Kappa Family Research Articles

Qualitative and quantitative analysis of carrageenan content in gametophytes of Mastocarpus stellatus (Stackhouse) Guiry along Galician coast (NW Spain)

Qualitative and quantitative differences in carrageenan composition of gametophytes of the rhodophyte Mastocarpus stellatus (Gigartinales) were observed in this study. Carrageenans in gametophytes belong to the kappa family (κ-, ι-, ν-, μ-carrageenan). The dominant fractions were κ- and ι-carrageenan (more than 80 % of the total carrageenans). Mean total carrageenan content in gametophytes was of 37.32 ± 1.21 % DW. Spatial and seasonal variations were observed, mainly related to changes on environmental and oceanographic factors and the role of carrageenans in adapting the fronds to these changes. Maximum values in carrageenan content were observed for San Roman (Biscay Bay) in May and for Laxe and Mougas (Atlantic coast) in June. The results of this study indicated that spatial differences in carrageenan content were due to interactions of different factors, rather than the effect of a single factor. Fronds from San Roman had higher carrageenan content (43.23 ± 1.87 % DW) than those collected at two sites of the Atlantic coast, Mougas and Laxe (32.20 ± 1.14 % DW). San Roman is exposed to the open sea, windy and oriented to the north, and the water temperature is higher in summer than in the Atlantic coast. However, seasonal variations in carrageenan content resulted to be more related to other factors directly correlated with the input of energy in the ecosystems (irradiance, sunshine hours and insolation). Thus, carrageenan content began to increase in early spring when the number of sunlight hours increased. Maximum values were reached in late spring or early summer, just before maximum values of irradiance and air temperature were achieved.

Spatial and temporal variations of Chondrus crispus (Gigartinaceae, Rhodophyta) carrageenan content in natural populations from Galicia (NW Spain)

Qualitative and quantitative differences in carrageenan composition of gametophytes and tetrasporophytes of Chondrus crispus were observed in this study. Carrageenans in gametophytes belong to the kappa family (κ-, ι-, ν- and μ-carrageenan). The dominant fractions were κ- and ι-carrageenan (more than 50% of the total carrageenans). In tetrasporophytes, the presence of λ-carrageenan was confirmed. Carrageenan content in gametophytes (37.4 ± 1.68% DW) was higher than in tetrasporophytes (29.13 ± 0.76% DW). Spatial and temporal variation in carrageenan content in both life cycle phases appears to be related mainly to seawater and air temperatures, insolation, water movement and desiccation. The highest values of carrageenan content were recorded in those localities where higher values of precipitation, wind speed or water movement occurred. A bimodal temporal pattern on carrageenan content was observed. Fronds showed a high carrageenan content in spring and autumn. During these seasons, the content was over 40% in gametophytes and 30% in the tetrasporophytes. In summer and winter, these values down in both life cycle phases below 30%. In general the highest carrageenan contents were related to highest seawater temperatures. On the contrary, high air temperature and high insolation appeared to be unfavourable for carrageenan production. GLM models were obtained to predict carrageenan production from natural C. crispus populations, along Galician coast.

Producing the recombinant CTB as a delivery/targeting vehicle

A recombinant single chain Fv (scFv) fragment with specific activity against platelet glycoprotein (GP) Ibα was developed and characterized. The scFv was generated from the SZ-2 hybridoma, which produced an anti-platelet antibody reactive to GPIbα. VH and VL gene segments were generated from the SZ-2 hybridoma by reverse transcribed-polymerase chain reaction (RT-PCR). After cloning into pUCm-T vector, the DNA sequences of both VH and VL genes were analyzed from two different clones, respectively, the same results were obtained. Comparison of SZ-2 variable region to the Kabat database showed that VH belonged to the mouse Ig heavy family XV while VL belonged to the mouse Ig kappa family XXVI. For assembly of the SZ-2 scFv, VH and VL fragments were cloned into pSW1-scFv successively. The scFv was arranged in VH–VL orientation, being joined together with a 15-amino-acid (Gly4Ser)3 linker. The scFv encoding sequence was amplified and cloned into pET22b vector in-frame with a pel B leader sequence to direct secretion of the protein. Escherichia coli strain BL-21(DE3)PlysS was transformed with the recombinant plasmid, and expression of the scFv was induced using isopropyl-β-d-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 31,000. By comparing band intensity on a Coomassie brilliant blue-stained SDS-PAGE, the production yield of SZ-2 scFv was about 25% of the total cellular proteins. The recombinant SZ-2 scFv antibody was successfully purified using Ni-NTA affinity chromatography with a yield of 120 mg/l. The SZ-2 scFv antibody could bind to platelets demonstrated by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Analyzed by Western blot, it could bind to platelet GPIb. It retained the binding capacity of its parental SZ-2 monoclonal antibody (MoAb). In functional studies, SZ-2 scFv inhibited platelet agglutination and aggregation induced by ristocetin and thrombin, respectively, but had no effect on ADP-induced platelet aggregation. Therefore, SZ-2 scFv has the potential to be used as an antithrombotic agent.

