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PubMed1992

Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: gene organization and implications for the evolution of light chains.

A full-length cDNA clone specifying sandbar shark (Carcharhinus plumbeus) immunoglobulin light chain has been isolated and sequenced. By alignment with human lambda chains, the leader, framework, complementarity-determining, joining, and constant regions are clearly identified in the shark light chain. Approximately 40-50% identity is shared between the human and shark sequences in the variable and constant regions. We have performed sequence comparisons of the individual segments and constructed phylogenetic trees for the variable region. These studies identify the shark protein as a lambda chain. In addition, the sandbar shark light chain is only distantly related to that of horned shark (Heterodontus francisci) [Shamblott, M. J. & Litman, G. W. (1989) Proc. Natl. Acad. Sci. USA 86, 4684-4688], demonstrating that the long evolutionary time of divergence among shark species has led to the generation of substantial differences in sequence. The positions of the variable, joining, and constant gene segments in 14 genomic clones have been mapped. The segments are linked in individual clusters (variable, joining, constant) occupying 3-7 kilobases. Cluster arrangement can be grouped into two patterns based upon spacing between the genes in the individual clones. This arrangement is fundamentally different from that observed in higher vertebrates.

Amino Acid Sequence↗
PubMed1986

Increased intrathecal immunoglobulin synthesis of both kappa and lambda types in patients with systemic lupus erythematosus and central nervous system involvement.

The concentration of immunoglobulins in paired serum and cerebrospinal fluid (CSF) specimens from 15 patients with systemic lupus erythematosus (SLE) and central nervous system (CNS) involvement (CNS-SLE) were determined based on their light chain properties. Eight patients with SLE without CNS involvement and 20 patients with noninflammatory neurological diseases were also studied for comparison. We found that both of the CSF Ig-k (immunoglobulin kappa) and Ig-1 (immunoglobulin lambda) indices were significantly elevated, indicating the increased intrathecal immunoglobulin synthesis of both kappa and lambda types in patients with CNS-SLE. Moreover, the CSF Ig-k and Ig-1 indices correlated well with each other, and both indices were found to decrease remarkably when the CNS manifestations subsided after successful treatment. In 4 of 15 patients, intrathecal synthesis of free light chains in addition to Ig bound light chains seemed to be increased. These observations provide substantial evidence for polyclonal B lymphocyte activation within the CNS.

Adolescent↗
PubMed1988

Addition of carbohydrate side chains at novel sites on influenza virus hemagglutinin can modulate the folding, transport, and activity of the molecule.

We have constructed and expressed a series of mutant influenza virus hemagglutinins, each containing a new consensus site for glycosylation in addition to the seven sites found on the wild-type protein. Oligosaccharide side chains were added with high efficiency at four of the five novel sites, located on areas of the protein's surface that are not normally shielded by carbohydrate. Investigations of the structure, intracellular transport, and biological activities of the mutant hemagglutinin molecules indicated that (a) supernumerary carbohydrate side chains can be used to shield or disrupt functional epitopes on the surface of hemagglutinin, and (b) the presence of an additional oligosaccharide may cause temperature-dependent defects in the transport of the glycoprotein. We discuss the addition of supernumerary oligosaccharides as a general tool for shielding chosen areas of the surface of proteins that enter or traverse the secretory pathway.

Amino Acid Sequence↗
PubMed1976

[Metabolic activity of Candida lipolytica adsorbed to bentonite with hydrophobic chains].

The respiration of Candida lipolytica on n-tetradecane is decreased in the presence of bentonite. This inhibition is less pronounced by the introduction of hydrophobic chains on the bentonite. Oxygen demand of resting cells varies with the length of hydrophobic chains. In fermentor, addition of adsorbant does not stimulate growth, but assimilation of n-tetradecane is enhanced with certain concentrations of the adsorbant. On glucose, addition of hydrophobic chains on the adsorbant does not change the effects of bentonite on exogenous respiration and substrate assimilation.

Adsorption↗
PubMed1990

Quantitative IR spectrophotometry of peptide compounds in water (H2O) solutions. II. Amide absorption bands of polypeptides and fibrous proteins in alpha-, beta-, and random coil conformations.

