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PubMed1979

Middle East lymphoma and alpha-chain disease. An immunohistochemical study.

An immunoperoxidase study of the small intestinal mucosa of three patients with alpha-chain disease showed heavy infiltration of the mucosa by plasma cells containing alpha-heavy chain and J-chain but no light chains. An additional band-like and nodular mucosal infiltrate was also present and consisted of cells showing no evidence of cytoplasmic immunoglobulin synthesis. The cells comprising this infiltrate invaded and destroyed intestinal crypts, and immunoperoxidase staining showed them to be sharply distinct from the alpha-chain-containing plasma cells. In two cases of Middle East lymphoma, immunohistochemistry revealed a normal plasma cell population in the lamina propria of the small intestine. These results show that alpha-chain disease can be diagnosed in routine paraffin sections which should permit clarification of its true incidence in Middle East lymphoma. The demonstration of sharp distinction between the two types of mucosal infiltrate in alpha-chain disease is in contrast to previous immunofluorescence results and enables more ready identification of mucosal changes that may be important in the management of the disease.

Child, Preschool↗

PubMed1997

Detection of antichlamydial antibodies in tears: a diagnostic aid?

OBJECTIVES: The antibody response in sera and tears of 167 patients with suspected chlamydial conjunctivitis was compared with the antibody response in sera and tears of 45 patients with symptoms of urogenital chlamydial infection to discover whether and which type of antichlamydial antibody detected in tears may be of diagnostic help in chlamydial conjunctivitis. METHODS: Diagnosis was based on chlamydial antigen detection from the conjunctiva and urogenital tract, done by a direct immunofluorescence assay, McCoy cell culture, and polymerase chain reaction. Additionally, antichlamydial immunoglobulin A (IgA) and immunoglobulin G (IgG) were determined in sera and tears of all patients by an immunoperoxidase assay. RESULTS: Two hundred twelve patients were examined--167 with conjunctivitis, 45 with symptoms of urogenital chlamydial infection. Cell culture, direct immunofluorescence assay, and polymerase chain reaction brought identical results. Conjunctival specimens taken from 33 (20%) of the patients with conjunctivitis were Chlamydia antigen positive; specimens taken from 134 (80%) were negative. Antichlamydial antibodies were found in tears of 29 (88%) of the patients with conjunctivitis whose specimens were Chlamydia antigen positive. Fifty-four (40%) of the patients with conjunctivitis whose specimens were Chlamydia antigen negative had antichlamydial antibodies in their tears. Twenty-five patients with urethritis (56%) were Chlamydia antigen positive in urethral swabs; 20 (44%) were negative. Antichlamydial antibodies were found in the tears of eight (32%) of the Chlamydia antigen-positive and two (10%) of the Chlamydia antigen-negative patients with urethritis. In contrast to patients with conjunctivitis, findings for patients with urethritis always were negative for antichlamydial IgG in the tears. CONCLUSION: Antichlamydial antibodies in tears were seen significantly more often in patients with conjunctivitis than in those with urethritis (P < or = 0.05). Antichlamydial IgG was found only in tears of patients with conjunctivitis. Therefore, the authors conclude that the detection of antichlamydial IgG in the tears might be helpful for diagnosis in patients with suspected chlamydial conjunctivitis who have antigen-negative conjunctival swabs.

Adolescent↗

PubMed1979

Heat-induced fragmentation of human alpha 2-macroglobulin.