Genomic structure and expression analysis of the RNase κ family ortholog gene in the insect Ceratitis capitata

Cc RNase is the founding member of the recently identified RNase kappa family, which is represented by a single ortholog in a wide range of animal taxonomic groups. Although the precise biological role of this protein is still unknown, it has been shown that the recombinant proteins isolated so far from the insect Ceratitis capitata and from human exhibit ribonucleolytic activity. In this work, we report the genomic organization and molecular evolution of the RNase kappa gene from various animal species, as well as expression analysis of the ortholog gene in C. capitata. The high degree of amino acid sequence similarity, in combination with the fact that exon sizes and intronic positions are extremely conserved among RNase kappa orthologs in 15 diverse genomes from sea anemone to human, imply a very significant biological function for this enzyme. In C. capitata, two forms of RNase kappa mRNA (0.9 and 1.5 kb) with various lengths of 3' UTR were identified as alternative products of a single gene, resulting from the use of different polyadenylation signals. Both transcripts are expressed in all insect tissues and developmental stages. Sequence analysis of the extended region of the longer transcript revealed the existence of three mRNA instability motifs (AUUUA) and five poly(U) tracts, whose functional importance in RNase kappa mRNA decay remains to be explored.

Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ibα

A recombinant single chain Fv (scFv) fragment with specific activity against platelet glycoprotein (GP) Ibα was developed and characterized. The scFv was generated from the SZ-2 hybridoma, which produced an anti-platelet antibody reactive to GPIbα. VH and VL gene segments were generated from the SZ-2 hybridoma by reverse transcribed-polymerase chain reaction (RT-PCR). After cloning into pUCm-T vector, the DNA sequences of both VH and VL genes were analyzed from two different clones, respectively, the same results were obtained. Comparison of SZ-2 variable region to the Kabat database showed that VH belonged to the mouse Ig heavy family XV while VL belonged to the mouse Ig kappa family XXVI. For assembly of the SZ-2 scFv, VH and VL fragments were cloned into pSW1-scFv successively. The scFv was arranged in VH–VL orientation, being joined together with a 15-amino-acid (Gly 4Ser) 3 linker. The scFv encoding sequence was amplified and cloned into pET22b vector in-frame with a pel B leader sequence to direct secretion of the protein. Escherichia coli strain BL-21(DE3)PlysS was transformed with the recombinant plasmid, and expression of the scFv was induced using isopropyl-β- d-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 31,000. By comparing band intensity on a Coomassie brilliant blue-stained SDS-PAGE, the production yield of SZ-2 scFv was about 25% of the total cellular proteins. The recombinant SZ-2 scFv antibody was successfully purified using Ni-NTA affinity chromatography with a yield of 120 mg/l. The SZ-2 scFv antibody could bind to platelets demonstrated by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Analyzed by Western blot, it could bind to platelet GPIb. It retained the binding capacity of its parental SZ-2 monoclonal antibody (MoAb). In functional studies, SZ-2 scFv inhibited platelet agglutination and aggregation induced by ristocetin and thrombin, respectively, but had no effect on ADP-induced platelet aggregation. Therefore, SZ-2 scFv has the potential to be used as an antithrombotic agent.

CARRAGEENANS BIOSYNTHESIZED BY CARPOSPOROPHYTES OF RED SEAWEEDS GIGARTINA SKOTTSBERGII (GIGARTINACEAE) AND GYMNOGONGRUS TORULOSUS (PHYLLOPHORACEAE)1

