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PubMed1980

Chinese hamster lung cells synthesize and confine to the cellular domain a collagen composed solely of B chains.

The acid-soluble collagen extracted from cultured Chinese hamster lung (CHL) cell layers has been isolated after limited pepsin digestion and differential salt fractionation. Polyacrylamide gel electrophoresis of this material under denaturing conditions showed the presence of collagen chains with an apparent molecular mass of 120,000 daltons both before and after reduction, indicating the absence of interchain disulfide bonds in the native molecule. When chromatographed on CM-cellulose under denaturing conditions, the majority (> 90%) of the CHL cell layer collagen chains eluted as relatively basic components slightly before the human alpha 2(I) chain and coincident with the human B chain. In addition, the CM-cellulose elution profiles of the cyanogen bromide peptides derived from the human B chain and from the CHL cell layer chain were essentially identical. Examination of CHL cells in culture by using affinity-purified antibody to human B chain revealed this collagen to be localized in an extracellular matrix surrounding the cells. Furthermore, analysis of the culture medium indicated the absence of any comparable collagen chain. These data provide additional evidence for the existence of a molecular form of collagen composed solely of B chains and suggest that this molecular form of collagen has an unusual affinity for the cell layer in this system.

Animals↗
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↗
PubMed2005

Analysis of minor hemoglobins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Hemoglobin (Hb) heterogeneity arises mainly from posttranslational modifications of the globin chains, and cation-exchange chromatography reveals falsely increased concentrations of some minor Hbs in the presence of abnormal Hbs. Here we describe a method for identification of the globin chains and their posttranslational modifications contained in the Hb fractions. METHODS: We used cation-exchange HPLC (PolyCAT A column) for separation of Hb fractions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for analysis of the separated globin chains. Globin chains were identified by their molecular masses. Posttranslational modifications of globin chains were identified by digestion of the proteins with endoproteinase V8 before MALDI-TOF MS of the resulting peptides. RESULTS: Analysis of the HbA2 fractions of patients with HbS revealed 4 different globin chains. We found, in addition to the expected alpha- and delta-chains, the carbamylated alpha- and the betaS-chains. Additionally, we analyzed HbH, Hb Barts, HbA 1b, pre-HbA 1c, HbA 1c, HbF1, HbF, HbA 1d3a, HbA 1d3b, HbA2, and HbC1 fractions from control and pathologic blood samples. We identified several posttranslational modifications of the globin chains, such as pyruvatization, glycation, acetylation, carbamylation, and acetaldehyde adduct formation. CONCLUSIONS: The native and posttranslationally modified globin chains in minor and major Hbs are unambiguously identified by MALDI-TOF MS. A minor Hb containing the carbamylated alpha- and the betaS-chain elutes at the same time as normal HbA2 (alpha2delta2) and thus leads to falsely increased HbA2 values in patients with HbS when blood is analyzed with PolyCAT A chromatography.

Chromatography, High Pressure Liquid↗
PubMed2006

Isolation of avian influenza virus A subtype H5N1 from internal contents (albumen and allantoic fluid) of Japanese quail (Coturnix coturnix japonica) eggs and oviduct during a natural outbreak.

Avian influenza virus (AIV) was recovered from the internal contents of eggs, including mixture of albumen and allantoic fluid, and from the oviduct of naturally infected Japanese quail (Coturnix coturnix japonica) flocks in the southern part of Thailand. The virus titers of 10(4.6)-10(6.2) ELD(50)/mL were directly measured from the internal content of infected eggs. The virus was isolated by chorioallantoic sac inoculation of embryonating chicken eggs. Infected allantoic fluid was identified as hemagglutinating virus and then was indicated the presence of H5 hemagglutinin. The virus was confirmed to be H5N1 subtype influenza A virus by reverse transcriptase-polymerase chain reaction. Additionally, real-time reverse transcriptase-polymerase chain reaction assay could specifically detect influenza virus subtype H5. Furthermore, indirect fluorescent antibody (IFA) test by using specific anti-influenza A monoclonal antibody indicated that virus antigens were detected in the parenchyma of multiple tissues. Systemic localization of viral antigen detected was certainly considered to be viremic stage. In addition, influenza virus antigen was also detected by IFA in allantoic fluid sediments isolated from internal content of egg or oviduct. The conclusion of isolated AIV type A subtype H5N1 from these two infected materials was correlated to the viremic stage of infection because the virus antigens could be observed in almost all tissues. Conclusively, the need for adequate safeguards to prevent contamination and spread of the virus to the environment during movement of eggs--including hatching eggs, cracked eggs, and other relevant infected materials-- or egg consumption from area of outbreak is emphasized and must not be ignored for the reasons of animal, public, and environmental health.

