TCRαß-depleted hematopoietic stem cell transplant and third-party CD45RA+ depleted adoptive cell therapy for treatment of post-transplant parvovirus B19 aplastic crisis.

This is a case report of a 6-year-old girl with relapsed B cell acute lymphoblastic leukemia in which adoptive cell therapy was applied successfully to treat refractory human parvovirus (HPV) B19 infection. Allogenic chimeric antigen receptor (CAR) T-cell therapy (bispecific CD19/CD22) was bridged to hematopoietic stem cell transplantation (HSCT) using a haploidentical paternal donor. However, HPV B19 DNAemia progressed and transfusion-related graft versus host disease occurred. After finding a third-party related donor with a better HLA match, haploidentical HPV B19-seropositive CD45RA+ depleted cells (16.5 × 106/kg) were administered and paternal TCRαß+ depleted stem cell were retransplanted. The HPV B19 DNAemia became negative within 1 week and the reticulocyte, neutrophil, hemoglobin, and platelet counts gradually normalized. The patient remained stable during the 1-year outpatient follow-up period. Thus, our case report highlights that persistent B19 infection can lead to pancytopenia, aplastic crisis, and graft rejection and TCRαß+ depleted haplo-HSCT is an effective means of hematopoiesis recovery. CD45RO memory T-cell therapy is the key to treating and preventing the development of refractory severe HPV B19 infection.

Droplet digital PCR: An effective method for monitoring and prognostic evaluation of minimal residual disease in JMML.

Juvenile myelomonocytic leukaemia (JMML) is a rare myeloproliferative neoplasm requiring haematopoietic stem cell transplantation (HSCT) for potential cure. Relapse poses a significant obstacle to JMML HSCT treatment, as the lack of effective minimal residual disease (MRD)-monitoring methods leads to delayed interventions. This retrospective study utilized the droplet digital PCR (ddPCR) technique, a highly sensitive nucleic acid detection and quantification technique, to monitor MRD in 32 JMML patients. The results demonstrated that ddPCR detected relapse manifestations earlier than traditional methods and uncovered molecular insights into JMML MRD dynamics. The findings emphasized a critical 1- to 3-month window post-HSCT for detecting molecular relapse, with 66.7% (8/12) of relapses occurring within this period. Slow MRD clearance post-HSCT was observed, as 65% (13/20) of non-relapse patients took over 6 months to achieve ddPCR-MRD negativity. Furthermore, bone marrow ddPCR-MRD levels at 1-month post-HSCT proved to be prognostically significant. Relapsed patients exhibited significantly elevated ddPCR-MRD levels at this time point (p = 0.026), with a cut-off of 0.465% effectively stratifying overall survival (p = 0.007), event-free survival (p = 0.035) and cumulative incidence of relapse (p = 0.035). In conclusion, this study underscored ddPCR's superiority in JMML MRD monitoring post-HSCT. It provided valuable insights into JMML MRD dynamics, offering guidance for the effective management of JMML.

Case Report: Unedited allogeneic chimeric antigen receptor T cell bridging to conditioning-free hematopoietic stem cell transplantation for a child with refractory Burkitt lymphoma.

