorescence properties of the rHDL were measured, as a function of temperature and guanidine hydrochloride concentration, to detect conformational differences in the apoA-I variants. In addition, the LCAT reaction kinetics were measured with all the rHDL, and the apparent kinetic constants were compared. In terms of the structure of the rHDL particles, all the normal variant apoA-Is had similar sizes (94, 96 A) and size distributions, and indistinguishable fluorescence properties, with the exception of the Lys107----0 mutant. This variant formed slightly larger particles that were resistant to rearrangements in the presence of LDL, and had an altered apoA-I conformation in the vicinity of the Trp residues. The kinetic experiments with LCAT indicated that the apoA-I variants, Lys107----0 and Pro165----Arg, in rHDL particles had statistically different (30 to 90%) kinetic constants from the corresponding normal allele products; however, the variability in the kinetic constants among the normal apoA-I products was even greater (40 to 430%). Therefore, we conclude that the effects of these six mutations in apoA-I on the activation of LCAT are minor, and that the structural effects on rHDL, and possibly native HDL, are insignificant with the exception of the Lys107----0 mutation.

Amino Acid Sequence↗
PubMed1990

Apolipoprotein A-I structure and lipid properties in homogeneous, reconstituted spherical and discoidal high density lipoproteins.

We prepared a spherical reconstituted high density lipoprotein (rHDL) particle in pure form and compared it with its homogeneous discoidal rHDL precursors, in terms of the structure and stability of the apolipoprotein A-I (apoA-I) component, the dynamics of the surface lipids, and the relative reactivity with lecithin-cholesterol acyltransferase. The apoA-I-structure was examined in the rHDL particles by circular dichroism and fluorescence spectroscopic methods, and the binding of monoclonal antibodies specific for apoA-I epitopes. The stability of apoA-I on the rHDL particles was assessed by the effects of guanidine hydrochloride on the wavelength of maximum intrinsic fluorescence of the apolipoprotein. Lipid dynamics in the acyl chain region and the polarity of the lipid-water interface were investigated by means of fluorescence probes. The conformation of apoA-I in the spherical 93-A rHDL particles was found to be very similar to that in the 96-A rHDL discs but distinct from the apoA-I structure in the 78-A rHDL discs. The stability of apoA-I to denaturation by guanidine hydrochloride was highest in the 93-A rHDL spheres. The experiments on the lipids indicate somewhat more ordered and motionally restricted acyl chains in the spheres, relative to the discs, but a similar surface polarity. These results suggest that the folding and organization of apoA-I on the three particles include protein domains consisting of interacting alpha-helical segments in the carboxyl-terminal region and a globular domain in the amino-terminal region of each apoA-I molecule. The reactivity with lecithin-cholesterol acyltransferase was highest for the 96-A rHDL disc, and 16- and 34-fold lower for the 78-A rHDL disc and the 93-A rHDL sphere, respectively, possibly as a result of differences in apoA-I structure and product inhibition in these particles.

Antibodies, Monoclonal↗
PubMed1990

Structure of apolipoprotein A-I in three homogeneous, reconstituted high density lipoprotein particles.

To elucidate further the conformation of human apolipoprotein A-I (apoA-I) in lipid-bound states and its effect on the reaction with lecithin cholesterol acyltransferase (LCAT), we prepared reconstituted HDL (rHDL) particles from a reaction mixture containing dipalmitoylphosphatidylcholine/cholesterol/apoA-I in the molar ratios of 150:7.5:1. The particles were separated by gel filtration into three classes of highly homogeneous and reproducible discs with diameters of 97, 136, and 186 A, containing 2, 3, and 4 molecules of apoA-I/disc, respectively, and increasing proportions of phospholipid and cholesterol. These three classes of particles were then investigated by a variety of fluorescence techniques, to probe the average environment and mobility of the tryptophan (Trp) residues in the structure of apoA-I. We found small, gradual changes in the fluorescence parameters with changes in the size of the rHDL, consistent with a shift of Trp residues to a more hydrophobic and more rigid environment, as well as an increased resistance of apoA-I to denaturation by guanidine hydrochloride in the larger particles. In contrast, circular dichroism measurements and binding studies with seven monoclonal antibodies indicated a similar alpha-helical structure (73%) for apoA-I in all the particles, and similar exposure of apoA-I epitopes in the COOH-terminal two-thirds of the apolipoprotein. Thus the structure of apoA-I is comparable for the three classes of particles and is consistent with the presence of eight alpha-helical segments per apoA-I in contact with the lipid. In addition, we obtained the apparent kinetic parameters for the reaction of the rHDL particles with lecithin cholesterol acyltransferase. The apparent Km values were similar but the apparent Vmax decreased almost 8-fold, going from the 97- to the 186-A particles; therefore, the decreasing reactivity for the larger particles can be attributed mainly to differences in the catalytic rate constant. The rate limiting step is probably affected by local structural differences in the apoA-I, or by the interfacial properties of the lipid.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1990

Investigation of the lipid domains and apolipoprotein orientation in reconstituted high density lipoproteins by fluorescence and IR methods.

The reconstituted high density lipoproteins (rHDL) that were described in the preceding paper (Hefele Wald, J., Krul, E. S., and Jonas, A. (1990) J. Biol. Chem. 265, 20037-20043) are used in this study to analyze the organization, conformation, and dynamics of the lipid phase, as well as the relative orientation of the apolipoprotein alpha-helices and the lipid hydrocarbon chains. Two fluorescence polarization probes and a fluorescence polarity probe were used to detect the lipid phase transition behavior of the various particles, and to estimate the lipid order, mobility, and environment polarity in their gel and liquid-crystalline states. Infrared attenuated total reflection spectroscopy was used to estimate the content of secondary structure of the apolipoprotein, and the orientation of its alpha-helices with respect to the lipid hydrocarbon chains. In addition, the infrared spectra were analyzed in terms of the conformation and organization of different regions of the lipid molecules in the rHDL particles. The results indicate that the overall organization and conformation of lipid molecules in a lipid bilayer is preserved in the rHDL particles, but that progressive increases in apolipoprotein content straighten the hydrocarbon chains and decrease their packing order in the gel state, and decrease their mobility in the liquid-crystalline state. The presence of apolipoprotein also affects the conformation of the lipids at the level of the ester bonds and the head group of the phospholipid. In all three particle classes the content and distribution of secondary structures of the apolipoprotein were similar, and the alpha-helical segments were parallel to the lipid hydrocarbon chains.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1990

Choledocholithiasis in infants: diagnostic and therapeutic problems.

During the 3-year period, January 1986 through December 1988, 7 of 24 infants evaluated for cholestatic liver disease were found to have choledocholithiasis with or without associated gallbladder disease. Five were born prematurely with associated predisposing factors, especially systemic infection and total parenteral nutrition. The clinical course varied from spontaneous recovery in two patients to ongoing liver disease requiring surgical or endoscopic intervention in five infants. Asymptomatic patients with normal liver function and persistently dilated extrahepatic biliary trees posed the most difficult therapeutic problems. Endoscopic retrograde cholangiography appeared to be an effective and rewarding therapeutic approach.

Biliary Tract↗