Biomedical subjects

Nil Ratan Saha

Publications and source records attributed to Nil Ratan Saha.

6 recordsLinked to original sources

PubMed2005

Variable lymphocyte receptors in hagfish.

A previously uncharacterized type of variable lymphocyte receptors (VLR) was identified recently in the Sea lamprey. This jawless vertebrate generates an extensive VLR repertoire through differential insertion of neighboring diverse leucine-rich repeat (LRR) cassettes into an incomplete germ-line VLR gene. We report here VLR homologs from two additional lamprey species and the presence of two types of VLR genes in hagfish, the only other order of contemporary jawless vertebrates. As in the Sea lamprey, the incomplete hagfish germ-line VLR-A and -B genes are modified in lymphocyte-like cells to generate highly diverse repertoires of VLR-A and -B proteins via a presently undetermined mechanism. This jawless-fish mode of VLR diversification starkly contrasts with the rearrangement of Ig V(D)J gene segments used by all jawed vertebrates to produce diverse repertoires of T and B lymphocyte antigen receptors. The development of two very different strategies for receptor diversification at the dawn of vertebrate evolution approximately 500 million years ago attests to the fitness value of a lymphocyte-based system of anticipatory immunity.

Amino Acid Motifs↗

PubMed2005

Analysis and characterization of the expression of the secretory and membrane forms of IgM heavy chains in the pufferfish, Takifugu rubripes.

We investigated the structure and expression of immunoglobulin genes in the pufferfish, Takifugu rubripes, a highly prized and economically important fish species. The cDNA fragment that partially encodes the constant region of the IgM heavy chain was isolated in these animals by RACE using degenerate primers after which it was used as a probe for screening IgM heavy chains in a fugu splenic cDNA library. The structural feature of the constant region of fugu sIgM was found to consist of four constant domains (CH1 to CH4), while mIgM was shown to contain a deletion of the CH4 domain, and its transmembrane domain was directly spliced to the CH3 domain as found in other teleosts. This feature may be common to all teleosts. In addition, five VH genes isolated in this study fell into two families based on their variability. Analysis of genomic sequences from the fugu genomic database also showed that there are only two VH families in the genome. The IgM gene was preferentially expressed in presumptive lymphoid tissues. Moreover, in situ hybridization revealed that large numbers of IgM positive cells were widely distributed throughout the spleen, head kidney, kidney, and thymus, confirming that these tissues were major sites of antibody production in fish. The expressions of IgM in the mucosal organs such as the skin, gills, and intestine suggest that they, too, contribute to humoral immunity in aquatic animals. The expression of IgM mRNA in the early development stages of this fish suggests that its larval form possesses a protective defense mechanism against foreign invaders.

Animals↗

PubMed2004

Fugu immunoglobulin D: a highly unusual gene with unprecedented duplications in its constant region.

We have isolated and characterized the cDNA that encodes IgD of fugu (Takifugu rubripes). Though the splicing of micro1 with the delta1 domain was similar to those reported for teleost IgDs, highly unusual and unprecedented domain duplications were found in the constant region of the fugu IgD. The structure of the fugu IgD is like VDJ-micro1-(delta1-delta2-delta3-delta4-delta5-delta6)(2)-delta7-deltam1-deltam2. Genomic sequence analysis of the fugu IgD gene supported the results of cDNA sequencing that the first six delta domains in the constant region are duplicated. Such a novel duplication pattern has not been reported in any other vertebrates. However, IgD secretory domains could not be identified in this study. The deduced amino acid sequence of the fugu IgD constant region showed high identity (35-55%) to the sequences of previously reported teleost IgDs. Gene expression analyses based on RT-PCR demonstrated that the IgD gene is preferentially expressed in presumptive lymphoid tissues; moreover, in situ hybridization showed that IgD-positive cells are distributed throughout the spleen and head kidney. The expression pattern is similar to that of IgM, corroborating the hypothesis that IgD plays an important role in the humoral immune system of this species.

Alternative Splicing↗

PubMed2004

Characterization and expression of the immunoglobulin light chain in the fugu: evidence of a solitaire type.

