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Biomedical subjects

B T Kurien

Publications and source records attributed to B T Kurien.

32 records · Page 2Linked to original sources
PubMed1998

Multiple nuclear dot antinuclear antibody in patients without primary biliary cirrhosis.

Multiple nuclear dot (MND), or pseudocentromere, anti-nuclear antibody (ANA) is an uncommon pattern associated primarily with primary biliary cirrhosis (PBC) and anti-mitochondrial antibody (AMA). A 53 kDa antigen with an apparent molecular mass of 100 kDa as found on sodium dodecyl sulphate-polyacrylamide gel electrophoresis is thought to be responsible for the uncommon pattern. This study analyzes sera from 21 patients without PBC or AMA that produced the uncommon MND ANA immunofluorescence pattern. Diseases present include lupus, rheumatoid arthritis and scleroderma. On immunoblotting nineteen of 21 (91%) bound a 70 kDa protein. Western blot analysis showed that this nuclear antigen was different from pyruvate dehydrogenase, p80 coilin and the antigen responsible for MND ANA in those with PBC. Affinity purified anti-70 kDa reproduced the MND ANA immunofluorescence pattern. Thus, the MND ANA in patients without PBC/AMA is associated with binding to a 70 kDa nuclear protein and not with a 53 kDa antigen (that runs at 100 kDa) found in those with MND and PBC/AMA. The data demonstrate the MND antigen without PBC/AMA is immunologically distinct from the pattern when found with PBC/AMA.

Antibodies, Antinuclear↗
PubMed1997

Multiple immunoblots after non-electrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens.

Western blotting is a very sensitive and powerful fundamental technique in immunology that has been used to detect and characterize proteins of low abundance. This technique employs the transfer of proteins separated on SDS-PAGE to nitrocellulose sheets for further detection using antibodies. Here we report the non-electrophoretic transfer of the 60-kDa Ro (or SSA) autoantigen, 240 and 220 kDa spectrin antigens and prestained molecular weight standards from SDS-PAGE gels to nitrocellulose to obtain multiple immunoblots. In fact, we have used this procedure to obtain 12 immunoblots from a single gel with multiple sera.

Antigens↗
PubMed1997

Efficient 5' end labeling of dephosphorylated DNA.

Efficient 5'-end labeling of DNA is an important procedure in recombinant DNA technology. Prior to labeling, it is important to inactivate alkaline phosphatase, used in the dephosphorylation of the DNA, by using proteinase K. Removal of proteinase K is usually performed by extracting twice with chloroform:isoamyl alcohol. In this report we show that extracting the sample four times with chloroform results in more efficient removal of sodium dodecyl sulfate (SDS), an important constituent of proteinase K buffer, which allows a 25- to 40-fold increase in labeling efficiency compared with extracting twice or once with chloroform, respectively. Unremoved SDS inhibits efficient labeling, possibly by inhibiting the activity of the kinase.

Bacteriophage T4↗
PubMed1997

Human anti-Ro autoantibodies bind multiple conformational epitopes of 60-kD Ro autoantigen.

A gel filtration method was developed to estimate the number of conformational epitopes on the 60-kD Ro antigen. Anti-Ro Fab or Fab' was incubated with native Ro antigen at different ratios and the Stokes radius molecular weight of complexes was estimated by gel filtration. Binding was saturated at 9 to 11 Fab molecules per bovine Ro molecule. Two additional Fab or Fab' were bound if human Ro was used as the antigen. Isolated Ro antigen/anti-Ro Fab complexes were evaluated for the relative proportion of antigen to antibody at saturation of antigen with antibody and thus stoichiometry was determined. This provided data supporting there being between 7 and 11 binding sites, results similar to those with the gel filtration method. Experiments carried out with anti-Ro monoclonal antibodies showed one binding site per molecule of 60-kD Ro. Therefore, we have developed methods to count conformational epitopes on autoantigens and have applied it to the Ro/anti-Ro system. The data indicate that multiple conformational epitopes can be bound simultaneously by polyclonal anti-Ro sera from patients with systemic lupus erythematosus.

Animals↗
PubMed1997

Anti-Ro fine specificity defined by multiple antigenic peptides identifies components of tertiary epitopes.

