Lymphocyte Crossmatch Testing or Donor HLA-DP and -DQ Allele Typing Effectiveness in Single Cord Blood Transplantation for Patients with Anti-HLA antibodies Other Than Against HLA-A, -B, -C, and -DRB1.

BACKGROUND: Anti-human leukocyte antigen (HLA) antibodies other than those against HLA-A, -B, -C, and DRB1 are a risk factor for engraftment delay and failure, especially in cord blood transplantation (CBT) OBJECTIVES: The primary objective of this study was to assess the impact of the presence of anti-HLA antibodies on CBT and to evaluate the utility of lymphocyte crossmatch testing or additional HLA-DP and HLA-DQ typing of CB units in improving transplant outcomes STUDY DESIGN: We retrospectively assessed the engraftment rates and transplant outcomes of 772 patients who underwent their first CBT at our hospital between 2012 and 2021. Donors were routinely typed for HLA-A, -B, -C, and-DRB1 alleles, and the anti-HLA antibodies of recipients were screened before donor selection in all cases. Among patients who had antibodies against other than HLA-A, -B, -C, and DRB1 (n=58), lymphocyte crossmatch testing (n=32) or additional HLA-DP/-DQ alleles typing of CB (n=15) was performed to avoid the use of units with corresponding alleles RESULTS: The median patient age was 57 years (16-77). Overall, 75.7% had a high-risk disease status at transplantation, 83.5% received myeloablative conditioning regimens, and > 80% were heavily transfused. Two hundred and twenty-nine of the 772 recipients (29.6%) were positive for anti-HLA antibodies. There were no statistical differences in the number of infused CD34-positive cells between the anti-HLA antibody-positive and the anti-HLA antibody-negative patients. Of the 229 patients with anti-HLA antibodies, 168 (73.3%) had antibodies against HLA-A, -B, -C, and-DRB1 (group A), whereas 58 (25.3%) had antibodies against HLA-DP, HLA-DQ, or -DRB3/4/5 with or without antibodies against HLA-A, -B, C, and-DRB1 (group B). No patients in both group A and B exhibited DSAs against HLA-A, -B, -C, -and DRB1. The neutrophil engraftment rate was lower in patients with anti-HLA antibodies than in those without antibodies (89.9% vs. 94.1%), whereas non-relapse mortality (NRM) before engraftment was higher in antibody-positive patients (9.6% vs. 4.9%). In patients who received two or more HLA allele-mismatched CB in the host-versus-graft (HVG) direction (n = 685), the neutrophil engraftment rate was lower in the anti-HLA antibody-positive recipients than in the antibody-negative recipients with significant differences (88.8% vs. 93.8%) (P = 0.049). Similarly, transplant outcomes were worse in the antibody-positive patients with respect to two-year overall survival (OS) (43.1% vs. 52.3%) and NRM (44.0% vs. 30.7%) than in the antibody-negative patients. In contrast, the results of group B were comparable to those of the antibodies-negative patients, while the results of group A were statistically worse than the antibody-negative patients in terms of all engraftment rate (88.6%), OS (34.2%), and NRM (49.0%) CONCLUSIONS: The presence of anti-HLA antibodies negatively impacts engraftment, NRM, and OS in CBT. However, HLA-DP/-DQ allele typing of CB units or lymphocyte crossmatch testing could be useful strategies to overcome poor engraftment rates and transplant outcomes, especially in patients with anti-HLA antibodies against HLA-DP, HLA-DQ, or -DRB3/4/5.