Previous studies have demonstrated that human plasma alpha 2-macroglobulin (alpha 2 M) possesses a single subunit chain (Mr approximately 185,000) when incubated with dodecyl sulfate and dithiothreitol at 37 degrees C and analyzed by dodecyl sulfate-gel electrophoresis. The present study details the observation that heating alpha 2 M to 90 degrees C under identical conditions produces at least two additional polypeptide chains, termed bands II and III, with apparent molecular weights of 125,00 and 62,000. The generation of these fragments is enhanced by increasing the time of incubation. The appearance of band II composition of the buffer, dodecyl sulfate concentrations, or alpha 2 M protein concentration in the incubation mixture. The electrophoretic bands II and III of alpha 2 M have dissimilar 125I-labeled tryptic peptide digests and also differ in their amino acid composition. The heat-induced fragmentation of alpha 2M is not affected by the inclusion of a variety of low molecular weight protease inhibitors, suggesting that the appearance of bands II and III is not due to enzyme-catalyzed hydrolysis. When the subunit chain of alpha 2M is first cleaved by trypsin into the previously described Mr = 85,000 derivative, neither band II nor III material, nor other lower molecular weight products are generated by heat treatment. Furthermore, preincubation of alpha 2M with methylamine prevents fragmentation of the subunit chain. These results indicate that these fragments are neither pre-existing subunits of alpha 2M nor derivatives formed prior to treatment for gel analysis. These data provide evidence that a covalent bond in the alpha 2M molecule is unusually susceptible to heat-induced cleavage.

Amino Acids↗

PubMed1990

Distinct Ig H chains in a primitive vertebrate, Eptatretus stouti.

Serum Ig from the Pacific hagfish, Eptatretus stouti, was isolated by affinity chromatography using a specific mAb (H.45). Analysis of the approximately 210-kDa molecule by SDS-PAGE under reducing conditions revealed two H chains of approximately 77 kDa (H1) and approximately 70 kDa (H2) and L chains of approximately 30 kDa. H1 and H2 were shown to differ with respect to their peptide maps, amino-terminal amino acid sequences, and reactivity to the mAb H.45, suggesting that they represent discrete H chain isotypes. Two-dimensional nonreducing/reducing SDS-PAGE demonstrated that H and L chains were covalently linked predominantly as H-H-L and H-L configurations. Noncovalently bound L chains were also found. H-H-L complexes were shown to contain H1-H2 heterodimers of H chains in addition to H1-H1 homodimers.

Amino Acid Sequence↗

PubMed1983

High incidence of free monoclonal lambda light chains in the sera of patients with Sjogren's syndrome.

Sjogren's syndrome presents a wide spectrum of disease manifestations ranging from benign to malignant lymphoproliferation. Sera from 21 patients with primary Sjogren's syndrome, 63 patients with other autoimmune rheumatic diseases, and 140 normal controls were studied by using high-resolution gel electrophoresis combined with immunofixation and specific absorption studies. Two-thirds of the sera from Sjogren's syndrome patients contained free monoclonal lambda light chains. In addition, one patient with Sjogren's syndrome and pseudolymphoma had a circulating monoclonal IgM-lambda immunoglobulin. In contrast, only 16% of the patients with other autoimmune diseases and 5% of normals had monoclonal bands; none of the other patients studied exhibited monoclonal-free lambda light chains in their serum. Our findings suggest that the monoclonal process in Sjogren's syndrome starts early in the disease process in a subset of B cells.

Antibodies, Monoclonal↗

PubMed1995

Chemical characterization of two lipopolysaccharide species isolated from Rhizobium loti NZP2213.

Phenol-water extraction of Rhizobium loti NZP2213 cells allowed a simultaneous isolation of two structurally different lipopolysaccharides from the aqueous (LPS-W) and phenol (LPS-P) phase that differed in their sodium deoxycholate-PAGE pattern and composition. LPS-W showed a profile indicating an R-type LPS; LPS-P had a cluster of poorly resolved bands in the high-molecular-weight region. LPS-P contained large amounts of 6-deoxy-L-talose (6dTal), and a small amount of 2-O-methyl-6-deoxy-talose (molar ratio approximately 30:1), both of which were completely absent in LPS-W. Methylation analysis gave only one major product, 2,4-di-O-methyl-6dTal, indicating that the O-chain is composed of a homopolymer of 1,3-linked 6dTal, having the methylated 6dTal (2-O-Me-6dTal) probably localized at the non-reducing end of the O-chain. This homopolymeric O-chain was additionally O-acetylated, as evidenced by GC-MS and by 13C NMR analysis. The lipid A moieties of both LPS-W and LPS-P showed almost identical composition, with six different 3-OH fatty acids and with two, so far not described, long-chain 4-oxo-fatty acids, all being amide-linked, and with 27-OH-28:0 as the main ester-linked fatty acid. Lipid A was of the lipid ADAG-type, i.e., having a (phosphorylated) 2,3-diamino-2,3-dideoxy-D-glucose-containing lipid A backbone. Lipid ADAG is widespread among species of the alpha-2 group of Proteobacteria, but has so far not been encountered in any other rhizobial or agrobacterial species.