Animals↗
PubMed1999

Characterization of the glucosyltransferases that assemble the side chains of the Indian Leishmania donovani lipophosphoglycan.

The life cycle of Leishmania parasites within its sand fly vector involves the development of extracellular promastigotes from a noninfective, "procyclic" stage into an infective, "metacyclic" stage that is adapted for transmission in the fly and survival in the mammalian host. Lipophosphoglycan (LPG), the predominant surface glycoconjugate in both procyclic and metacyclic stages, is a critical virulence determinant. LPG is a multidomain molecule; the structural polymorphisms among species lie in branching from the backbone 6Galbeta1,4Man(alpha1)-PO(4) repeat units and in the composition of the small oligosaccharide caps. We have recently demonstrated that the LPG from an Indian isolate of Leishmania donovani differs from a Sudanese strain by possessing one or two side chain beta(1,3)-linked glucose residues. We now have characterized the glucosyltransferase activities responsible for glucosylating the LPG. When incubated with UDP-[(3)H]glucose and Mn(2+), microsomal membranes from the Indian isolate transferred [(3)H]glucose to the repeat units of the exogenous acceptor Sudanese L. donovani LPG, which does not contain any side chain branching. Glucose addition was maximal at 28 degrees C, the optimal growth temperature of procyclic L. donovani. Consistent with the lack of side chain branching in its LPG, Sudanese L. donovani showed minimal glucosyltransferase activity. Indian metacyclic promastigotes, in contrast to procyclic promastigotes, express no glucose side chains off the repeat units. Therefore, we compared the relative activity of the glucosyltransferases in microsomes from procyclic and metacyclic promastigotes and observed approximately 80% less activity in the latter. These results provide evidence that the glucose side chain addition to LPG is developmentally regulated during the parasite's life cycle and that the glucosyltransferases of L. donovani are strain specific.

Animals↗
PubMed2006

In vitro interactions with the Cytochrome P450 system, toxicity, and local anaesthetic effects of Fomocaine Alkylmorpholine derivatives in rats.

Fomocaine (CAS 56583-43-8) is a basic ether-type local anaesthetic used in dermatological practice for surface anaesthesia. For many years, modifications of the fomocaine molecule have been pursued, e.g. to improve its affinity to the sodium channel and also in view of possible new (systemic) applications. In the present study fomocaine and eight fomocaine derivatives with an additional alkyl chain in 2- or 3-position of different length (C1 up to C4), or with a branched C3 chain in 3-position, respectively, at the morpholine ring were evaluated in vitro for possible structure-activity relationships with respect to the interactions with cytochrome P450 (CYP) mediated monooxygenase and oxidase functions using rat liver 9000 g supernatants or microsomes. Results were compared to in vivo data from rats on toxicity (LD50), paresis of the N. ischiadicus and surface and conduction anaesthesia (cornea, N. ischiadicus). In general, the influence of the derivatives on the CYP system was less than that of fomocaine, showing a further decline with enlarging chain length. Toxicity of the derivatives was comparable to that of fomocaine and lower only with the compound with a C4 alkyl chain in 2-position. The derivatives caused a stronger surface anaesthesia than fomocaine, exhibiting an additional increase with enlarging chain length. No clear-cut structure-activity relationships were observed with respect to paresis of the N. ischiadicus and to conduction anaesthesia. Especially the derivatives having a C2 or C4 chain in 2- or a C3 chain in 3-position, respectively, may be of interest for further investigations. In comparison to fomocaine they caused a stronger surface anaesthesia combined with a lower interaction capacity with the CYP system.

Anesthesia, Conduction↗
PubMed1982

Phosphorylation-dependent activated tension in skinned gizzard muscle fibers in the absence of Ca2+.

A Ca2+-insensitive myosin light chain kinase was prepared and used in experiments with skinned gizzard fibers. In the absence of Ca2+, this kinase activated isometric force development. The force development was associated with phosphorylation of the 20,000-dalton myosin light chains and addition of Ca2+ did not activate the fibers further. Pretreatment of the fibers with Ca2+-insensitive myosin light chain kinase and adenosine 5'-O-(3-thiotriphosphate) in the absence of Ca2+ caused thiophosphorylation of the light chains and, on the addition of ATP, an activation of isometric tension was observed. The subsequent addition of Ca2+ did not increase force development. A comparison of Ca2+-activated tension in the skinned gizzard muscle fibers with Ca2+-insensitive myosin light chain kinase-activated tension suggests that the phosphorylation of the myosin light chains is the dominant factor in the development of tension in smooth muscle.

