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IgG half-molecules: clinical and immunologic features in a patient with plasma cell leukemia.

The clinical manifestations and immunologic features of a patient with plasma cell leukemia who produced k, IgG half-molecules are described. His serum contained both 7S myeloma protein and 4.3S half-molecules, whereas his urine contained predominantly half-molecules. The half-molecules were discovered because the serum and urine formed double precipitin lines when analyzed by commercially available IgG radial immunodiffusion plates that contained antibodies to determinants on both the Fab and Fc fragments. Immunoelectrophoresis also revealed double precipition lines with such antisera. In contrast, when antisera specific for the IgG Fc fragment were used, the serum showed only a single line formed by intact IgG, and the urine failed to react, indicating that the half-molecule was antigenically deficient in the Fc fragment. The half-molecule consisted of one covalently linked heavy and light chain, both having about normal molecular weights, suggesting that they did not have a large deletion which could have caused the half-molecule production. Comparison of the clinical manifestations of the patient with those of four other known patients who produced half-molecules suggested that half-molecule formation is not associated with a distinct clinical syndrome.

Aged↗

GammaG-globulin production and light-chain metabolism in patients with metastatic cancer.

GammaG-Globulin and excess light-chain metabolism were studied in eight subjects with progressive metastatic malignant disease by determining the plasma radioactivity curves following the administration of appropriately labeled substances. In addition to the plasma die-away curves, which required about 3 weeks for full expression for gamma-globulin, but only 3 to 4 days for light-chain, urinary excretion of the label from metabolized protein was determined. The data are compared to similar studies in control individuals. The metabolism of excess light chain was similar to normal in all respects. The total synthesis of gammaG-globulin was increased with a mean value about twice normal. The mean survival time of a circulating immunoglobulin molecule was short, indicating rapid loss from the system. Other aspects of immunoglobulin metabolism were similar to normal with a normal percentage of the labeled protein appearing in the urine, suggesting no abnormality in the utilization pattern but simply an increased rate of turnover. The capability of malnourished patients with cancer to produce large quantities of immunoglobulin is not specific for this disease, since similar patterns may be seen in response to infections in protein-depleted individuals. However, there is the possibility that the cancer itself acts as an inciting agent in these subjects. Furthermore, such sustained protein synthesis may place an additional burden on already compromised host metabolism.

Humans↗

The primary structure of a human lambda II chain.

The human myeloma protein Boh (gamma 2, lambda) was isolated and completely reduced and aminoethylated. The light chain was obtained by chromatography on Sephadex G-100 in 4 M guanidine HC1. The amino-terminal sequence on the blocked light chain could be determined by automatic sequence degradation after PCAase treatment. Twenty-one peptides were isolated from a tryptic digest and 12 peptides from a chymotryptic digest. The sequence determination on these peptides was performed by automatic sequencing methods. The light chain of Boh protein belongs to the lambda II subgroup. Unique substitutions have been found at position 8 (Arg) and position 62 (Tyr). Furthermore, the Boh light chain has six cysteine residues, the additional (sixth) cysteine being adjacent to the invariable intrachain-S-S linking cysteine at position 91. Sequence comparison of lambda II proteins reveals a high degree of homology emphasizing the biologic significance of the hypervariable region sequences;

Amino Acid Sequence↗

Evidence for the absence of noncovalent bonds in the Fcmu region of IgM.

IgM isolated from the sera of five patients with Waldenström's macroglobulinemia was subjected to tryptic digestion at 60 degrees C. The Fc5mu fragments recovered from the digests were reduced by 0.05 M cysteine and alkylated by iodoacetamide, producing large quantities of an Fcmu fragment having a sedimentation velocity (see article) of 2.9S and a molecular weight of 33,500 by sedimentation equilibrium in neutral buffer. Further studies on the Fcmu fragment from one of the proteins demonstrated that it was not dissociated into smaller fragments by 5 M guanidine-HC1, even after reduction with 0.1 M 2-mercaptoethanol in 5 M guanidine at pH 7.5. The number of sulfhydryl groups released by the latter treatment indicated the presence of two intrachain disulfide bonds. These observations provide evidence that this portion of the mu-chain demonstrates minimal noncovalent interactions. Tryptic digestion of the Fcmu fragment at 37 degrees C resulted in the production of several lower molecular weight fractions. The three major fractions demonstrated apparent molecular weights of 21,000, 13,800 and 6800 by sedimentation equilibrium in 5 M guanidine-HC1. The latter fraction (fraction C) had no detectable carbohydrate and consisted of two disulfide-bonded peptides having molecular weights of approximately 3800 and 2200. Studies on the amino acid composition and amino-terminal sequences indicated that fraction C was derived from the Cmu4 homology region and consisted of residues 468 through 546 of the mu-chain with the tryptic peptides encompassing residues 492 through 514 missing.

Amino Acid Sequence↗

Distribution and association of heavy and light chain variable region subgroups among human IgA immunoglobulins.