Purpose: Burkitt lymphoma (BL) is the most common tumor of non-Hodgkin's lymphoma (NHL) in children, accounting for about 40% of cases. Although different combined short-course chemotherapies have achieved a good effect, refractory/relapsed BL has a poor prognosis with cure rates less than 30%. Chimeric antigen receptor T cell (CAR-T) therapy has developed rapidly in recent years and achieved excellent results in acute lymphoblastic leukemia (ALL). However, in some cases, there is a failure to produce autologous CAR-T cells because of T-cell dysfunction. In such cases, allogeneic CAR-T therapy has to be considered. Methods: A 17-year-old boy with stage II BL did not respond to extensive chemotherapy and sequential autologous CAR-T therapy. Lentiviral vectors containing anti-CD20-BB-ζ (20CAR) and anti-CD22-BB-ζ (22CAR) transgenes were used to modify the T cells from an HLA-identical matched unrelated donor. Flow cytometry was used to assess the cytokine analyses and CAR-T cell persistence in peripheral blood, enumerated by qPCR as copies per ug DNA. Informed consent for autologous/allogeneic CAR-T therapy was obtained from the patient and his legal guardian. Results: Unedited HLA-matched allogeneic CD20 and CD22 CAR-T cells were infused after lymphodepletion chemotherapy with cyclophosphamide and fludarabine. The patient experienced Grade IV cytokine release syndrome (CRS) and went into complete remission (CR) after anti-inflammatory treatment including tocilizumab. Because of persistent pancytopenia and full donor chimerism, the same donor's conditioning-free peripheral blood stem cells were successfully transplanted 55 days post CAR-T. Neutrophils were engrafted at day +11 and platelets were rebuilt at day +47 without obvious acute graft-versus-host disease (GVHD), but there was mild chronic GVHD in the skin and eyes. Currently, active anti-rejection therapy is still underway. Conclusion: Unedited HLA-matched allogeneic CAR-T cell therapy could be an innovative, effective, and safe treatment for children with refractory/relapse BL without obvious acute GVHD. Conditioning-free allogeneic hematopoietic stem cell transplantation (HSCT) from the same donor is feasible for a patient with full donor T-cell chimerism after allogeneic CAR-T. It cannot be ignored that close GVHD monitoring is needed post HSCT.

Toxoplasma gondii infection in children after allogeneic hematopoietic stem cell transplantation: A case report and literature review.

INTRODUCTION: Toxoplasmosis is a life-threatening complication after hematopoietic stem cell transplantation (HSCT). However, for several reasons, clinicians know little about Toxoplasma infection. CASE PRESENTATION: We report a case of toxoplasmosis that was diagnosed by bone marrow smear and metagenomic next-generation sequencing (mNGS) after HSCT in a boy. Additionally, we summarize the characteristics of toxoplasmosis after pediatric HSCT reported in the literature published in PubMed. CONCLUSION: Clinicians should increase their awareness of toxoplasmosis in children after HSCT and implement pre-transplant screening and post-transplant monitoring and prevention in future according to the national conditions of our country.

Isolated and assembled silver aggregates on the Si(001) surface: the initial stage of film formation.

We examined the dynamics of adsorption and the subsequent growth of submonolayered silver on Si(001) from 100 K to 230 K, using scanning tunneling microscopy and density functional theory. The dynamics is demonstrated to depend on substrate temperature, as described in the following three stages: (I) at 100-140 K, silver is adsorbed as isolated aggregates (regular-Ag4, variant-Ag4 and Ag2), in the absence of single silver adatoms. The spontaneous formation of silver aggregates arises from the hot-atom motion upon the initial impingement of individual silver atoms onto the Si(001) substrate. (II) At 140-190 K, the migration of isolated Ag-aggregates is sufficiently activated, leading to the formation of Ag-chains by surface polymerization. (III) At 190-230 K, there is implication that the Ag-chains become mobile on Si(001), en route to forming patches of 2×2 Ag-films by agglomeration.

Novel GLP-1 analog supaglutide improves glucose homeostasis in diabetic monkeys.

Glucagon-like peptide 1 (GLP-1) is an insulinotropic hormone and plays an important role in regulating glucose homeostasis. GLP-1 has a short half-life (t1/2 < 2 min) due to degrading enzyme dipeptidyl peptidase-IV and rapid kidney clearance, which limits its clinical application as a therapeutic reagent. We demonstrated recently that supaglutide, a novel GLP-1 mimetic generated by recombinant fusion protein techniques, exerted hypoglycemic and ß-cell trophic effects in type 2 diabetes db/db mice. In the present study, we examined supaglutide's therapeutic efficacy and pharmacokinetics in diabetic rhesus monkeys. We found that a single subcutaneous injection of supaglutide of tested doses transiently and significantly reduced blood glucose levels in a dose-dependent fashion in the diabetic monkeys. During a 4-week intervention period, treatment of supaglutide of weekly dosing dose-dependently decreased fasting and random blood glucose levels. This was associated with significantly declined plasma fructosamine levels. The repeated administration of supaglutide remarkably also decreased body weight in a dose-dependent fashion accompanied by decreased food intake. Intravenous glucose tolerance test results showed that supaglutide improved glucose tolerance. The intervention also showed enhanced glucose-stimulated insulin secretion and improved lipid profile in diabetic rhesus monkeys. These results reveal that supaglutide exerts beneficial effects in regulating blood glucose and lipid homeostasis in diabetic rhesus monkeys.