Infrared spectra of poly(D,L-alanine), poly(L-glutamic acid), poly(L-lysine), silk fibroin, and tropomyosin have been registered for various conformations of the polypeptide chain. Assuming additivity of the main- and side-chain absorption, spectral parameters of amide I and II absorption bands corresponding to alpha-, beta-, and random coil conformations have been derived. The amide I band parameters for H2O and D2O have been compared.

Amides↗
PubMed1991

Comparative specificity of plasma lecithin:cholesterol acyltransferase from ten animal species.

The molecular specificities of plasma lecithin:cholesterol acyltransferase (LCAT) from ten animal species have been compared. Using a reassembled high density lipoprotein containing a mixture of phosphatidylcholines, the relative rates of liberation of different species of cholesteryl ester were measured. All but two species of LCAT clustered according to one of three patterns of substrate specificity. The LCAT from six species, including human, did not transfer highly polyunsaturated fatty acyl chains. In addition, human LCAT transesterified saturated fatty acyl chains more effectively than unsaturated fatty acyl chains. We conclude that the structures of the active sites of the enzymes differ, and that this may be related to size constraints that prevent efficient binding of large bulky phosphatidylcholines.

Animals↗
PubMed1989

Biosynthesis of high molecular weight polylactosamine-type glycopeptides in rat Zajdela hepatoma ascites cells.

The first steps of the biosynthetic pathway of high molecular weight polylactosamine-type glycopeptides from rat Zajdela hepatoma cells were studied by pulse-chase experiments, biochemical analysis and by inhibition of N-glycosylation. It is clear that this process involves firstly the transfer of a lipid-linked high-mannose oligosaccharide precursor to a protein moiety in a similar way to that of N-linked glycopeptides of a more common size range according to the classical 'cycle of dolichol'. In the presence of enzymes which are inhibitors of the processing of high-mannose oligosaccharide chains, this class of oligosaccharides was considerably increased, whereas polylactosamine chains and lower complex N-linked glycopeptides were concomitantly decreased in the same kinetics and the same ratio. As expected in the presence of N-methyldeoxynojirimycin, which is an alpha-glucosidase inhibitor, high-mannose oligosaccharides remained glycosylated and are mostly of the Glc1-3Man9GlcNAc type. In the presence of swainsonine, which is an alpha-mannosidase (EC 3.2.1.24) inhibitor, these chains were devoid of glucose residues. In addition, some chains displayed hybrid structures. It appears, therefore, that the first steps of the biosynthesis of polylactosamine-type and N-linked oligosaccharides of a more common size range proceed similarly and that differences between their biosynthetic pathways occur during the elongation phase, which leads to their final respective structures. Glycopeptides prepared from the cell surface by mild trypsin treatment as well as from entire cells, previously treated or not by processing inhibitors, display the same gel filtration patterns indicating that modifications in protein glycosylation do not prevent glycoprotein insertion into the cell membrane.

Amino Sugars↗
PubMed1986

Analysis of DR beta and DQ beta chain cDNA clones from a DR7 haplotype.

A cDNA library was constructed from a DR7, DRw53, DQw2 homozygous cell line, cDNA clones corresponding to DR beta and DQ beta chains were isolated, and the nucleotide sequences of the polymorphic first domains of these chains were determined. A novel screening strategy allowed rapid and simple identification of cDNA clones corresponding to both DR beta chains (DR7 beta1 and DR7 beta2): DR7 beta2 clones have a recognition site for the enzyme BssHII, whereas DR7 beta1 clones do not. The DR7 beta 1 sequence differs significantly from all previously described DR beta chains. As predicted by the presence of the BssHII site in DR7 beta 2 clones, the DR7 beta 2 sequence differs from the DR7 beta 1 sequence. The sequence of the DRw53-associated DR7 beta 2 chain is identical to the reported sequence of the DRw53-associated DR4 beta 2 chain. In addition, the sequence of the DQ beta chain from the DR7, DQw2 cell line is identical to the reported sequence of a DQ beta chain from a DR3, DQw2 cell. These findings raise interesting questions about the evolution of the DR3, DR4, and DR7 haplotypes.

