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Biomedical subjects

L Pilström

Publications and source records attributed to L Pilström.

At least 19 recordsLinked to original sources
PubMed2001

Immune parameters of immunised cod (Gadus morhua L.).

The immune response of cod (Gadus morhua L.) is unusual in that specific antibody response is limited or absent. In the present study cod was immunised with haptenated and non-haptenated protein antigen at two different temperatures and the antibody response monitored over a period of 18 months. Other humoral parameters of immunological importance were also analysed, namely total immunoglobulin concentration, anti-protease and spontaneous haemolytic activity. No specific antibody response was detected but increased activity of non-specific anti-TNP antibodies was observed 10-12 weeks after immunisation, irrespective of the antigen used. This antibody activity was attributed to the adjuvant used (FCA) and did not cross react with other antigens tested. Other parameters were probably not influenced by the immunisation but seasonal fluctuations were indicated. The immunoglobulin level appeared to peak in August-September and the anti-protease activity and the haemolytic activity in October-January.

Animals↗
PubMed2001

T-cell antigen receptors in Atlantic cod (Gadus morhua l.): structure, organisation and expression of TCR alpha and beta genes.

By using short degenerate primers complementing conserved T-cell antigen receptor (TCR) variable and constant region segments for PCR, we were able to isolate putative TCRalpha and beta chain full length cDNAs in Atlantic cod. The Valpha and Vbeta domains have the canonical features of known teleost and mammalian TCR V domains, including conserved residues in the beginning of FR2 and at the end of FR3. The Jalpha and Jbeta region possess the conserved Phe-Gly-X-Gly motif found in nearly all TCR and immunoglobulin light chain J regions. Similar to other vertebrates, the Atlantic cod Calpha and Cbeta sequences exhibit distinct immunoglobulin, connecting peptide, transmembrane and cytoplasmic regions. The Atlantic cod Cbeta sequence lacks a cysteine in its connecting peptide region, but other motifs proposed to be important for dimerisation and cell surface expression are observed. Four different cod Cbeta sequences were identified, two of which share 3' untranslated regions different from one of the other two sequences, suggesting the existence of isotypic gene variants of Cbeta. Based on Southern blot analyses, the TCRalpha and beta gene loci appear to be arranged in translocon organisation (as opposed to multicluster) with multiple V gene segments, some (D) and J gene segments and a single or few C gene segments. Northern blot analyses show expression of the TCRalpha and beta chains in thymus, spleen and head kidney, expression of the TCRbeta chain was also detected in the ovary. Interestingly, no expression was detected in intestine even though the existence of T-cells in intestine has been proposed in other teleost species.

Amino Acid Sequence↗
PubMed2000

Transcriptional enhancers of immunoglobulin light chain genes in Atlantic cod (Gadus morhua).

The organization of immunoglobulin heavy (H) chain genes in teleosts resembles that of mammals and amphibians, whereas light (L) chain genes are arranged in multiple clusters of variable (VL), joining (JL), and constant (CL) region segments. Sequence analysis of two Atlantic cod genomic clones (14,966 and 13,116 bp in length) revealed a very compact IgL chain locus with the VL genes in opposite transcriptional orientation to the JL and the CL genes. This suggests the possibility of rearrangements between clusters by inversion. Each cluster spans approximately 2.1 kb and distances between clusters vary between 2.1 and 4.8 kb. To gain insight into the transcriptional regulation of this complex, multiclustered locus, chloramphenicol acetyl transferase reporter constructs containing 14 different DNA segments from the two genomic clones were transfected into channel catfish B and non-B-cell lines, as well as into mouse B-cell lines. These studies showed strong enhancer activity downstream of the CL region in three out of six L chain gene clusters when assayed in fish, but not in mouse B cells. Interestingly, both mouse and human lambda enhancers exhibited strong activity in the fish B cells, while the mouse 3' kappa enhancer did not. This suggests that transcription factors similar to those involved in mammalian lambda expression are present in B cells from teleosts.

Animals↗
PubMed2000

Variable region diversity of the Atlantic cod (Gadus morhua L.) immunoglobulin heavy chain.