Carbohydrates↗

PubMed1998

Luminescence resonance energy transfer measurements in myosin.

Myosin is thought to generate force by a rotation between the relative orientations of two domains. Direct measurements of distances between the domains could potentially confirm and quantify these conformational changes, but efforts have been hampered by the large distances involved. Here we show that luminescence resonance energy transfer (LRET), which uses a luminescent lanthanide as the energy-transfer donor, is capable of measuring these long distances. Specifically, we measure distances between the catalytic domain (Cys707) and regulatory light chain domain (Cys108) of the myosin head. An energy transfer efficiency of 21.2 +/- 1.9% is measured in the myosin complex without nucleotide or actin, corresponding to a distance of 73 A, consistent with the crystal structure of Rayment et al. Upon binding to actin, the energy transfer efficiency decreases by 4.5 +/- 1.0%, indicating a conformational change in myosin that involves a relative rotation and/or translation of Cys707 relative to the light chain domain. Addition of ADP also alters the energy transfer efficiency, likely through a rotation of the probe attached to Cys707. These results demonstrate that LRET is capable of making accurate measurements on the relatively large actomyosin complex, and is capable of detecting conformational changes between the catalytic and light chain domains of myosin.

Actins↗

PubMed1989

Oxygen diffusion and mitochondrial respiration in neuroblastoma cells.

Suspensions of human neuroblastoma cells consume oxygen at a constant rate when the oxygen pressure is greater than approximately 11 Torr. The rate of oxygen consumption, however, becomes dependent on the oxygen pressure below this level. The falling respiratory rate at the lower pressures gives rise to an oxygen pressure for half-maximal respiration (P50) of approximately 0.8 Torr, which is consistent with the 0.5 Torr value for suspensions of isolated mitochondria in the presence of ATP (J. Biol. Chem. 263: 2712-2718, 1988). When the cellular metabolic energy state is lowered by addition of an uncoupler of mitochondrial oxidative phosphorylation, the respiratory rate increases up to fivefold, but the P50 decreases to approximately 0.6 Torr. In the cells treated with uncoupler, the P50 decreases further when the mitochondrial respiratory rate is inhibited with amobarbital (amytal), an inhibitor of the respiratory chain. The additional decrease in P50 is proportional to the decrease in respiratory rate. Thus, for cells treated with uncoupler, the P50 appears to be limited by oxygen diffusion from the external medium to the mitochondria. When the respiratory rate of the uncoupled cells is inhibited to the level of coupled cells, the P50 for the former is less than 0.15 Torr. This indicates that for coupled cells the difference in oxygen pressure from the external medium to the mitochondria is less than 0.15 Torr at half-maximal respiratory rate and does not significantly affect the P50 for oxygen that occurs at 0.8 Torr.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗

PubMed1976

[Beta-Hydroxybutyrate dehydrogenase of rat liver inner mitochondrial membrane. Its isolation, characterization, and reactivation by lecithins differing in their apolar regions. The influence of the addition of cholesterol on its level of reactivation (author's transl)].

The beta-hydroxybutyrate dehydrogenase has been isolated and purified from the inner mitochondrial membrane of the rat liver. It consists in a dimer of molecular weight 77 000 composed by two subunits of molecular weight 38 000 each. The level of its reactivation by lecithin is influenced by the length and degree of unsaturated of their aliphatic chains. The addition of cholesterol inhibits the reactivation.

Animals↗

PubMed2001

Two simple NMR experiments for measuring dipolar couplings in asparagine and glutamine side chains.