Molecular characterization and functional analysis of four teleostean K+ channels in macrophages of sea perch (Lateolabrax japonicas).

Potassium ion channels are one of the most diversely and widely distributed channels, which are involved in all kinds of physiological functions in both excitable and non-excitable cells. The expression of voltage-gated potassium ion (Kv) channels is highly variable according to the state of macrophages activation. Macrophages have an important function in innate immunity against intruding pathogens. They produce a variety of inflammatory and immunoactive molecules that modulate imflammatory responses. Here we show that blockade of K+ channels by non-selective Kv channel inhibitor tetraethylammonium chloride (TEA), and 4-aminopyridine (4-AP) inhibited proinflammatory cytokines expression, cell proliferation, and reactive oxygen species (ROS) production in LPS-stimulated macrophages of Sea perch (Lateolabrax japonicas). Then we isolated four Kv channels genes (spKv1.1, spKv1.2, spKv1.5 and spKv3.1) in LPS-activated fish macrophages. These channels genes were up-regulated after LPS stimulation except spKv3.1, which remained unchanged during the test. The results of this study indicate that Kv channels could be required for modulating the immune function of fish macrophages.

Transcriptome-wide analysis of DEAD-box RNA helicase gene family in an Antarctic psychrophilic alga Chlamydomonas sp. ICE-L.

DEAD-box RNA helicase family proteins have been identified in almost all living organisms. Some of them play a crucial role in adaptation to environmental changes and stress response, especially in the low-temperature acclimation in different kinds of organisms. Compared with the full swing study in plants and bacteria, the characters and functions of DEAD-box family proteins had not been surveyed in algae. To identify genes critical for freezing acclimation in algae, we screened DEAD-box RNA helicase genes from the transcriptome sequences of a psychrophilic microalga Chlamydomonas sp. ICE-L which was isolated from Antarctic sea ice. Totally 39 DEAD-box RNA helicase genes had been identified. Most of the DEAD-box RNA helicase have 1:1 homologous relationships in Chlamydomonas reinhardtii and Chlamydomonas sp. ICE-L with several exceptions. The homologous proteins in ICE-L to the helicases critical for cold or freezing tolerance in Arabidopsis thaliana had been identified based on phylogenetic comparison studies. The response of these helicase genes is not always identical in the Chlamydomonas sp. ICE-L and Arabidopsis under the same low-temperature treatment. The expression of several DEAD-box RNA helicase genes including CiRH5, CiRH25, CiRH28, and CiRH55 were significantly up-regulated under freezing treatment of ICE-L and their function in freezing acclimation of ICE-L deserved further investigation.

Negative feedback-defective PRPS1 mutants drive thiopurine resistance in relapsed childhood ALL.

Relapse is the leading cause of mortality in children with acute lymphoblastic leukemia (ALL). Among chemotherapeutics, thiopurines are key drugs in ALL combination therapy. Using whole-exome sequencing, we identified relapse-specific mutations in the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1), which encodes a rate-limiting purine biosynthesis enzyme, in 24/358 (6.7%) relapsed childhood B cell ALL (B-ALL) cases. All individuals who harbored PRPS1 mutations relapsed early during treatment, and mutated ALL clones expanded exponentially before clinical relapse. Our functional analyses of PRPS1 mutants uncovered a new chemotherapy-resistance mechanism involving reduced feedback inhibition of de novo purine biosynthesis and competitive inhibition of thiopurine activation. Notably, the de novo purine synthesis inhibitor lometrexol effectively abrogated PRPS1 mutant-driven drug resistance. These results highlight the importance of constitutive activation of the de novo purine synthesis pathway in thiopurine resistance, and they offer therapeutic strategies for the treatment of relapsed and thiopurine-resistant ALL.