This study was undertaken to determine whether a lack of VH domain diversity could explain, in part, the failure of Atlantic cod to respond to immunization with the production of specific antibodies. The variability of cod VH regions was studied in 113 cDNA and 2 genomic clones. A fourth VH family and a second putative JH element were identified. The expressed VH repertoire showed a clear bias in the pattern of VH family utilization, with about 80% of the clones belonging to the VH-III family. Furthermore, the VH-III family was complex and could be subdivided into several subfamilies, while little variation was seen within the other families. The VH family bias gives a somewhat reduced variability of the VH gene region of cod, but not lower than that of the rabbit IgM repertoire. The H chain CDR3 region of cod was longer than that of trout, frog and mouse, and also highly variable in sequence, probably reflecting a relative importance of this region in cod. On the other hand, the CDR3 length variability was restricted, and this may reduce the diversity of the cod VH region.

Amino Acid Sequence↗
PubMed2000

Different genomic organization and expression of immunoglobulin light-chain isotypes in the rainbow trout.

cDNA studies have distinguished two isotypes of the rainbow trout (Oncorhynchus mykiss) immunoglobulin (Ig) light chain (designated L1 and L2). This study characterized genomic clones of these isotypes. L1 genes are arranged in clusters with single copies of variable (V), joining (J), and constant (C) segments. The transcriptional orientation of the V genes is opposite to that of the J and C segments, indicating that the V genes must be rearranged by inversion. L2 is also organized in clusters, consisting of two or three V, one J, and one C exon, all in the same transcriptional orientation. L1 and L2 of rainbow trout are similar to the previously identified cod and catfish clusters. Repeat sequences were found upstream of each J segment in the L2 genes, each of which includes a 16-bp sequence similar to the conserved kappa sequence motifs of mammalian Jkappa1 genes. Sequence analyses showed that the regions upstream of L1 and L2 genes have several putative cis-acting elements also present in the promoter regions of Ig genes of other organisms. Octamer motifs, a TATA box, and an E-box were found in the 5' region of an IgL1V gene. A kappa-Y element, a CCCT element, a TATA box, an E-box but no classical octamer were found in the 5' region of the IgL2 gene. Northern blot analyses showed that L1 and L2 are expressed in spleen, head kidney, excretory kidney, thymus, and heart. The expressed ratio of L1 and L2 is estimated to 85:15% in blood and lymphoid tissues.

Amino Acid Sequence↗
PubMed2000

Evolution of adaptive immunity.

Antigen receptors and major histocompatibility molecules, key elements required for adaptive immunity, are first seen in jawed fish. So, how did they evolve and have they changed?

Animals↗
PubMed1999

TGF-beta3 exists in bony fish.

A partial nucleotide sequence of transforming growth factor-beta3 (TGF-beta3) has been isolated from the Siberian sturgeon (Acipenser baeri), rainbow trout (Oncorhynchus mykiss) and European eel (Anguilla anguilla), confirming a ubiquitous presence in the ray-finned (Actinopterygian) bony fish. The bony fish TGF-beta3 is highly conserved, with some 83-84% nucleotide identity (coding region) and 90-95% predicted amino acid identity to known homeotherm TGF-beta3's. Far lower homologies are apparent with other known TGF-beta isoforms in fish (e.g. 64-66% and 81-82% amino acid identity to trout TGF-beta 1/5 and carp TGF-beta2 respectively). Phylogenetic tree analysis showed that the fish TGF-beta3's clustered with the known homeotherm TGF-beta3's. The relatively tight clustering of TGF-beta1, TGF-beta2 and TGF-beta3 was in contrast to the TGF-beta5's, which are clearly a more heterogenous group.

Amino Acid Sequence↗
PubMed1999

Characterization of MHC class I and beta(2)-microglobulin sequences in Atlantic cod reveals an unusually high number of expressed class I genes.