A Nationwide Retrospective Analysis of Allogeneic Hematopoietic Stem Cell Transplantation for Adult Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening disorder characterized by systemic hyperinflammation. Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only potentially curative treatment for primary and relapsed/refractory HLH, the optimal strategy has not been established. We retrospectively analyzed 56 adult patients (≥18 years) with primary and secondary HLH (mainly consisting of Epstein-Barr virus-associated HLH) who underwent allo-HSCT using the registry database of the Japanese Society for Transplantation and Cellular Therapy, including 26 patients who underwent cord blood transplantation (CBT). One-fourth of patients received myeloablative conditioning (MAC), mainly consisting of total body irradiation-based regimens. The 3-year overall survival (OS) was 40.6%, while the 3-year cumulative incidences of relapse and non-relapse mortality (NRM) were 19.8% and 39.6%, respectively. In univariable analysis, age at allo-HSCT (the 3-year OS: 27.5% for ≥ 25 years old vs 58.0% for < 25 years old, P = .025), conditioning intensity (7.1% for MAC vs 51.8% for reduced-intensity conditioning (RIC), P = .002), and donor source (26.0% for CBT vs 52.9% for bone marrow or peripheral blood stem cell transplantation (BMT/PBSCT), P = .030) were associated with significantly inferior OS. In multivariable analysis, older age at allo-HSCT (≥ 25 years old) (Hazard ratio [HR], 2.37; 95% CI, 1.01 to 5.58; P = .048), MAC (HR, 2.45; 95% CI, 1.09 to 5.53; P = .031), and CBT (HR, 2.21; 95% CI, 1.04 to 4.71; P = .040) were independently associated with worse OS. In addition, only conditioning intensity predicted higher NRM (the 3-year NRM: 78.6% for MAC vs 26.6% for RIC), while no factors were associated with the relapse rate. This study includes the largest number of adult HLH patients undergoing CBT. Although the use of CBT is acceptable, BMT/PBSCT are more favorable strategies in allo-HSCT in adult HLH. Regarding conditioning intensity, RIC regimens are more beneficial in this setting.

Immunomodulatory Effect of Proteasome Inhibitors via the Induction of Immunogenic Cell Death in Myeloma Cells.

Several anti-cancer drugs are known to have immunomodulatory effects, including immunogenic cell death (ICD) of cancer cells. ICD is a form of apoptosis which is caused by the release of damage-associated molecular patterns (DAMPs), the uptake of cancer antigens by dendritic cells, and the activation of acquired immunity against cancer cells. ICD was originally reported in solid tumors, and there have been few reports on ICD in multiple myeloma (MM). Here, we showed that proteasome inhibitors, including carfilzomib, induce ICD in myeloma cells via an unfolded protein response pathway distinct from that in solid tumors. Additionally, we demonstrated the potential impact of ICD on the survival of patients with myeloma. ICD induced by proteasome inhibitors is expected to improve the prognosis of MM patients not only by its cytotoxic effects, but also by building strong immune memory response against MM cells in combination with other therapies, such as chimeric antigen receptor-T cell therapy.

Steroidogenic potential of the gonad during sex differentiation in the rice field frog Hoplobatrachus rugulosus (Anura: Dicroglossidae).

Prior studies demonstrated that gonadal differentiation in the rice field frog, Hoplobatrachus rugulosus, was of an undifferentiated type since all individuals had ovaries at complete metamorphosis. However, the steroidogenic potential of the gonad is still unknown. In this study, H. rugulosus were obtained by stimulating fertilization in the laboratory under natural light and temperature conditions. The gonads were collected and their steroidogenic potential was evaluated by determining the expression level of messenger RNA (mRNA) encoding for cytochrome P450 17-hydroxylase/C17-20 lyase (CYP17) and cytochrome P450 aromatase (CYP19) using quantitative real-time RT-PCR and the localization of CYP17 mRNA in tissues by in situ hybridization. The CYP17 mRNA levels in males at 4-11 weeks postmetamorphosis were higher than in female and intersex gonads. This corresponded to their localization in the gonadal tissues, where CYP17 signals were specifically detected in the Leydig cells of the testis at 5-16 weeks postmetamorphosis but was undetectable in all ovary samples. The CYP19 mRNA levels in females at 4-11 weeks postmetamorphosis was higher than in male and intersex gonads, which corresponded with gonadal development, indicating the potential steroidogenic function of the ovary. Based on the present results, the role of CYP17 and CYP19 mRNA in sex differentiation in H. rugulosus may occur after gonadal sex differentiation and the steroidogenic potential of the gonads exhibited a sexual dimorphic pattern. These results provide a crucial basis for further research on the developmental biology in anuran species.