Residual dipolar couplings are now widely used for structure determination of biological macromolecules. Until recently, the main focus has been on measurement of dipolar couplings in the protein main chain. However, with the aim of more complete protein structure, it is also essential to have information on the orientation of protein side chains. In addition, residual dipolar couplings can potentially be employed to study molecular dynamics. In this Communication, two simple NH(2) and spin-state edited experiments are presented for rapid and convenient determination of five residual dipolar couplings from (15)N, (1)H correlation spectrum in asparagine and glutamine side chains. The pulse sequences are demonstrated on two proteins, 30.4-kDa Cel6A in diluted liquid crystal phase and 18-kDa human cardiac troponin C in water.

Anisotropy↗

PubMed1996

Protein stabilization by removal of unsatisfied polar groups: computational approaches and experimental tests.

The role of polar and charged side chains in partially buried protein environments has been probed in a variant of Arc repressor (MYL) in which hydrophobic interactions between Met31, Tyr36, and Leu40 replace the wild-type salt-bridge interactions between Arg31, Glu36, and Arg40. In the absence of this salt-bridge triad, three additional side chains were identified by continuum electrostatic calculations as incurring larger desolvation penalties during folding than were recovered in favorable electrostatic interactions in the folded state. These side chains (Asn29, Ser44, and Glu48) were mutated singly and collectively to alanine in the MYL background, and the thermodynamic stabilities of the resulting mutant proteins were found to be increased by 0.1 to 1.3 kcal/mol of dimer. All of the mutants displayed cooperative thermal melts and appeared to have well-packed hydrophobic cores by near-UV circular dichroism spectroscopy, indicating that conformational specificity is maintained. The Arc variant (MYL-NA29/SA44/EA48) in which the entire six-residue polar network is replaced by nonpolar groups is 5.1 kcal/mol of dimer more stable than wild type, indicating that the strategy of replacing buried or partially buried charged and polar side chains with hydrophobic residues can lead to substantial stabilization.

Calorimetry↗

PubMed1983

Biosynthesis of Trypanosoma brucei variant surface glycoproteins - analysis of carbohydrate heterogeneity and timing of post-translational modifications.

The variant surface glycoproteins from two cloned populations of Trypanosoma brucei brucei which were known to migrate as multiple bands on SDS gels have been studied. The heterogeneity present was located in those oligosaccharide side chains the addition of which is tunicamycin-sensitive. The time required for the trypanosome to synthesize and express a variant surface glycoprotein molecule in vitro was found, from pulse-chase and limited trypsinisation experiments, to be approximately 40 min. In the light of these data, pulse-chase experiments on the two antigens known to have heterogeneity in their oligosaccharide side chains demonstrated that the heterogeneity probably arose by two different mechanisms. Pulse-chase experiments on three different clones of trypanosomes have also been used to investigate the timing of cleavage of the carboxyl-terminal extension, known to be encoded on variant surface glycoprotein mRNA. Similar pulse-chase experiments followed by immunoprecipitation using affinity purified antiserum have been used to investigate the addition of the cross-reacting determinant. The timing of both these events has been discussed in relation to the time necessary for the synthesis and expression of the variant surface glycoprotein on the surface of the trypanosome.

Animals↗

PubMed1985

Amino acid sequence of the phosphorylation site of bovine cardiac myosin light chain.

Amino acid sequences of peptides containing the phosphorylation site of bovine cardiac myosin light chain (L2) were determined. The site was localized to a serine residue in the tentative amino terminus of the light chain and is homologous to phosphorylation sites in other myosin light chains. Phosphorylation of bovine cardiac light chain by chicken gizzard myosin light chain kinase was Ca2+-calmodulin dependent. Kinetic data gave a Km of 107; microM and a Vmax of 23.6 mumol min-1 mg-1. In contrast to what has been observed with smooth muscle light chains, neither the phosphorylation site fragment of the cardiac light chain nor a synthetic tetradecapeptide containing the phosphorylation site were effectively phosphorylated by the chicken gizzard kinase. Phosphorylation of cardiac myosin light chains by chicken gizzard myosin light chain kinase, therefore, requires other regions of the light chain in addition to a phosphate acceptor site.