[The abnormal expression of IKZF1 encoded protein-IKAROS in B-ALL children].

OBJECTIVE: To analyze the isoforms of IKAROS in the bone marrow samples from children with acute B-lineage lymphoblastic leukemia (B-ALL) and to investigate the relationship between frequency of dominant-negative (DN) IKAROS isoforms and prognosis of B-ALL, and to preliminarily study the relevant mechanism. METHODS: A total of 137 children with newly diagnosed B-ALL, who sequentially entered the Department of Hematology and Oncology, Shanghai Children's Medical Center between January 2005 and September 2010, were included in the study. Nest-PCR, Sanger sequencing, and TA cloning were used to analyze the expression of IKAROS isoforms in these children. The relationship between frequency of DN IKAROS isoforms and prognosis of B-ALL was investigated. RESULTS: Of the 137 children with newly diagnosed B-ALL, 16 had expression of IK6, 14 had expression of IK4, and 2 had expression of IK7. There was significant difference in 2.5-year event-free survival between the cohorts of DN IKAROS and non-DN IKAROS (P=0.01). Analysis of the 10 paired of diagnosis/relapse samples from 10 patients with recurrence showed that 8 of 10 paired diagnosis and relapse samples had inconsistent expression of IKAROS isoforms. The rate of IK6 expression in relapse samples from 21 relapse ALL patients was significantly higher than in the 137 children with newly diagnosed ALL (62% vs 12%, P<0.01). CONCLUSIONS: Expression of DN IKAROS isoforms can be a poor prognostic factor in B-ALL and is closely associated with recurrence of B-ALL.

[Expression of zinc finger protein X-linked in childhood B lineage acute lymphoblastic leukemia].

OBJECTIVE: To study the expression of zinc finger protein X-linked (ZFX) in bone marrow mononuclear cells (BMMCs) of children with B lineage acute lymphoblastic leukemia (B-ALL) and its relationship with prognosis. METHODS: The expression of ZFX in human leukemia cell lines (REH, HL-60, NB(4) and K562) was measured by Western blot. ZFX gene was cloned by PCR from one patient and DNA sequencing technology was used to confirm it. Real-time PCR was used for detecting ZFX mRNA expression in the BMMCs of 82 children with newly-diagnosed B-ALL, 24 children with complete remission (CR) after induction therapy and 64 control children (fracture or congenital heart disease patients). According to the presence of bone marrow or central nervous system relapse during a follow-up of 3 years, the patients were identified as having a good or poor prognosis. Their ZFX mRNA levels in BMMCs at diagnosis were compared. RESULTS: ZFX protein was expressed in human leukemia cell lines REH, HL-60, NB(4) and K562. ZFX mRNA expression was significantly higher in the newly-diagnosed ALL group than in the control group (P < 0.01). ZFX mRNA expression in the ALL CR group was significantly reduced compared with the newly-diagnosed ALL group (P < 0.01). Children with a poor prognosis had significantly higher ZFX mRNA levels at diagnosis than those with a good prognosis (P < 0.05). CONCLUSIONS: ZFX is over-expressed in children with B-ALL and its levels are higher in those with a poor prognosis than those with a good prognosis, which suggests that ZFX is important in the prognosis evaluation of B-ALL.

Next-generation sequencing-based transcriptome profiling analysis of Pohlia nutans reveals insight into the stress-relevant genes in Antarctic moss.