Hemocytes of Yesso scallop characterized by cytological, molecular marker, and functional analyses.

Bivalve hemocytes have pivotal role as cellular biodefense. However, no information is available for cytological parameters, marker gene and function of the hemocytes in Yesso scallop, a commercially important aquaculture species worldwide. Due to their extremely strong cell aggregation ability, the scallop hemocytes were not able to assess as a single cell so far. In the present study, we established methodologies for studying the hemocytes of Yesso scallop, assessed cell morphology, measured seasonal fluctuation, and analyzed transcriptomes and cellular behavior during the immune response. Our results showed that the Yesso scallop possesses a single type of leukocyte-type hemocytes similar to other bivalve granulocytes circulating at an average of 1 × 107 cells/ml throughout the year. In addition, we identified five molecular marker genes specific to the scallop hemocytes. These hemocyte markers enabled us to precisely detect the hemocyte localization. Using these markers, we confirmed that tissue transplantation can experimentally induce an immune response, leading to the mobilization of circulating hemocytes for encapsulation. This study provides a comprehensive understanding of scallop hemocytes and their role in the cellular biodefense system of bivalves and various methods for cytological analysis.

Expression and functional analyses for estrogen receptor and estrogen related receptor of Yesso scallop, Patinopecten yessoensis.

Estrogen receptors (ERs) were known as estrogen-activated transcription factors and function as major reproduction regulators in vertebrates. The presence of er genes had been reported in Molluscan cephalopods and gastropods. However, they were considered as constitutive activators with unknown biological functions since reporter assays for these ERs did not show a specific response to estrogens. In this study, we tried characterization of ER orthologues from the Yesso scallop, Patinopecten yessoensis, in which estrogens had been proven to be produced in the gonads and involved in the spermatogenesis and vitellogenesis. Identified ER and estrogen related receptor (ERR) of Yesso scallops, designated as py-ER and py-ERR, conserved specific domain structures for a nuclear receptor. Their DNA binding domains showed high similarities to those of vertebrate ER orthologues, while ligand binding domains had low similarities with them. Both the py-er and py-err expression levels decreased in the ovary at the mature stage while py-vitellogenin expression increased in the ovary by quantitative real-time RT-PCR. Also, the py-er and py-err showed higher expressions in the testis than ovary during the developing and mature period, suggesting both genes might function in the spermatogenesis and testis development. The py-ER showed binding affinities to vertebrate estradiol-17ß (E2). However, the intensity was weaker than the vertebrate ER, indicating scallops might exist endogenous estrogens with a different structure. On the other hand, the binding property of py-ERR to E2 was not confirmed in this assay, speculating that py-ERR was a constitutive activator as other vertebrate ERRs. Further, the py-er was localized in the spermatogonia in the testis and in the auxiliary cells in the ovary by in situ hybridization, indicating its potential roles in promoting spermatogenesis and vitellogenesis. Taken together, the present study demonstrated that py-ER was an authentic E2 receptor in the Yesso scallop and might have functions for the spermatogonia proliferation and vitellogenesis, while py-ERR was involved in the reproduction by undiscovered manners.

Negative E-cadherin expression on bone marrow myeloma cell membranes is associated with extramedullary disease.