Amino Acid Sequence↗

PubMed1977

Amino acid sequence of the first 217 residues of a mouse heavy chain (MOPC 47A) with a domain deletion.

Cyanogen bromide cleavage of the deleted heavy (alpha) chain of mouse IgA 47A yielded five peptides, CNBr 1-5, consisting of 34, 50, 28, 70, and 160 residues. A 217-residue NH2-terminal sequence, which comprises the variable region and the first domain of the constant region (CH1), and a 19-residue COOH-terminal sequence of the chain were obtained from the sequences of CNBr 1-4 and the NH2-terminal and COOH-terminal sequences of CNBr 5. The tryptic peptides of the remainder of the chain have been partially characterized. Comparison of these data with the sequences of other, nondeleted, chains reveals that the 47A chain terminates exactly at the end of the CH2 domain and that there are no deletions up to that point. It is concluded that the deletion in the chain is a single, large deletion consisting of the entire CH3 domain. The 47A chain also differs from other, nondeleted, mouse alpha chains derived from BALB/c strains in that it contains a labile cysteine at position 135, which is believed to participate in the light-heavy chain bond, and contains galactosamine in the hinge region. This may mean that the 47A alpha chain, in addition to being deleted, represents a mouse IgA subclass analogous to human IgA1.

Amino Acid Sequence↗

PubMed2001

Regulation of tight junction permeability and occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms.

In epithelial and endothelial cells, tight junctions regulate the paracellular permeability of ions and proteins. Disruption of tight junctions by inflammation is often associated with tissue edema, but regulatory mechanisms are not fully understood. Using ECV304 cells as a model system, lysophosphatidic acid and histamine were found to increase the paracellular permeability of the tracer horseradish peroxidase. Cytoskeletal changes induced by these agents included stimulation of stress fiber formation and myosin light chain phosphorylation. Additionally, occludin, a tight junction protein, was a target for signaling events triggered by lysophosphatidic acid and histamine, events that resulted in its phosphorylation. A dominant-negative mutant of RhoA, RhoA T19N, or a specific inhibitor of Rho-activated kinases, Y-27632, prevented stress fiber formation, myosin light chain phosphorylation, occludin phosphorylation, and the increase in tracer flux in response to lysophosphatidic acid. In contrast, although RhoA T19N and Y-27632 blocked the cytoskeletal events induced by histamine, they had no effect on the stimulation of occludin phosphorylation or increased tracer flux, indicating that occludin phosphorylation may regulate tight junction permeability independently of cytoskeletal events. Thus, occludin is a target for receptor-initiated signaling events regulating its phosphorylation, and this phosphorylation may be a key regulator of tight junction permeability.

Actins↗

PubMed1984

New structural model for mixed-chain phosphatidylcholine bilayers.

Multilamellar suspensions of a mixed-chain saturated phosphatidylcholine with 18 carbon atoms in the sn-1 chain and 10 carbon atoms in the sn-2 chain have been analyzed by X-ray diffraction techniques. The structural parameters for this lipid in the gel state are quite different than usual phosphatidylcholine bilayer phases. A symmetric and sharp wide-angle reflection at 4.11 A indicates that the hydrocarbon chains in hydrated C(18):C(10)PC bilayers are more tightly packed than in usual gel-state phosphatidylcholine bilayers and that there is no hydrocarbon chain tilt. The lipid thickness is about 12 A smaller than would be expected in a normal bilayer phase, and the area per molecule is 3 times the area per hydrocarbon chain. In addition, the bilayer thickness increases upon melting to the liquid-crystalline state, whereas normal bilayer phases decrease in thickness upon melting. On the basis of these data, we propose a new lipid packing model for gel-state C(18):C(10)PC bilayers in which the long C(18) chain spans the entire width of the hydrocarbon region of the bilayer and the short C(10) chain aligns or abuts with the C(10) chain from the apposing molecule. This model is novel in that there are three hydrocarbon chains per head group at the lipid-water interface. Calculations show that this phase is energetically favorable for mixed-chain lipids provided the long acyl chain is nearly twice the length of the shorter chain. In the liquid-crystalline state C(18):C(10)PC forms a normal fluid bilayer, with two chains per head group.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗

PubMed2001

Overexpression of type IV collagen in chorionic villi in hydatidiform mole.