Degenerate polymerase chain reaction (PCR) primers based on conserved residues from alignments of species with already characterized major histocompatibility complex (MHC)-encoded sequences were used in the search for class I and beta(2)-microglobulin (b(2)m) genes in Atlantic cod (Gadus morhua L. ). After PCR amplification and subsequent sequencing a putative class I sequence was identified, from which a probe was designed and used to screen a spleen cDNA library from one single individual. The full-length clone obtained was sequenced and shown to be a classical Mhc class I-encoded sequence. It revealed the characteristic alpha1-, alpha2-, and alpha3-domains and transmembrane and cytoplasmic region, with several conserved amino acids. A PCR amplification from the alpha2-domain to the CY-region was performed on the same library, using a proof-reading enzyme. At least 11 unique additional sequences were isolated. Moreover, sequencing of the additional cDNA clones resulted in a total of 17 different Mhc class I sequences in this individual. A Southern hybridization of DNA from four different individuals using an alpha3-specific probe confirmed this large number of genes. Interestingly, based on differences mainly in their transmembrane region, the sequences obtained could be divided into two distinct groups. Within the groups no support could be obtained for any further subdivision. Southern experiments using an alpha1-specific probe gave almost the same restriction fragment length polymorphism with a high number of hybridizing bands, suggesting a low divergence in this part of the gene. Sequencing of PCR clones obtained with a proof-reading enzyme confirmed this at the nucleotide level. PCR amplification to isolate and characterize the b(2)m gene resulted in a sequence which was used to screen a thymus cDNA library. Two different alleles were obtained and these showed the characteristic features of known teleostean beta(2)m sequences. A Southern hybridization with genomic DNA from four different individuals suggested the presence of one b(2)m locus in Atlantic cod.

Amino Acid Sequence↗
PubMed1999

Light chain variable region diversity in Atlantic cod (Gadus morhua L.).

This study was undertaken to determine if a lack of V(L) domain variability could explain, in part, the failure of Atlantic cod to respond to immunization with the production of specific antibodies. The variability of cod V(L) regions was studied in 33 cDNA and two genomic clones. The variability of the CDRs was estimated by the Shannon entropy method and compared with that in other species. It was found to be lowest in the little skate (Raja erinacea), higher in cod, and highest in Xenopus and mouse. While the variability of the CDRs is slightly lower in cod than in Xenopus and mouse, it is spread over broader areas of the amino acid sequence. The length of CDR1 and CDR3 in cod is equal to or exceeds that found in skate, Xenopus, chicken and mammals. Isoelectric points and hydrophobicity vary substantially among the studied Ig light chain domains. Thus, neither the length, nor the variability, nor the physicochemical properties (pI and hydrophobicity) of the L chain CDRs can explain the absence of antibody response to immunization in cod.

Amino Acid Sequence↗
PubMed1999

Humoral immune parameters in Atlantic cod (Gadus morhua L.) I. The effects of environmental temperature.

The effects of environmental temperature on certain humoral immune parameters in Atlantic cod (Gadus morhua L.) were studied. Serum samples were collected from captive cod, of wild origin, kept at different temperatures for 12 months. It was found that immunoglobulin and natural antibody levels increased with increasing temperature whereas the total serum protein concentration, anti-protease activity, iron concentration, unsaturated and total iron binding capacity decreased with increasing temperature. Haemolytic activity and percentage iron saturation also tended to decrease with increasing temperature although this was not statistically significant.

Adaptation, Physiological↗
PubMed1999

Humoral immune parameters in Atlantic cod (Gadus morhua L.) II. The effects of size and gender under different environmental conditions.

The effects of size and gender on several humoral immune parameters in cod were examined under different environmental conditions. Serum samples were collected from wild cod of different sizes. Two samplings were undertaken: In the spring in relatively cold waters off the north west coast of Iceland and in the fall in relatively warm waters off the west coast of Iceland. Most of the parameters increased with increasing cod size, except the haemolytic activity which decreased. Higher serum protein levels were seen in cod sampled in the fall than in the spring. In cod sampled in the spring there was an apparent difference between specimens < 75 cm in length and the larger specimens with respect to haemolytic activity and iron concentration. None of the parameters were influenced by the gender of the cod.

Adaptation, Physiological↗
PubMed1999

Influence of the mu-chain C-terminal sequence on polymerization of immunoglobulin M.