Animals↗
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↗
PubMed1998

Addition of side-chain interactions to 3(10)-helix/coil and alpha-helix/3(10)-helix/coil theory.

An increasing number of experimental and theoretical studies have demonstrated the importance of the 3(10)-helix/ alpha-helix/coil equilibrium for the structure and folding of peptides and proteins. One way to perturb this equilibrium is to introduce side-chain interactions that stabilize or destabilize one helix. For example, an attractive i, i + 4 interaction, present only in the alpha-helix, will favor the alpha-helix over 3(10), while an i, i + 4 repulsion will favor the 3(10)-helix over alpha. To quantify the 3(10)/alpha/coil equilibrium, it is essential to use a helix/coil theory that considers the stability of every possible conformation of a peptide. We have previously developed models for the 3(10)-helix/coil and 3(10)-helix/alpha-helix/ coil equilibria. Here we extend this work by adding i, i + 3 and i, i + 4 side-chain interaction energies to the models. The theory is based on classifying residues into alpha-helical, 3(10)-helical, or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation plus i, i + 3 and i, i + 4 side-chain interactions. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices and for predicting the locations and stabilities of these structures in peptides and proteins. We conclude that strong i, i + 4 side-chain interactions favor alpha-helix formation, while the 3(10)-helix population is maximized when weaker i, i + 4 side-chain interactions are present.

Chemical Phenomena↗
PubMed1996

Laminins, tenascin and type VII collagen in colorectal mucosa.

The distribution of different laminin polypeptides, type VII collagen and tenascin has been studied in adult and foetal colorectal mucosa by using the indirect immunofluorescence technique. Immunoreactivity for laminin alpha 1 chain was located to basement membranes of epithelia, muscularis mucosae, and blood vessels, respectively in different segments of adult colon and rectum. Laminin beta 1 and gamma 1 chains were additionally expressed in lamina propria. Laminin alpha 2 chain was also found in lamina propria around the pericryptal fibroblasts. Immunoreactivity for laminin beta 2 chain was restricted to basement membranes in the muscularis mucosae and arteries. Laminin alpha 3 and beta 3 chains, suggestive for laminin-5, were confined especially to surface epithelial basement membranes. Immunoreactivity for type VII collagen was confined to basement membrane of surface epithelium in a punctate manner, while that for tenascin was seen slightly more broadly in the basement membrane zone and also in the muscular layer. The distribution of laminin chains in 16-week-foetal colon mostly resembled that of corresponding adult tissue, although immunoreactivities for laminin alpha 2 and beta 2 chains were lacking. Type VII collagen and the high molecular weight isoform of tenascin were also absent from the foetal colon. The results show that the basement membrane of the surface epithelium of colon and rectum express the components of epithelial adhesion complex, laminin-5 (alpha 3-beta 3-gamma 2) and type VII collagen, resembling in this respect small intestine and stomach while laminin-2 (alpha 2-beta 1-gamma 1) appears to be associated with pericryptal fibroblasts, and laminin-1 (alpha 1-beta 1-gamma 1) widely in most basement membranes.

Adult↗
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↗
PubMed1994

T cell receptor beta chain gene rearrangement and selection during thymocyte development in adult mice.

The identification is made in normal mice of the stages in T cell development at which the rearranged beta chain of the T cell receptor (TCR) is utilized to promote T cell maturation, independent of the TCR alpha chain. In addition, evidence is provided that utilization of beta chains in T cell progenitors does not preclude differentiation to TCR gamma delta + T cells. This is consistent with the view that an initial consequence of beta chain expression by early thymocytes is clonal expansion, increasing the size of the pool of useful precursors. This allows the proposal to be made that allelic exclusion may be a byproduct of cell cycle regulation during early thymocyte differentiation, which may in turn explain why the efficiency of allelic exclusion varies at different TCR or immunoglobulin loci.

Animals↗
PubMed2006

Influence of N-acylation of a peptide derived from human lactoferricin on membrane selectivity.