In this study, we characterized the immunoglobulin light (IgL) chain gene and examined its expression in the fugu (Takifugu rubripes). The cDNA fragment that partially encodes the IgL chain was isolated by RACE and used as a probe for screening for IgL in a fugu splenic cDNA library. The IgL cDNA sequence that we found consisted of a variable (V(L)) and a constant (C(L)) segment. Its structural features were similar to the IgL isotype commonly found in teleosts. Genomic sequence analysis revealed that the IgL gene was organized as two V(L) gene segments (designed V(L1) and V(L2)) followed by single joining (J(L)) and C(L) segment. In addition, an unusual duplicate V(L1) gene segment was found downstream of the C(L) segment. The transcriptional orientation of the V(L) exons was found to be opposite to that of the J(L) and C(L) segments. Genomic blot hybridizations with V(L) and C(L) probes gave multibands, supporting the contention that the teleost IgL forms a multicluster. Both genomic and cDNA sequences analyses showed that all of the constant segments found in the fugu are identical, suggesting that no other isotypes could be found in this species. Comparison of the deduced amino acid sequence of the fugu C(L) domain with those of other species showed a high degree of identity (from 40 to 77%). IgL mRNAs were found to be expressed primarily in the lymphoid tissues. In situ hybridization revealed the presence of IgL-positive cells widely distributed throughout the spleen, head kidney, kidney, and thymus. These results support the contention that the lymphoid tissues are the major sites of antibody production in fish. Since IgL mRNA was also expressed in the skin and gill that are exposed to external antigens, it is likely that mucosal Ig plays an important role in immune protection.

Amino Acid Sequence↗

PubMed2004

In vitro effects of steroid hormones on IgM-secreting cells and IgM secretion in common carp (Cyprinus carpio).

The effects of steroid hormones on in vitro IgM-secreting cells (IgMSC) and IgM secretion by lymphocytes of the lymphoid organs in common carp, Cyprinus carpio were examined by ELISPOT and ELISA assay, respectively. Cells isolated from peripheral blood (PB), spleen and head kidney were cultured for 12, 24 and 48 h either in the absence or in the presence of steroid hormones, i.e. cortisol, testosterone, 11-ketotestosterone (11-KT), and estradiol-17beta (E(2)) at doses of 1, 10 and 100 ng/ml. Cortisol reduced the IgMSC numbers and IgM secretion by cells from all organs. In addition, cortisol induced apoptosis in lymphocytes from all organs. High dose of testosterone showed tissue-specific functions; it reduced the number of IgMSC and amount of IgM secretion by cells from spleen and head kidney, but not in PB, though IgM secretion was suppressed. However, no effects of sex steroids were observed in this study. The results show that sex-specific steroid hormones may have no immunosuppressive effects in common carp.

Animals↗

PubMed2003

A double staining flow cytometric assay for the detection of steroid induced apoptotic leucocytes in common carp (Cyprinus carpio).

Steroid hormones play an important role in the regulation of the immune system through different ways. In this in vitro study, the effects of steroid hormones on the apoptosis of leucocytes were evaluated to understand the involvement of this process in the immunocompetence of common carp. Prior to the investigation, a double staining flow cytometric assay using fluorescein diacetate (FDA), which reacts with esterases of viable cells, and propidium iodide (PI), an acid dye that binds with nuclear DNA, was established. FDA and PI negative cells were regarded as apoptotic. The FDA-PI technique is comparable to the Annexin V-PI technique and can be used in the quantification of the apoptosis of fish leucocytes accurately. The results suggest that the disappearance of esterases and externalization of phosphatidylserine (PS) may be common to many apoptotic pathways. Cells collected from peripheral blood, spleen, head kidney, and thymus were cultured for 16 h either in the absence or presence of steroid hormones, i.e. cortisol (F), testosterone, 11-ketotestosterone, and estradiol-17beta, and analyzed by flow cytometry followed by the FDA-PI method. Results showed that F induced apoptosis in leucocytes from blood and other lymphoid organs suggesting the role of F as an immune regulator. The participation of sex steroids to the immunocompetence of carp was not found, since they did not induce apoptosis of leucocytes in any organ.

Animals↗