To investigate the characteristic structure of hydatidiform mole, type IV collagen expression was determined in human villous tissues obtained from normal pregnancies (n = 17) and complete hydatidiform moles (n = 10). Indirect immunofluorescent staining was performed to detect type IV collagen with specific monoclonal antibody, and Northern blot analysis was performed to assess expression of messenger ribonucleic acid for the alpha1(IV) chain. In addition, serum levels of type I, III, and IV collagen were measured by RIA. Immunohistochemical staining for type IV collagen revealed stronger staining of the trophoblastic basement membrane in hydatidiform mole than in normal pregnancy. Northern blot analysis revealed that the villous expression of messenger ribonucleic acid for the alpha1(IV) chain was significantly increased in hydatidiform moles compared with normal pregnancy (P < 0.01). Although there were no differences in the serum type I and III collagen levels between hydatidiform mole and normal pregnancy, the type IV collagen level was significantly higher in patients with hydatidiform mole than in normal pregnancy (P < 0.05). These results suggest that type IV collagen might play an important role in determining the pathophysiology and structure of hydatidiform mole.

Adult↗

PubMed1977

Vibrational Raman spectra of lipid systems containing amphotericin B.

Resonance-enhanced and normal vibrational Raman spectra were observed for both multilamellar and single-wall vesicle assemblies of dimyristoyl phosphatidylcholine containing amphotericin B, a channel-forming polyene antibiotic, and cholesterol. The decrease in the frequency of the polyene antibiotic C = C stretching mode at 1556 cm-1 and the increase in intensity of the C-C-H in-plane deformation mode at 1002 cm-1 indicate that amphotericin B is ordered in a lipid-cholesterol medium similarly to the solid, but is surrounded by a slightly more polar environment. The intensity of the C = C stretching mode I1556 decreases 4-fold during the broadened gel to liquid crystalline phase transition (16--32 degrees C) of dimyristoyl lecithin-cholesterol (4 : 1) multilayers. Other resonance-enhanced vibrations of amphotericin B exhibit similar behavior. For amphotericin B in pure dimyristoyl lecithin multilayer or vesicle systems, however, the vibrational intensity associated with the C = C stretching mode remains constant during the melting of lipid hydrocarbon chains. In addition, a third effect occurs in liquid crystalline egg lecithin-cholesterol (4 : 1, mol ratio) multilayers in which I 1556 first increases by 25% between 3 and 25 degrees C, in parallel with the loss of active channels, and then remains constant as the temperature increases from 25 to 42 degrees C. This latter intensity pattern is masked in the dimyristoyl lecithin-cholesterol system by the overwhelming effect upon the C = C mode from changes in the lipid chain packing characteristics which occur during the phase transition. The broadened phase transition in 4 : 1 dimyristoyl lecithin-cholesterol multilayers (16--32 degrees C), as followed by the ratio of intensities at 2880 and 2850 cm-1 (asymmetric and symmetric methylene C-H stretching modes, respectively) is slightly narrowed by the addition of amphotericin B, and effect from which a binding stoichiometry at 24 degrees of 1 : 1 amphotericin B: cholesterol is estimated. This stoichiometry was confirmed by differential calorimetric scans, which also show the presence of a peak proportional to cholesterol content. Raman I2880/2850 peak height ratios in pure dimyristoyl lecithin bilayers were increased over the 14--38 degrees C range by amphotericin B, a spectral effect which suggests an ordering of the lipid matrix perhaps as a consequence of the polyene binding to the bilayer surface. For bilayers containing cholesterol, the ratios of intensities of the 2935 cm-1 feature, composed mainly of acyl chain terminal methyl and underlying methylene C-H stretching modes, to the 2850 cm-1 feature are significantly increased by amphotericin B. This effect indicates that the antibiotic penetrates the bilayer in the lipid-sterol system.

Amphotericin B↗