Genome-wide characterization of the Pohlia nutans transcriptome is essential for clarifying the role of stress-relevant genes in Antarctic moss adapting to the extreme polar environment. High-throughput Illumina sequencing was used to analyze the gene expression profile of P. nutans after cold treatment. A total of 93,488 unigenes, with an average length of 405 bp, were obtained. Gene annotation showed that 16,781 unigenes had significant similarity to known functional protein-coding genes, most of which were annotated using the GO, KOG and KEGG pathway databases. Global profiling of the differentially expressed genes revealed that 3,796 unigenes were significantly upregulated after cold treatment, while 1,405 unigenes were significantly downregulated. In addition, 816 receptor-like kinases and 1,309 transcription factors were identified from P. nutans. This overall survey of transcripts and stress-relevant genes can contribute to understanding the stress-resistance mechanism of Antarctic moss and will accelerate the practical exploitation of the genetic resources for this organism.

Hydrogen-producing microflora and Fe-Fe hydrogenase diversities in seaweed bed associated with marine hot springs of Kalianda, Indonesia.

Microbial fermentation is a promising technology for hydrogen (H(2)) production. H(2) producers in marine geothermal environments are thermophilic and halotolerant. However, no one has surveyed an environment specifically for thermophilic bacteria that produce H(2) through Fe-Fe hydrogenases (H(2)ase). Using heterotrophic medium, several microflora from a seaweed bed associated with marine hot springs were enriched and analyzed for H(2) production. A H(2)-producing microflora was obtained from Sargassum sp., 16S rRNA genes and Fe-Fe H(2)ase diversities of this enrichment were also analyzed. Based on 16S rRNA genes analysis, 10 phylotypes were found in the H(2)-producing microflora showing 90.0-99.5 % identities to known species, and belonged to Clostridia, Gammaproteobacteria, and Bacillales. Clostridia were the most abundant group, and three Clostridia phylotypes were most related to known H(2) producers such as Anaerovorax odorimutans (94.0 % identity), Clostridium papyrosolvens (98.4 % identity), and Clostridium tepidiprofundi (93.1 % identity). For Fe-Fe H(2)ases, seven phylotypes were obtained, showing 63-97 % identities to known Fe-Fe H(2)ases, and fell into four distinct clusters. Phylotypes HW55-3 and HM55-1 belonged to thermophilic and salt-tolerant H(2)-producing Clostridia, Halothermothrix orenii-like Fe-Fe H(2)ases (80 % identity), and cellulolytic H(2)-producing Clostridia, C. papyrosolvens-like Fe-Fe H(2)ases (97 % identity), respectively. The results of both 16S rRNA genes and Fe-Fe H(2)ases surveys suggested that the thermophilic and halotolerant H(2)-producing microflora in seaweed bed of hot spring area represented previously unknown H(2) producers, and have potential application for H(2) production.

Molecular cloning and functional analysis of a voltage-gated potassium channel in lymphocytes from sea perch, Lateolabrax japonicus.

Voltage-gated potassium (Kv) channels on cell plasma membrane play an important role in both excitable cells and non-excitable cells and Kv1 subfamily is most extensively studied channel in mammalian cells. Recently, this potassium channel was reported to control processes inside mammalian T lymphocytes such as cell proliferation and volume regulation. Little is known about Kv1 channels in fish. We have postulated the presence of such a channel in lymphocytes and speculated its potential role in immunoregulation in fish. Employing specific primers and RNA template, we cloned a segment of a novel gene from sea perch blood sample and subsequently obtained a full cDNA sequence using RACE approach. Bioinformatic analysis revealed structural and phylogenetic characteristics of a novel Kv channel gene, designated as spKv1.3, which exhibits homologous domains to the members of Kv1.3 family, but it differs notably from some other members of that family at the carboxyl terminus. Full-length of spKv1.3 cDNA is 2152 bp with a 1440 bp open reading frame encoding a protein of 480 amino acids. SpKv1.3 gene is expressed in all of the tested organs and tissues of sea perch. To assess the postulated immune function of spKv1.3, we stimulated lymphocytes with LPS and/or channel blocker 4-AP. Expression levels of messenger RNA (mRNA) of spKv1.3 under stimulation conditions were measured by quantitative RT-PCR. The results showed that LPS can motivate the up-regulation of spKv1.3 expression significantly. Interestingly, we found for the first time that 4-AP with LPS can also increase the spKv1.3 mRNA expression levels in time course. Although 4-AP could block potassium channels physically, we speculated that its effect on blockage of potassium channel may start up an alternative mechanism which feed back and evoke the spKv1.3 mRNA expression.