Amino Acid Sequence↗
PubMed1991

Indications that branched chain amino acids, in addition to glucagon, affect the glomerular filtration rate after a high protein diet in insulin-dependent diabetes.

Hormonal changes and whole blood free amino acid levels and their relation to renal function were measured in 12 insulin-dependent diabetic patients after two 10-day periods with a diet consisting of 10% and 20% respectively of the energy as protein. The patients were 15-21 years old and mean duration of diabetes was 12 (5-20) years. Glomerular filtration rate, renal plasma flow, and albumin excretion rate were measured together with plasma concentrations of glucagon, growth hormone, insulin-like growth factor 1 (IGF-1), somatostatin, serum insulin and free amino acids in blood. Glomerular filtration rate was 123 +/- 3 ml/min/1.73 m2 on high protein diet and 113 +/- 3 ml/min/1.73 m2 on low protein diet (p = 0.02). Renal plasma flow was unchanged. Glucagon, IGF-1, branch chained amino acids (BCAA), tyrosine, phenylalanine, lysine, and methionine were increased after the high protein diet. Growth hormone, somatostatin, insulin, and other amino acids remained unchanged. The increase in glomerular filtration rate was significantly correlated to the increase in glucagon, isoleucine, and valine (glucagon r = 0.71, p = 0.01, isoleucine r = 0.59, p = 0.04, valine r = 0.62, p = 0.03). In a multiple regression model the increase in glomerular filtration correlated most strongly to the increase in isoleucine, followed by valine and glucagon. Together these variables explained 88% of the total variance of the change in glomerular filtration rate (r2 = 0.88, p = 0.001). Albumin excretion rate was correlated to IGF-1 (r = 0.86, p less than 0.001) on the high protein diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1976

The relationship between chain elongation of palmitoyl-CoA and phospholipid content in rat liver microsomes.

The relationship between the chain elongation of palmitoyl-CoA and phospholipid content in rat liver microsomes was studied. When liver microsomes were incubated with phospholipase C, microsomal phospholipids were linearly hydrolyzed during 10 min of incubation under the present experimental conditions. Coincident with the decrease in microsomal phospholipid content by phospholipase C treatment, the chain elongation activity also decreased linearly. The decreased chain elongation activity in phospholipase C-treated microsomes was completely or partially recovered by the addition of a sonicated dispersion of phosphatidylcholine, microsomal phospholipids or phosphatidylcholine/phosphatidylethanolamine mixtures. The extent of recovery of decreased activity by a sonicated dispersion of phosphatidylcholine/phosphatidylethanolamine mixture was gradually reduced by increasing amounts of phosphatidylethanolamine in the dispersion. In addition, the chain elongation activity in native nicrosomes was more stimulated by the addition of a sonicated dispersion of phosphatidylcholine alone than by that of phosphatidylcholine/phosphatidylethanolamine mixtures. The chain elongation activity of palmitoyl-CoA was inhibited by the addition of stearoyl-CoA which is the end-product of this reaction. The inhibitory effect of stearoyl-CoA was partially eliminated by the addition of a sonicated dispersion of phosphatidylcholine. The increase of the chain elongation activity in native and phospholipase C-treated microsomes by the addition of a sonicated dispersion of phosphatidylcholine was not related to the activity of fatty acyl-CoA hydrolase.

Animals↗
PubMed1992

Addition of long-chain polyunsaturated fatty acids to formula for very low birth weight infants.

Thirty-four premature infants who were appropriate for gestational age and weighing less than 1500 g at birth were fed "preemie" SMA-24 formula, "preemie" SMA-24 formula manufactured to contain C20 and C22 omega 6 and omega 3 fatty acids (LCPE-SMA), or expressed milk (EBM). Blood samples were drawn from a small arm vein during the first week of life and after 28 days of feeding. The fatty acid content of plasma phospholipids was determined. Infants fed SMA-24 had a high content of 18:2 omega 6 in plasma phospholipids. Feeding LCPE-SMA normalized plasma phospholipid levels of C20 and C22 omega 6 and omega 3 fatty acids to be similar to levels of C20 and C22 omega 6 and omega 3 fatty acids found in infants fed EBM, and significantly higher than characteristic levels for infants fed SMA-24. Feeding LCPE-SMA or EBM results in a balanced incorporation of C20 and C22 omega 6 and omega 3 fatty acids into phospholipids derived from the liver or perhaps the small intestine.