A series of randomly selected human IgA myeloma proteins were examined for the presence of the VHIII subgroup as defined by the possession of an unblocked amino terminal amino acid and characteristic linked residues along the heavy chain. Blocked heavy chains were classified as VHB proteins. The data showed that 20 of 30 such random alpha chains (67%) were classifiable as members of the VHIII subgroup. Similarly, 75% of heavy chains isolated from pools of normal serum IgA contained a VHIII variable region. The pattern stands in marked contrast to the situation in human IgG proteins where approximately 20% of heavy chains from both pools and myeloma proteins are VHIII. There was thus a clear divergence of the pattern of variable region: constant region association between these two classes of immunoglobulin. Some more limited data were obtainable for the light chains of the IgA myeloma proteins. Certain predilections for light chain subgroup:heavy chain subgroup (VHIII or VHB) associations were discernable, but more data are required for definite conclusions. Overall, this study suggests that although the pool of available variable region sequences is indeed shared among human IgA and IgG proteins, the partitioning is not exactly equivalent between the two immunoglobulin classes. The pattern is particularly apparent at present for the VHIII subgroup which comprises approximately 70% of human alpha chains and only about 20% of human gamma chains.

Amino Acid Sequence↗

Initiation by methionine of mouse immunoglobulin light chain containing NH-2terminal pyroglutamic acid.

The mechanism of biosynthesis of NH2-terminal pyroglutamic acid has been studied in a mouse plasmacytoma (RPC-20) which produces an immunoglobulin light (lambda) chain containing NH2-terminal pyroglutamic acid. To this end, initation of lambda chain synthesis in plasmacytoma cell suspensions has been investigated. The analysis of radioactive lambda chain synthesis by these cells was accomplished with an antibody preparation specific for the precipitation of lambda chain protein from total plasmacytoma protein. NH2-terminal analysis of plasmacytoma cells labeled with [35S]methionine showed that the ratio of radioactivity in NH2-terminal methionine to total incorporation in lambda chain was greater at 2 min of labeling than at 60 min. However, such a pattern of transient labeling of the NH2 terminus of the lambda chain was not obtained when cells were incubated with tritiated leucine, arginine, or tryptophan. The data indicate that methionine is the initiator amino acid for the synthesis of lambda chain containing NH2-terminal pyroglutamic acid.

Amino Acid Sequence↗

[Production of rabbit precipitating antisera to subclasses of human IgG].

Precipitating antisera to human subclasses IgG were obtained by immunization of rabbits by whole molecules IgG2, IgG3, IgG4 and gamma 1-chains derived from IgG1H (Pr). Analysis of the antisera obtained demonstrated that rabbits produced specific antibodies to the antigenic subclass determinants IgG3 well, to IgG2, IgG4--much worse, and failed to produce specific antibodies to subclass IgG1 (in immunization with whole molecules of this protein). Antisera contained antibodies to the antigenic determinants common of IgG, and antibodies to light chains which were removed by immunosorption, for which purpose a sorbent on the basis of BrCN sepharose conjugated with IgG of the three other subclasses and Fab-fragment was used.

Animals↗

IgD myeloma protein with "unreactive" light chain determinants.

Serum from a patient with multiple myeloma showed a monoclonal protein, classified by immunoelectrophoresis as IgD. Immunofixation electrophoresis and immunoelectrophoresis failed to demonstrate a precipitation reaction between the paraprotein and antisera to immunoglobulin light chains. The light chains of the monoclonal protein, immunologically inaccessible in the intact molecule, reacted with anti-lambda chain antisera only after reduction and alkylation of the paraprotein. Moreover, interpretation of the immunoelectrophoretic patterns was hampered by the presence in patient's serum of free lambda chains having about the same mobility as that of the paraprotein.

Aged↗

[Contribution of new methods of quantitative cytology and immunology to the understanding of lymphoproliferative syndromes].

Seventy one patients with a lymphoid hemopathy, three with agammaglobulinemia and six normal controls were investigated with regard to their blood lymphocyte membrane-associated light chains. Detection and quantitation of antigenic determinants were performed by means of peroxidase-labeled antibodies. Compared to normal controls, values found in chronic lymphocytic leukemia (CLL) were very low (tenfold decrease). The number of antigenic determinants on lymphoid cells from patients with blast crises supervening in CLL, prolymphocytic leukemia, Waldenstrom's macroglobulinemia and Burkitt cell acute leukemia were significantly higher than those seen in patients with CLL. The data obtained in this investigation through quantitative immunocytology constitutes a new parameter for the classification of lymphoid hemopathies and for an approach to their pathogenesis, in particular if the quantity of membrane immunoglobulin correlates with the stage of cell maturation.

Antibodies↗

[Participation of rat immunoglobulin light chains of the kappa and lambda type in formation of antibodies to the polysaccharide of group A streptococcus].