Anti-Ro (or SSA) is a clinically important autoantibody that is found in 25-40% of patients with systemic lupus erythematosus as well as an even greater proportion of patients with Sjögren's syndrome or subacute cutaneous lupus. We have studied the binding of anti-Ro sera to multiple antigenic peptides constructed from the sequence of the 60-kD Ro molecule. The results demonstrate that sera bind these peptides in solid-phase assay. Surprisingly, some of these peptides also form a precipitin line in double immunodiffusion with anti-Ro sera. Formation of lines of identity in double immunodiffusion as well as absorption studies indicate that peptides distant in the primary amino acid sequence and without shared sequence are bound by the same antibody. In addition, data from surface plasmon resonance demonstrate that peptides identified in this manner have protein-protein interactions. Thus, these techniques may identify the components of conformational epitopes.

Amino Acid Sequence↗
PubMed1997

Immunologically restricted and inhibitory anti-Ro/SSA in monozygotic twins.

A pair of monozygotic twins with Sjögren's syndrome are described who both have large amounts of anti-Ro/SSA in their sera by ELISA, but no precipitin in double immunodiffusion. Unique among previously encountered specimens, sera from the twins inhibit the formation of Ro/SSA-precipitins in double immunodiffusion by known anti-Ro/SSA positive SLE patients. The twins have near identical, clonally restricted anti-Ro/SSA autoantibodies as evaluated by isoelectric focusing and bind the same 60 kD Ro/SSA peptides. Thus, this pair of monozygotic twins has an identical fine specificity in their immune response to 60 kD Ro/SSA, despite potential differences in their immune response generated by random processes in the formation of immunoglobulin molecules and T cell receptors. These data imply that the anti-Ro/SSA found in these sera binds less than three epitopes such that soluble antigen/antibody complexes are formed instead of an insoluble complex that precipitates.

Adult↗
PubMed1996

Immunization with short peptides from the sequence of the systemic lupus erythematosus-associated 60-kDa Ro autoantigen results in anti-Ro ribonucleoprotein autoimmunity.

Systemic lupus erythematosus is characterized by the production of Abs to ribonucleoproteins, including Ro (or SS-A) and La (or SS-B). The mechanism by which these autoantibodies arise is unknown. We have immunized rabbits with peptides from the sequence of the 60-kDa Ro. Animals so immunized not only produce Abs to the immunogen peptide, but also develop an autoimmune response to the entire 60-kDa Ro Ag. In about 20% of these animals, autoimmunity also spreads to the La Ag. Anti-native DNA was present in one of nine rabbits. All animals had anti-nuclear Ab, but even among those immunized with the same peptides, different patterns of immunofluorescence were found. Thus, among these outbred animals immunized with peptides from 60-kDa Ro, distinct serologic manifestations were present, showing heterogeneity in the development of autoimmunity.

Amino Acid Sequence↗
PubMed1996

Development of the anti-Ro autoantibody response in a patient with systemic lupus erythematosus.

OBJECTIVE: To characterize the initial events in anti-Ro production by a patient with systemic lupus erythematosus, in whom this autoantibody is developing. METHODS: The immune response to the Ro ribonucleoprotein and other autoantigens were studied by enzyme-linked immunosorbent assay for IgG and IgM, by isoelectric focusing, and by inhibition studies to determine apparent avidity. RESULTS: The patient's sera showed an oligoclonal response to Ro that increased in complexity and affinity with time. IgM anti-Ro appeared shortly before IgG anti-Ro, and disappeared as IgG anti-Ro increased in titer and affinity. IgG antiribosomal P autoantibodies also appeared during the patient's course, but in contrast to anti-Ro, were not preceded by IgM antiribosomal P. CONCLUSION: These data are consistent with the Ro autoantigen being presented and processed in a manner similar to heterologous antigen, and with differences in the mechanisms that lead to the production of IgG anti-Ro autoantibodies as opposed to antiribosomal P autoantibodies.

Adult↗
PubMed1995

Polyethylene glycol-mediated bacterial colony transformation.

A moderately efficient and quick method of bacterial colony transformation is described. Plasmid DNA was added to bacteria suspended in a solution of polyethylene glycol/calcium chloride (PEG/CaCl2). After a brief incubation and heat shock, the cells were directly plated. Transformation efficiencies up to 8.6 +/- 1.28 x 10(6) transformants per microgram of pUC18 were obtained. We have found that the reverse of the transformation process could also take place. Suspending a bacterial pellet harboring the plasmid of interest in PEG/CaCl2 results in the release of the plasmid DNA, and thus indirectly lends support to the transformation process.