Fatty Acids↗
PubMed1986

The role of the VL- and VH-segments in the preferential reassociation of immunoglobulin subunits.

Competitive reassociation experiments, in which equimolar amounts of two different L-chains were allowed to compete for a limiting amount of H-chain, were performed to assess the role of the V kappa- and J kappa-segments on the ability of an L-chain to compete. Using H- and L-chains from the murine anti-phosphorylcholine (PC) myelomas, TEPC15, MOPC167 and MCPC603, and a series of V kappa 21 L-chains, it was found that the V kappa 21 L-chains competed uniformly better than the anti-PC L-chains, when the anti-PC H-chains were used, despite any differences in the J-segments of the competing L-chains. In addition, when the anti-PC L-chains, which all employ identical J kappa-segments but very diverse V kappa-segments, were in competition against each other, a hierarchy of competitive ability existed which was independent of whether the chains were autologous or heterologous and independent of antigen binding activity. Competitive reassociation experiments between the V kappa 21 and anti-PC L-chains were also performed using the heterologous anti-lysozyme monoclonal HyHEL-10 H-chain or the anti-galactan J539 H-chain, and it was found that the relative competitive ability of the V kappa 21 L-chains with respect to the anti-PC L-chains was dependent on which H-chain was employed. The results suggested that the main factor favouring preferential reassociation by any particular L-chain was the V kappa-segment and that the effects of the J kappa-segment could not be observed where a high degree of diversity in the V-segments existed. Furthermore, while the results implied that specific pairs of VH- and VL-domains had a higher affinity for each other, this was not a necessary criterion in the formation of autologous pairs of H- and L-chains as demonstrated by the preferential heterologous reassociation of the V kappa 21 L-chains over the autologous anti-PC L-chains. These results were consistent with the independent, random rearrangement of immunoglobulin H- and L-chain V-domain gene segments and predict that the hypothetical repertoire of antibodies is not limited by the selection of specific pairs of high-affinity VH-VL domains.

Amino Acid Sequence↗
PubMed1986

Addition of branched-chain amino acids to parenteral nutrition of stressed critically ill patients.

We compared the efficacy of a total parenteral nutrition solution supplemented with 44.6% branched-chain amino acids (BCAA) to standard total parenteral nutrition (19.0% BCAA) in the support of critically ill patients. After a 4-day administration of a randomly assigned solution, amino acids were assayed from femoral venous and arterial samples. The BCAA-supplemented solution caused increases in arterial concentrations of valine, isoleucine, and leucine compared to the control solution. The femoral arteriovenous differences for these BCAA were more positive in patients given the control solution. There were no significant differences in nitrogen balance. Since previous studies showing promotion of nitrogen balance with BCAA did not use a balanced substrate for nonprotein caloric support, the lack of a significant difference in the present study may be due to effective utilization of lipid as a fuel source by both groups.

Adult↗
PubMed1992

Comparison of two preterm formulas with or without addition of medium-chain triglycerides (MCTs). I: Effects on nitrogen and fat balance and body composition changes.

Medium-chain triglycerides (MCTs) are included in the fat blend of several preterm formulas because of their complete absorption and rapid oxidation. The effects of two different fat blend compositions on nitrogen and fat balances and macronutrient oxidation were investigated in 28 healthy very-low-birth weight infants at 4 weeks of age. A preterm formula with a traditional corn oil/MCT blend containing 38% MCTs (MCT group) was compared to a new fat blend, designed to resemble human milk more, containing 6% MCTs (LCT group). There were no differences in nitrogen absorption or in excretion. Median nitrogen retention was 74% (MCT) vs. 71% (LCT) of intake. Fat absorption was higher (p less than 0.05) in the MCT group (88%) vs. 79% in the LCT group (median values). MCTs did not stimulate fat oxidation as measured by indirect calorimetry, so fat deposition was also higher on the MCT formula. As weight gain was not different between groups, the percentage of weight gain consisting of fat accretion was significantly (p less than 0.005) higher with the MCT formula (24% vs. 21%). On the other hand, there was no increase in percent protein accretion (both 15% of weight gain). We conclude that the existence of a slightly lower fat absorption in the healthy growing neonate fed a LCT formula compared with a MCT formula does not impair growth or nitrogen retention, but merely induces a slight decrease in the high relative fat accretion encountered in the preterm neonate.