Validation of housekeeping genes for gene expression studies in an ice alga Chlamydomonas during freezing acclimation.

Antarctic ice alga Chlamydomonas sp. ICE-L can endure extreme low temperature and high salinity stress under freezing conditions. To elucidate the molecular acclimation mechanisms using gene expression analysis, the expression stabilities of ten housekeeping genes of Chlamydomonas sp. ICE-L during freezing stress were analyzed. Some discrepancies were detected in the ranking of the candidate reference genes between geNorm and NormFinder programs, but there was substantial agreement between the groups of genes with the most and the least stable expression. RPL19 was ranked as the best candidate reference genes. Pairwise variation (V) analysis indicated the combination of two reference genes was sufficient for qRT-PCR data normalization under the experimental conditions. Considering the co-regulation between RPL19 and RPL32 (the most stable gene pairs given by geNorm program), we propose that the mean data rendered by RPL19 and GAPDH (the most stable gene pairs given by NormFinder program) be used to normalize gene expression values in Chlamydomonas sp. ICE-L more accurately. The example of FAD3 gene expression calculation demonstrated the importance of selecting an appropriate category and number of reference genes to achieve an accurate and reliable normalization of gene expression during freeze acclimation in Chlamydomonas sp. ICE-L.

A novel omega-3 fatty acid desaturase involved in acclimation processes of polar condition from Antarctic ice algae Chlamydomonas sp. ICE-L.

The ability of Antarctic ice algae, Chlamydomonas sp. ICE-L, to survive and proliferate at low temperature and high salinity implies that they have overcome key barriers inherent in Antarctic environments. A full-length complementary DNA (cDNA) sequence of omega-3 fatty acid desaturase, designated CiFAD3, was isolated via reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. The full-length of CiFAD3 cDNA contained an open reading frame of 1,302 bp with 5'-terminal untranslated region (UTR) of 36 bp and 3'-terminal UTR of 507 bp encoding a fatty acid desaturase protein of 434 amino acids. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known chloroplastic omega-3 fatty acid desaturase. Meanwhile, CiFAD3 sequence showed typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane spanning regions that were universally present among plant desaturases. Under different stress conditions, messenger RNA (mRNA) expression levels of CiFAD3 were measured by quantitative RT-PCR. The results showed that both temperature and salinity could motivate the upregulation of CiFAD3 expression. The mRNA accumulation of CiFAD3 increased 2.6-fold at 0°C and 1.8-fold at 12°C compared to the algae at 6°C. Similarly, mRNA expression levels of CiFAD3 increased 3.8-fold after 62‰ NaCl treatment for 2 h. However, CiFAD3 mRNA expression levels were partially decreased after UV radiation. These data suggest that CiFAD3 is the enzyme responsible for the omega-3 fatty acid desaturation involved in ice algae Chlamydomonas sp. ICE-L acclimatizing to cold temperature and high salinity in Antarctic environment.

Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Antarctic ice algae Chlamydomonas sp. ICE-L.