Carrageenans biosynthesized by gametophytic and tetrasporic plants of seaweeds belonging to the Gigartinaceae and Phyllophoraceae are different: gametophytes produce carrageenans of the kappa family, whereas lambda‐carrageenans are extracted from tetrasporophytes. For Gigartina skottsbergii Setchell and Gardner and Gymnogongrus torulosus Hooker et Harvey, mature cystocarps were isolated and carrageenans were extracted. Structural determination by methylation analysis, Fourier transform infrared spectroscopy, and 13C‐NMR spectroscopy showed that they were kappa/iota‐carrageenans. For the extract obtained from cystocarps of Gigartina skottsbergii with water at room temperature, the ratio kappa:iota was 1:0.30 and at 90° C was 1:0.43; significant amounts of precursors were also present. The extract obtained from cystocarps of Gymnogongrus torulosus at 90° C showed prevalence of iota‐carrageenans (ratio kappa:iota 1:1.21). These extracts are similar to the polysaccharides produced by gametophytes of these seaweeds. For Gigartina skottsbergii, it was possible to separate the pericarpic tissue from the carposporophyte. Thus, they were extracted separately, and the carrageenans isolated were studied as described before, obtaining similar conclusions. These results clearly show that whereas the carposporophytes are located inside the cystocarp, they produce carrageenans of the kappa family despite of being diploid cells.

Sequence analysis suggests the presence of an IG-like domain in the N-terminal region of alpha-dystroglycan which was crystallized after mutation of a protease susceptible site (Arg168-->His).

Recently, we demonstrated that the N-terminal region of mouse alpha-dystroglycan represents an autonomously folding globular domain, organized into at least two subdomains (Brancaccio et al., Eur. J. Biochem. 246, 166-172, 1997). We have now found a similarity between a part of the alpha-dystroglycan N-terminal sequence (approximately from position 80 to 180) and several protein sequences belonging to the immunoglobulin kappa family. Moreover, we have recombinantly expressed and purified a 31 kDa protein fragment which matches the entire alpha-dystroglycan N-terminal globular domain. To prevent the action of bacterial endogenous proteases and/or thrombin, which cleaves the protein into two fragments at an Arg-Ala trypsin-sensitive site in positions 168-169, we have introduced a single mutation (Arg168-->His), thus making the whole domain more stable and suitable for crystallization. Crystals of this mutant protein were obtained by vapor diffusion using the hanging drop technique, and they diffract to 0.28 nm Bragg spacing.

Molecular cloning of the complementary DNA for an additional member of the family of aortic aneurysm antigenic proteins.

We have purified and partially sequenced a protein from the adventitia of the human aorta (aortic aneurysm antigenic protein 40 kDa; AAAP-40) that has homologies to bovine aortic microfibril-associated glycoprotein (MAGP-36). It is immunoreactive with immunoglobulin G (IgGs) purified from the serum and aortic wall of patients with abdominal aortic aneurysms. AAAP-40 and MAGP-36 have fibrinogen-like and vitronectin-like motifs. Screening an expression library constructed from human aortic adventitial messenger RNA has resulted in the cloning of three complementary DNAs whose gene products are immunoreactive with immunoglobulin G from patients with abdominal aortic aneurysms. Two strongly resemble each other and have been described separately. The purpose of this article is to report the third clone. Messenger RNA from a specimen of human aortic aneurysmal adventitia was reverse-transcribed for insertion into the phagemid Uni Zap XR (Stratagene). A strain of Escherichia coli, engineered for expression (XL 1-Blue MFR', Stratagene), was transfected, and rabbit antihuman vitronectin antibody as used to identify positive clones. Sequencing of the positive clones was performed by the Core Laboratories at Columbia University. The hypothetical protein of rAAAP-CL4 (clone 4) shares sequence motifs with known microfibril-associated glycoproteins (MAGPs). The recombinant protein (rAAAP-CL4) is immunoreactive with serum from patients (three of four abdominal aortic aneurysm sera). In addition, similarities have been detected with immunoglobulins of the kappa family and with a protein from cytomegalovirus that is a potential molecular mimic. There may several members of a novel family of human aortic autoantigenic proteins implicated in abdominal aortic aneurysm disease.

Analysis of the kappa light chain variable region in multiple myeloma.