Body Composition↗
PubMed1992

Comparison of two preterm formulas with or without addition of medium-chain triglycerides (MCTs). II: Effects on mineral balance.

Medium-chain triglycerides (MCTs) are included in the fat blend of several preterm formulas because of their complete absorption and rapid oxidation. The effects of two different fat blend compositions on calcium (Ca), phosphorus (P), and magnesium (Mg) balances and plasma levels and on plasma levels of parathyroid hormone (PTH), alkaline phosphatase (AP), and 1,25-dihydroxyvitamin D [1,25-(OH)2D] were investigated in 28 healthy very-low-birth weight infants at 4 weeks of age. A preterm formula with a traditional corn oil/MCT blend containing 38% MCTs (MCT group) was compared to a new fat blend, designed to resemble human milk more, containing 6% MCTs (LCT group). There was a higher absorption of Ca in the MCT group (73% vs. 60%. p less than 0.005), and an equal absorption of P (both 92%). The excretion of Ca correlated with the excretion of fat (p less than 0.00005). The LCT group showed a higher median PTH level (MCT: 2.1 pmol/L, LCT: 4.7 pmol/L, p less than 0.01) and a higher urinary P excretion (p less than 0.001). Mg absorption was also lower with LCT, but retention of Mg exceeded intrauterine values in both groups. Mineral plasma levels were in the normal range in both groups. AP was not different between groups and in the upper part of the reference range, whereas 1,25-(OH)2D levels were above the normal range and also not different between groups. We conclude that with the LCT formula, Ca absorption is slightly lower than with the MCT formula, whereas P absorption is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1979

Characterization of the ribonucleic acid primers and the deoxyribonucleic acid product synthesized by the DNA polymerase and gene 4 protein of bacteriophage T7.

The DNA polymerase and gene 4 protein of phage T7, in the presence of helix-destabilizing protein (DNA binding protein), catalyze DNA synthesis on duplex templates. As has been previously shown (Kolodner, R. D., and Richardson, C. C. (1978) 4. Biol. Chem. 253, 574-584), in the absence of ribonucleoside 5'-triphosphates DNA synthesis is initiated at nicks, and all of the newly synthesized DNA is covalently attached to the template. In this paper we characterize the DNA synthesized in the presence of ribonucleoside 5'-triphophates and show that, in contrast, the major portion of the newly synthesized DNA is not attached to the template, having an average chain length of 5000 to 6000 nucleotides. In addition, each chain of newly synthesized DNA is terminated at its 5'-end by a covalently attached tetranucleotide RNA primer whose sequence is predominantly pppApCpCpC and pppApCpCpA. The results of isotope transfer experiments are in agreement with the number of initiation events determined by the incorporation of [gamma-32P]ATP and indicate that each of the four deoxyribonucleotides is present at the RNA-DNA junction.

DNA Helicases↗
PubMed1990

In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways.

Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2(+)-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Km for C3 was 2.6 microM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis.

Amino Acids↗
PubMed1975

Comparative studies on monotypic IgMlambda and IgGkappa from an individual patient. I. Evidence for shared VH idiotypic determinants.

A comparative idiotypic antigenic analysis of an IgMlambda and an IgGkappa paraprotein obtained from sera of an individual patient, Br, revealed the presence of very similar idiotypic determinants associated with the VH regions of the Br mu- and gamma-chains. In addition, the IgGkappa protein expressed light (L) chain-associated idiotypic determinants which were not evident on the IgMlambda protein or its isolated L chains. Extensive tests of specificity revealed that the shared VH idiotypic determinants were not present in large weight amounts of heterologous nonspecific IgM and IgG, nor on Ig molecules contained in a large number of normal and myeloma sera.

Animals↗