A cDNA encoding heat shock protein 70 of Antarctic ice algae Chlamydomonas sp. ICE-L (designated as CiHsp70) was identified by RT-PCR and rapid amplification of cDNA ends approaches. The full-length cDNA of CiHsp70 was 2,232 bp, consisting of a 5'-terminal untranslated region (UTR) of 76 bp, a 3'-terminal UTR of 203 bp with a poly (A) tail, and an open reading frame of 1,953 bp. The CiHsp70 cDNA encoded a polypeptide of 651 amino acids with an ATPase domain of 388 amino acids, the substrate peptide binding domain of 246 amino acids and a C-terminus domain of 17 amino acids. The inducible CiHsp70 cDNA was highly homologous to other plant cytosolic Hsp70 genes and clustered together with green algae and higher plant rather than brown algae, diatom and Cryptophyta. Antarctic ice algae were treated with different stress conditions and messenger RNA (mRNA) expression levels of CiHsp70 were quantified by quantitative RT-PCR. The results showed that both cold and heat shock treatments could stimulate CiHsp70 mRNA expression. Meanwhile, CiHsp70 mRNA expression level increased 2.9-fold in response to UV-B radiation for 6 h, while the expression levels of CiHsp70 were remarkably increased after removing the UV-B radiation and immediately providing additional 6 h visible light. Furthermore, treating with 62 or 93 per thousand NaCl for 2 h, CiHsp70 mRNA expression level increased 3.0- and 2.1-fold, respectively. Together, our observations revealed that CiHsp70 as a molecular chaperone might play an important role in Antarctic ice algae Chlamydomonas sp. ICE-L acclimatizing to polar environment.

Molecular cloning and tissue expression patterns of a small conductance calcium-activated potassium channel gene in turbot (Scophthalmus maximus L.).

Calcium-activated potassium channels on plasma membrane enable potassium influx into the cell with ensuing changes in plasma membrane potential and consequent effects on cellular metabolic functions. Recently, this potassium channel was reported to regulate the cellular responses of mammalian immune cells. We have postulated the presence of such a channel in fish immune cells and its potential role in immunoregulation in fish. Employing specific primers and RNA template, we cloned a segment of a novel gene from turbot blood sample and subsequently obtained a full cDNA sequence using RACE approaches. Bioinformatic analysis revealed structural and phylogenetic characteristics of a novel small conductance calcium-activated potassium channel gene, we called TSKCa, which exhibits homologous domains to other species particularly in the transmembrane regions. Full-length TSKCa cDNA is 1698 bp with a 1632 bp open reading frame encoding a protein of 544 amino acids. TSKCa gene is expressed in majority of the tested organs and tissues of turbot. To assess the postulated immune function of TSKCa, we infected turbot with the pathogen Vibrio anguillarum. Here, semi-quantitative RT-PCR analysis demonstrated increased mRNA expression of TSKCa in head kidney, spleen and blood, indicating an important role of TSKCa in these organ tissues that mediate the immune defense response of turbot. In contrast, there was less change in expression in the turbot intestines and liver which were less implicated in the immune response in present study.

Cloning and expression of lipP, a gene encoding a cold-adapted lipase from Moritella sp.2-5-10-1.

A gene (lipP, 837 bp in length) coding for a cold-adapted lipase of psychrophilic bacterium Moritella sp. 2-5-10-1 isolated from Antarctic region was cloned and sequenced in this study. The deduced amino acid sequence revealed a protein of 278 amino acid residues with a molecular mass of 30,521. The primary structure of the lipase deduced from the nucleotide sequence showed consensus pentapeptide containing the active serine [Gly-Trp-Ser-Leu-Gly] and a conserved His-Gly dipeptide in the N-terminal part of the enzyme. These sequences were involved in the lipase active site conformation. Structure factors that would allow proper enzyme flexibility at low temperatures were discussed. It was suggested that the changes in the primary structure of the psychrophilic lipases compared to the thermophilic ones could account for their ability to catalyze lipolysis at temperatures close to 0 degrees C. For expression, the sequence corresponding to the cold-adapted lipase of strain 2-5-10-1 was subcloned into the pET-28a expression vector to construct a recombinant lipase protein. Expression of the lipase by Escherichia coli BL21 (DE3) cells was observed as clear halos on 1% (vol/vol) tributyrin upon induction with IPTG at 25 degrees C.