Immunoglobulin (IgM) is found in various states of covalent polymerization (microL)n, where n is typically 8, 10, or 12. The usual form of IgM of bony fish is tetrameric (8 microL units) as compared to the pentameric form (10 microL units) observed in cartilaginous fish and mammals. Two hypotheses were tested in this study. First, that the length of the mu-chain C terminus following Cys575 determines whether an IgM polymerizes as a tetramer or as a pentamer. This was tested by examining the covalent polymerization state of mouse IgM mutated to contain a series of mu-chain C-termini from bony and cartilaginous fish. The results proved this hypothesis wrong: mouse IgM bearing the C-terminal sequence of shark, salmon and cod mu-chain behaved identically to native mouse IgM, forming predominantly (microL)10 and (microL)12 forms. The second hypothesis was that an additional Cys residue near the C terminus of the mu-chain is responsible for the multiple covalent structures seen in IgM of the channel catfish. The addition of a catfish C terminus to the mouse mu-chain resulted, as predicted, in the production of a series of covalently bonded forms, with the major species being (microL)4. When a Ser-Cys unit was removed from the catfish C terminus added to the mouse mu-chain, this resulted in production of IgM indistinguishable in structure from that of wild-type mouse IgM.

Animals↗
PubMed1998

Characterization of the gene for the membrane and secretory form of the IgM heavy-chain constant region gene (C mu) of the cow (Bos taurus).

Our present understanding of the evolution of immunoglobulins is derived from a few vertebrate species. In order to obtain additional information on the development of the humoral immune system, we cloned and determined the nucleotide sequence of the bovine cDNA and genomic IgM heavy-chain constant region gene (C mu). The gene contains four constant region domain-encoding exons (CH1 to CH4) and two exons encoding the transmembrane domain (TM1, TM2), expressed in the membrane-bound receptor form of the IgM. The sequence of a cDNA clone encoding the 3' portion of the membrane form of the mu-chain revealed that the TM1 exon is spliced to the CH4 exon, as occurs in other mammals. Comparison of deduced amino acid sequence data from different vertebrates revealed a high similarity to sheep C mu (88%) and a lower degree of similarity to pig (62%), rat (62%), rabbit (58%) human (56%), hamster (55%), mouse (54%), chicken (28%) and horned shark (22%) C mu.

Amino Acid Sequence↗
PubMed1998

The immunoglobulin light chain in poikilothermic vertebrates.

The immunoglobulin light chains are classified as kappa or lambda in mammals and birds (homeothermic vertebrates), but the traditional criteria for this classification are not applicable to the light chains found in poikilothermic vertebrates. Still it is possible to find some relationships between Ig light chain sequences in these animals and in those of the homeothermic animals. It is generally accepted that the Ig light chains contribute to the antigen binding capacity of antibodies and the variability is approximately similar in all studied vertebrate species except the elasmobranchs. This might be explained by the organisation of the Ig light chain locus in these animals and the fact that the variable and joining DNA segments are joined in the genome. These conclusions are limited by the small number of species studied in this respect.

Animals↗
PubMed1996

Ig light chain gene in the Siberian sturgeon (Acipenser baeri).

Elasmobranch and teleost fish have their Ig light (L) chain loci organized in multiple clusters (VL-JL-CL). The VL segments of teleosts are in opposite transcriptional orientation to the CL genes, suggesting that in teleosts and elasmobranchs there may have been separate evolutionary events leading to this organization. To address this problem, the IgL locus from the Siberian sturgeon (Acipenser baeri) (representative of a branch between elasmobranchs and teleosts) was investigated. Sequence analysis of cDNA clones shows that sturgeon VL genes are most similar to those of teleosts, but that sturgeon CL genes are more similar to those of the sharks. Southern blot analyses of sturgeon erythrocyte DNA with VL- and CL-specific probes showed that there are more than 20 VL segments in both the tetraploid Siberian sturgeon and the diploid sterlet (Acipenser ruthenus), but only a few CL segments in the genome of the Siberian sturgeon and up to four CL segments in that of the sterlet. Screening of an unamplified genomic library gave more than 300 VL-positive and four CL-positive clones. None of these contained inserts positive for both probes. PCR analysis of a genomic CL clone using IC and CL-specific primers suggested that upstream of the CL segment there are at least seven JL segments. it is concluded that sturgeons have a kappa-like organization of their IgL locus and that the clustered organization of IgL loci in bony fish and sharks arose from two distinct evolutionary events.

Amino Acid Sequence↗