Costs and Cost Analysis↗
PubMed1995

Immunization with vesicular stomatitis virus nucleocapsid protein induces autoantibodies to the 60 kD Ro ribonucleoprotein particle.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disorder of unknown etiology that is characterized by antibodies binding ribonucleoprotein complexes. The epitopes of SLE associated 60 kd Ro (or SSA) autoantigen share sequence with vesicular stomatitis virus (VSV) nucleocapsid (N) protein and SLE patients with anti-Ro are more likely to bind the N-protein than anti-Ro negative patients. METHOD: We immunized New Zealand white (NZW) rabbits with purified VSV N-protein to examine the relationship between the immune response to N-protein and anti-Ro. RESULTS: After multiple immunizations with VSV N-protein, NZW rabbits produced not only IgG antibodies to VSV N-protein but also an autoimmune response to Ro antigen. The IgG fraction of the rabbit immune serum precipitates 60 kd Ro protein as well as its associated Y RNAs. Antibodies elicited by 10 immunizations of VSV N-protein bind to at least 20 linear epitope groups on VSV N-protein and over 25 linear epitope groups on 60 kd Ro protein. These antibodies also bound 5 of the 6 shared sequences between 60 kd Ro protein and VSV N-protein. CONCLUSIONS: Our data demonstrate that an immune response initiated towards a foreign antigen, selected on the basis of sharing short sequence similarity with the autoantigenic epitopes of an autoantigen, can lead to an autoimmune response.

Animals↗
PubMed1994

Phosrestin I undergoes the earliest light-induced phosphorylation by a calcium/calmodulin-dependent protein kinase in Drosophila photoreceptors.

Activation of PI-PLC initiates two independent branches of protein phosphorylation cascades catalyzed by either PKC or Ca2+/calmodulin-dependent protein kinase (CaMK). We find that phosrestin I (PRI), a Drosophila homolog of vertebrate photoreceptor arrestin, undergoes light-induced phosphorylation on a subsecond time scale which is faster than that of any other protein in vivo. We determine that a CaMK activity is responsible for in vitro PRI phosphorylation at Ser366 in the C-terminal tryptic segment, MetLysSer(P)IleGluGlnHisArg, in which Ser(P) represents phosphoserine366. We also demonstrate that Ser366 is the phosphorylation site of PRI in vivo by identifying the molecular species resulting from in-gel tryptic digestion of purified phospho-PRI using HPLC-electrospray ionization tandem quadrupole mass spectroscopy. From these data, we conclude that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event following activation of PI-PLC in living Drosophila photoreceptors.

Amino Acid Sequence↗
PubMedDate not supplied

Protein-protein interaction of the Ro-ribonucleoprotein particle using multiple antigenic peptides.

Protein protein interactions play a significant role in maintaining the structural and functional integrity of the cell. We used multiple antigen peptides (MAPs) to analyze such interactions within the Ro (or SSA) ribonucleoprotein complex. Our data showed that 60 kD Ro and La colocalize in the nucleus of the cell. Previous data have indicated that 60 kD Ro and La co-exist via interactions with the hYRNAs. We were interested to see whether 60 kD Ro and La interact with each other through protein protein interactions. MAPs were produced with sequences derived from the autoepitopes of 60 kD Ro. When used in agarose immunodiffusion certain MAPs formed precipitin lines specifically with Ro and La antigens. Used in affinity chromatography the Ro MAPs purified the Ro ribonucleoprotein particle from lymphocyte extract. Solid phase immunoassay and surface plasmon resonance (SPR) confirmed the observations obtained with agarose diffusion. Using SPR, kinetic analyses gave an apparent affinity constant of about 1 x 10(7) M(-1) for Ro-MAP-60 kD Ro interactions. The autoantigens Ro and La are specific targets in autoimmune diseases, particularly systemic lupus erythematosus (SLE) and Sjögren's syndrome, and are known to exist together as a complex with hYRNAs. The present data indicate that there are protein-protein interactions between Ro and La.

Amino Acid Sequence↗