The study of immunoglobulin heavy chain gene rearrangements in multiple myeloma has revealed extensive divergence from the germline sequences, but no intraclonal diversity with disease evolution. Our study investigated the state of the rearranged kappa light chain variable region (V kappa) gene segments as well as abortive V kappa family gene usage in cases of multiple myeloma expressing lambda light chain. We studied 11 cases of kappa and five cases of lambda light chain-expressing multiple myeloma. Total cellular RNA was extracted from the bone marrow of patients with overt disease and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to amplify clonally rearranged variable region sequences. Direct nucleotide sequencing by the dideoxy-chain termination method was performed on the RT-PCR products. We did not observe preferential usage of certain V kappa gene families. Mutation frequencies of the V kappa segments varied in number. In the majority of cases, extensive somatic mutations occurred within the complementarity determining regions (CDRs) of V kappa, whereas only a limited degree of divergence from the germline was observed in others. In all cases studied. replacement mutations tended to cluster in the CDRs, a finding compatible with an antigen-driven somatic hypermutation process. In 3/5 cases of lambda light-chain expressing multiple myeloma, abortively rearranged V kappa gene segments were amplified from genomic DNA; in two cases a non-templated nucleotide insertion rendering the V kappa sequences out-of-frame was observed, and in the third a stop codon was identified in the open reading frame of the V kappa sequence. Somatic mutations were observed in all cases of abortive V kappa genes studied; however, their distribution does not suggest selection by antigen. We conclude that somatic mutations observed in the V kappa regions of myeloma cells are of variable extent and suggest operation of the antigen selection process. Lack of or minimal somatic hypermutation in a few cases may be in some way implicated in the biological heterogeneity of the disease.

Ig V kappa family repertoire of plasma cells derived from autoimmune MRL mice.

V kappa gene family usage was determined in the resident in vivo-activated plasma cells of individual diseased MRL mice by using in situ hybridization. In this way, the entire autoimmune repertoire could be analyzed. Autoantibody levels and extent of glomerulonephritis were also measured, so that the severity of disease could be assessed. It was found that V kappa expression was highly variable from mouse to mouse. Some animals displayed a V kappa family repertoire similar to mitogen-stimulated cells and consistent with the size of the families. These animals tended to have lower disease indices. Other animals, which had higher disease indices, displayed considerable over- or underutilization of individual V kappa families. However, no particular V kappa families were repeatedly biased in their expression, as was found at the VH level with J558. Importantly, in the 10% of animals that expressed VH J558 exclusively, four or more V kappa families were expressed and multiple antiself specificities were produced. The data are most consistent with a number of J558 genes being expanded in a variety of self-specificities. However, because only VH J558 is expressed in these sicker animals, nonspecific polyclonal activation is highly unlikely. These results underscore the continuing evolution of the autoimmune repertoire, with considerable diversity at early stages followed by a highly selected repertoire in which a potential role for nonspecific polyclonal activation is virtually excluded.

Identification of a new V kappa gene family that is highly expressed in hybridomas from an autoimmune mouse strain.

We have identified a new murine V kappa family that contains five to seven members, one member of which encodes the L chain V region of an anti-dsDNA antibody produced by a BALB/c hybridoma, C8.5. The cloned C8.5 V kappa gene exhibits highest homology with a human V kappa gene that was cloned from a nonproductive rearrangement but has never been seen in an expressed repertoire. Because this family was first identified in an autoantibody, we studied its expression in an autoimmune mouse strain. This V kappa family is expressed in 20% of hybridomas from NZB mice.

The Mouse Immunoglobulin Kappa Light-Chain Genes Are Located in Early- and Late-Replicating Regions of Chromosome 6

The murine immunoglobulin kappa (kappa) light-chain multigene family includes the constant region (C kappa), joining-region genes, and approximately 30 kappa-variable (V kappa) region families. The entire region occupies an estimated 1,000 to 3,000 kilobases, and some V kappa families have been linked by recombinant inbred mapping. The C kappa gene and 14 V kappa families replicated differently among cell lines of lymphoid and nonlymphoid origin. In nonlymphoid cells, the C kappa gene replicated earlier than the V kappa families. A transition from replication during the second third of S phase for the C kappa gene to later replication during S for V kappa families was observed. The V kappa family (V kappa 21) that maps closest to the C kappa gene, replicated during the first half of the S phase; most of the other V kappa families replicated during the second half of S, and some replicated during the last quarter of the S phase. In lymphoid cells, the kappa locus replicated earlier in the pre-B than in the B-cell lines. In one pre-B-cell line, 22D6, the kappa genes examined replicated at the beginning of the S phase. In the B-cell lines, the EcoRI segment containing the transcribed gene replicated near the beginning of the S phase. Other V kappa families replicated within the first two-thirds of S phase. Some linked V kappa families replicated at similar times. In the B-cell lines, a transition from replication at the beginning of S for the transcribed C kappa and V kappa genes and surrounding DNA sequences to later replication for the other V kappa families was observed. However, in contrast to the non-lymphoid cell lines, the replication of this locus occurred predominantly during the first half of S. The kappa locus contains both early- and late-replicating genes, and early replication is usually associated with transcriptional activity. The results are discussed with respect to the organization of transcriptionally active chromatin domains.