Increasing numbers of bacterial strains being resistant to conventional antibiotics emphasize the urgent need for new antimicrobial agents. One strategy is based on host defence peptides that can be found in every organism including humans. We have studied the antimicrobial peptide LF11, derived from the pepsin cleavage product of human lactoferrin, known for its antimicrobial and lipid A-binding activity, and peptide C12LF11, the N-lauryl-derivative of LF11, which has owing to the attached hydrocarbon chain an additional hydrophobic segment. The influence of this hydrocarbon chain on membrane selectivity was studied using model membranes composed of dipalmitoylphosphatidylglycerol (DPPG), mimicking bacterial plasma membranes, and of dipalmitoylphosphatidylcholine (DPPC), a model system for mammalian membranes. A variety of biophysical techniques was applied. Thereby, we found that LF11 did not affect DPPC bilayers and showed only moderate effects on DPPG membranes in accordance with its non-hemolytic and weak antimicrobial activity. In contrast, the introduction of the N-lauryl group caused significant changes in the phase behaviour and lipid chain packing in both model membrane systems. These findings correlate with the in vitro tests on methicillin resistant S. aureus, E. coli, P. aeruginosa and human red blood cells, showing increased biological activity of C12LF11 towards these test organisms. This provides evidence that both electrostatic and hydrophobic interactions are crucial for biological activity of antimicrobial peptides, whereas a certain balance between the two components has to be kept, in order not to loose the specificity for bacterial membranes.

Acylation↗
PubMed1982

Evidence for the regulation of the branched chain alpha-keto acid dehydrogenase multienzyme complex by a phosphorylation/dephosphorylation mechanism.

The regulation of the branched chain alpha-keto acid dehydrogenase complex by covalent modification was investigated in rat liver mitochondria. Depletion of intramitochondrial calcium and magnesium caused an inactivation of the branched chain alpha-keto acid dehydrogenase complex. Following inactivation of the branched chain complex, addition of calcium or magnesium ions separately to incubations of mitochondria only partially reactivated the enzyme complex. However, simultaneous addition of calcium and magnesium activated the branched chain enzyme complex rapidly and nearly completely. Mitochondrial incubations were performed in the presence of [32P]phosphate under conditions known to activate or to inactivate the branched chain alpha-keto acid dehydrogenase complex. Evidence demonstrating that [32P]-phosphate was incorporated into two major protein bands separated in sodium dodecyl sulfate-polyacrylamide gels of the mitochondrial incubations is presented. Migration of the labeled mitochondrial protein bands in the gel system corresponded exactly to the migration of the alpha subunit of the purified heart-derived pyruvate dehydrogenase (decarboxylase, E1) and the alpha subunit of the purified kidney-derived branched chain alpha-keto acid dehydrogenase (decarboxylase, E1). Furthermore, when the measured activity of the branched chain complex was minimized, the amount of [32P]phosphate incorporated into the alpha chain of the branched chain enzyme was maximal. Conversely, incubation conditions which activated maximally the enzyme complex minimized the [32P]phosphate incorporation into the alpha subunit of the branched chain dehydrogenase.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1997

Partial volumes and compressibilities of extended polypeptide chains in aqueous solution: additivity scheme and implication of protein unfolding at normal and high pressure.

An empirical additivity method for calculation of the partial volumes and adiabatic compressibilities of extended oligo- and polypeptides having arbitrary amino acid compositions has been developed and tested by comparison with available experimental data. Its accuracy is the best among the known empirical approaches. Comparison of experimental data on protein denaturation with the results of calculation allows one to discriminate between the unfolded and molten globule states of globular proteins and to estimate the extent of unfolding. For the first time, experimental nonlinear data for the volume-pressure relationship in proteins and model compounds have been used to interpret the high-pressure denaturation of proteins. It has been shown that the two denatured states, molten globule and unfolded ones, can be attained by a pressure rise: the molten globule state by moderate pressure and the unfolded one by high pressure. The relationship between volumetric properties and hydration is briefly discussed.

Peptides↗
PubMed2002

Differential distribution of basement membrane type IV collagen alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) chains in colorectal epithelial tumors.

In this study, we examined the relationship between the histopathological grade and immunohistochemical localization of six genetically distinct type IV collagen alpha chains, the major component of basement membrane (BM), in normal and neoplastic colorectal tissues. In the normal colorectal mucosa, alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were stained in all epithelial BM. However, alpha3/alpha4(IV) chains were restrictively immunostained in the BM of the apical surface epithelium. Similar immunostaining profiles for alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were observed in tubular adenomas with mild/moderate atypia. However, in intramucosal carcinomas, both alpha1/alpha2(IV) chains were linearly stained in the BM of cancer cell nests, while the assembly of alpha5/alpha6(IV) chains into the BM was inhibited in a discontinuous or negatively stained pattern. The normal colorectal mucosa forms a second network of BM composed of alpha5/alpha6(IV), partly alpha3/alpha4(IV) chains, in addition to the classic network of alpha1/alpha2(IV) chains. The differential immunohistochemical localization of the type IV collagen alpha5/alpha6 chains could be one diagnostic marker for the invasiveness of colorectal cancer.

Adenoma↗