The role of light kappa and lambda chains and also allelic variants of kappa chains of rat immunoglobins in the formation of antibodies to beta-N-acetyl-glucosamine polysaccharides of streptococcus group A of inbred rat strains MSU, WAG, August and hybrids of the first generation (MUS X WAG)F1 and (MSU X August)F1 was studied. From individual sera of immune rats fractions of specific antibodies to beta-N-acetyl-glucosamine were isolated. These antibodies differ in their affinity to antigenes. The retio of molecules with kappa and lambda light chain types was determined for the fraction of specific antibodies. The ratio of molecules kappa and lambda depends on the affinity of antibodies to beta-N-acetyl-glucosamine and on the genotype of the animals studied. Data obtained allow to conclude that differences in the functional activity of lambda chains between strains WAG and August, on one hand, and strain MSU, on the other, do exist. Functional differences releaved between these rat strains were confirmed by analyzing corresponding antibody fractions to beta-N-acetylglucosamine in F1 hybrids. Differences between allelic variants of kappa chains in the formation of antibodies to beta-N-acetylglucosamine of polysaccharides were not found.

Acetylglucosamine↗

Cryoglobulinemia and amyloidosis associated with intestinal lymphoma.

A case of lymphocytic lymphoma of the small intestine was associated with cryoglobulinemia and amyloidosis. The neoplastic lymphoid cell demonstrated surface IgG membrane markers by immunofluorescence and immunoelectron microscopy. The cryoglobulins were characterized as monoclonal IgG3 proteins with lambda light chains. Amyloidosis of the small intestine and regional lymph nodes was found in association with lymphatic infiltration of these organs, suggesting the production of amyloid in situ.

Aged↗

Immunohistochemical characterization of Burkitt's lymphoma.

Cytoplasmic immunoglobulins and muramidase (lysozyme) were demonstrated in formalin-fixed tissues by an immunoperoxidase procedure in 3 cases of Burkitt's lymphoma. The Burkitt cells were strongly positive with the full panel of monospecific antisera against human immunoglobulin components (kappa and lambda light chains, gamma, alpha and micron heavy chains). The 'starry-sky' macrophages were weakly positive with antimuramidase antiserum and strongly positive with the antisera against immunoglobulins, thus demonstrating their phagocytic and histiocytic nature. The reasons for the polyclonal increase in immunoglobulins are discussed.

B-Lymphocytes↗

Normal values for free light chains in serum different age groups.

The concentration of free light chains from the immunoglobulins was measured in twelve paired sera from mothers and newborns and from 149 sera from normal individuals in various age groups. Variations in concentration during life are correlated to the variations in the concentration of 'regular' immunoglobulins. A concentration of light chains in cord blood of 35% of mean normal adult level (MNA) together with a rapid passage of light chains across the placenta is interpreted as indicating catabolization of maternal light chains in the fetus. This is further supported by the finding of a lower concentration of light chains in maternal serum than in normal adult serum. The investigation shows that the concentration of light chains falls rapidly from 35 to 24% of MNA during the first few days of life. From the first week of life the concentration of light chains increases and low normal adult values are attained by one year of age. Except for difference in concentration, the elution pattern for light chains from Sephadex G-100 columns was similar for normal, adult and cord blood. The relationship between kappa and lambda chains--the K/L ratio--is 1.2 for normal and maternal serum and 1.0 for cord serum.

Adolescent↗

[Determination of the subclasses of monoclonal human immunoglobulins G and of the light chain type with the aid of specific antisera].

Sera of 86 patients suffering from G-myeloma were studied for the purpose of determination of subclasses of monoclone IgG. Investigations were carried out by means of antisera to subclasses IgG by the double diffusion method in gel after Ouchterlony. The following distribution of myeloma Ig was revealed: G1--70%, G2--17%, G3--11%,and G4--2%. In typing of the light igG chains by the method of immunoelectrophoresis, using antisera to the light chains of immunoglobulins of the chi and lambda type it was found that IgG1 chi was encountered more frequently than IgG1 lambda (3:1 ratio). The amount of the sera with the IgG2, IgG3, and IgG4 was insufficient for the reliable conclusion of their distribution by the type of light chains.

Humans↗

[Rule of antibody structure. Primary structure of a human monoclonal IgAl-immunoglobulin (myeloma protein Tro). VI. Amino acid sequence of the L-chain, lambda-type, subgroup II].

The primary structure of the L-chain of an IgA1-immunoglobulin (Myeloma protein Tro) has been determined by means of cleavage with trypsin and, if necessary, with alpha-chymotrypsin. The tryptic peptides of the variable part were characterized by amino acid analysis, Dansyl-Edman degradation and cleavage with carboxypeptidase; the peptides of the constant part were identified by amino acid analyses and determination of its N- and C-terminal residues. The sequence of the remaining amino acids and the arrangement of the peptides were established in homology to known structures. The protein comprises 216 amino acids. The homology of the variable part clearly characterizes it as belonging to subgroup II of lambda-chains. In positions 27a, b and c, there are the subgroup-specific additional residues and in position 96 is the characteristic deletion. The constant part of the chain is Kern- and Oz- which indicates that it has serine in position 154 and arginine in position 191.

Amino Acid Sequence↗