The mouse immunoglobulin kappa light-chain genes are located in early- and late-replicating regions of chromosome 6.

The murine immunoglobulin kappa (kappa) light-chain multigene family includes the constant region (C kappa), joining-region genes, and approximately 30 kappa-variable (V kappa) region families. The entire region occupies an estimated 1,000 to 3,000 kilobases, and some V kappa families have been linked by recombinant inbred mapping. The C kappa gene and 14 V kappa families replicated differently among cell lines of lymphoid and nonlymphoid origin. In nonlymphoid cells, the C kappa gene replicated earlier than the V kappa families. A transition from replication during the second third of S phase for the C kappa gene to later replication during S for V kappa families was observed. The V kappa family (V kappa 21) that maps closest to the C kappa gene, replicated during the first half of the S phase; most of the other V kappa families replicated during the second half of S, and some replicated during the last quarter of the S phase. In lymphoid cells, the kappa locus replicated earlier in the pre-B than in the B-cell lines. In one pre-B-cell line, 22D6, the kappa genes examined replicated at the beginning of the S phase. In the B-cell lines, the EcoRI segment containing the transcribed gene replicated near the beginning of the S phase. Other V kappa families replicated within the first two-thirds of S phase. Some linked V kappa families replicated at similar times. In the B-cell lines, a transition from replication at the beginning of S for the transcribed C kappa and V kappa genes and surrounding DNA sequences to later replication for the other V kappa families was observed. However, in contrast to the non-lymphoid cell lines, the replication of this locus occurred predominantly during the first half of S. The kappa locus contains both early- and late-replicating genes, and early replication is usually associated with transcriptional activity. The results are discussed with respect to the organization of transcriptionally active chromatin domains.

Stochastic pairing of heavy-chain and kappa light-chain variable gene families occurs in polyclonally activated B cells.

Frequencies of 25 immunoglobulin heavy-chain and kappa light-chain variable (VH + V kappa) gene-family pairings expressed in splenic B-cell populations were determined by hybridization of VH- and V kappa-family-specific DNA probes to mitogen-induced B-cell colonies from C57BL/6 mice or hybridomas derived from BALB/c and NZB mice. Both analyses support the conclusion that VH and V kappa gene families pair without bias; as would be expected for random association, the frequencies of specific VH + V kappa pairs may be estimated by the product of the independent VH and V kappa frequencies. Based upon the frequencies at which 9 VH and 12 V kappa gene families are expressed, we calculated the expected usage for approximately 100 VH + V kappa family pairings in neonatal and adult C57BL/6 mice. Variability in the expression of such VH + V kappa pairings is considerable; pairs representing greater than 10% to less than 0.01% of the splenic B-cell population occur. This variability is most pronounced in the neonate, where 6 VH + V kappa family pairs account for nearly 40% of all mitogen-reactive B cells. As the neonate matures, the distribution of frequencies for VH + V kappa pairings becomes more nearly uniform. This process may underlie the patterned acquisition of humoral immune responsiveness.

Reassessment of opioid binding sites in the rat brain

Opioid binding sites have been characterized pharmacologically in membranes from different areas of the rat brain. Delta,mu and sites belonging to the kappa family (K1, K2, K3) have been detected. Delta sites were more abundant in cortex and striatum,mu sites in striatum and hypothalamus, while kappa binding site concentration was higher in deeper enkephalic structures (brainstem, cerebellum, hypothalamus) and the pituitary gland. A distinct distribution of each subtype of the kappa site was found: kappa 1 sites were higher in the spinal cord, kappa 2 sites in the brainstem and kappa 3 sites in cerebellum. The distribution of delta and kappa sites in the central nervous system was correlated with the distribution of proenkephalin-A derived peptides and precursors, suggesting that these peptides could be their endogenous ligands.

A Look at the Burr and Related Distributions

Summary The Burr distribution (Burr type XII), which yields a wide range of values of skewness and kurtosis, can be used to fit almost any given set of unimodal data. The Burr distribution has appeared in the literature under different names. The relationship between the Burr distribution and the various other distributions, namely, the Lomax, the Compound Weibull, the Weibull-Exponential, the logistic, the log logistic, the Weibull and the Kappa family of distributions is summarized. Also it is shown that the 'reciprocal Burr' distribution (Burr type III) covers a wider region in the (V,f fi2) plane and includes all the region covered by the Burr type XII distribution.