Background: The loss of E-cadherin expression and the induction of N-cadherin are known as hallmarks of the epithelial-to-mesenchymal transition, an essential initial step in the process of metastasis in solid tumors. Although several studies have reported expressions of these cadherins in patients with multiple myeloma (MM), their clinical significance is unknown as MM cells are non-epithelial. Methods: In this study, we examined the expression of E- and N-cadherins by immunohistochemistry using bone marrow (BM) biopsy specimens from 31 newly diagnosed MM patients and in subsequent biopsy specimens from six of these. Results: Negative E-cadherin expression on BM myeloma cell membranes was significantly associated with the presence of soft-tissue masses arising from bone lesions and breaking through the cortical bone, referred to as extramedullary disease (EMD). Conclusions: Given the aggressive nature of EMD, our study suggests that screening for E-cadherin using BM immunohistochemistry is one measure that could predict the development of EMD in patients with MM.

Gene delivery available in molluscan cells by strong promoter discovered from bivalve-infectious virus.

Understanding gene functions in marine invertebrates has been limited, largely due to the lack of suitable assay systems. Such a system requires investigative methods that are reproducible and can be quantitatively evaluated, such as a cell line, and a strong promoter that can drive high expression of a transgene. In this study, we established primary cell culture from a marine bivalve mollusc, Mizuhopecten yessoensis. Using scallop primary cells, we optimized electroporation conditions for transfection and carried out a luciferase-based promoter activity assay to identify strong promoter sequences that can drive expression of a gene of interest. We evaluated potential promoter sequences from genes of endogenous and exogenous origin and discovered a strong viral promoter derived from a bivalve-infectious virus, ostreid herpesvirus-1 (OsHV-1). This promoter, we termed OsHV-1 promoter, showed 24.7-fold and 16.1-fold higher activity than the cytomegalovirus immediate early (CMV IE) promoter and the endogenous EF1α promoter, the two most commonly used promoters in bivalves so far. Our GFP assays showed that the OsHV-1 promoter is active not only in scallop cells but also in HEK293 cells and zebrafish embryos. The OsHV-1 promoter practically enables functional analysis of marine molluscan genes, which can contribute to unveiling gene-regulatory networks underlying astonishing regeneration, adaptation, reproduction, and aging in marine invertebrates.

[PAX5-positive plasma cell leukemia presenting as lymphocytosis].

An 82-year-old Japanese male patient was initially diagnosed with lymphocytosis. His complete blood count revealed a white blood cell count of 30.9×109/l with 81% abnormal lymphocytes. The abnormal lymphocytes included monoclonal clones of CD38+ and CD138+cytoplasmic κ+ and IgG-κ M-protein, which led to the final diagnosis of plasma cell leukemia (PCL). Bortezomib and dexamethasone therapy was initiated, but the patient succumbed to the disease on the 8th day of hospitalization. A cytogenetic examination revealed a t (9;14)(p13;q32) translocation and the Western blotting confirmed high PAX5 expression. Similar to our present case, PCL cases with "lymphocytosis" have been widely reported, which some speculating the involvement of PAX5 overexpression in the pathogenesis. Such cases, including ours, may be classified as a unique group of disorders (PCL presenting as "lymphocytosis"), which requires accurate differential diagnosis and subsequent urgent multidisciplinary intensive treatment.

Rare evolution of CSF3R-mutated chronic neutrophilic leukemia to t(4;12)(q12;p13) acute myeloid leukemia with SETBP1 mutation.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative disease accompanied by mutations in CSF3R. Here, we present a patient with CNL who developed to acute myeloid leukemia (AML) at the same time that a t(4;12)(q12;p13) translocation appeared. The uniqueness of this cytogenetic abnormality led us to delineate the molecular aberrations relevant for clonal evolution. While the CSF3R mutation was present throughout the course of the disease, the SETBP1 mutation was newly acquired at the AML transformation. The present case suggests that careful monitoring of t(4;12)(q12;p13) and SETBP1 is crucial to predict AML evolution in CNL patients.

Loss of Axdnd1 causes sterility due to impaired spermatid differentiation in mice.

Purpose: Spermiogenesis, the process of deformation of sperm head morphology and flagella formation, is a phenomenon unique to sperm. Axonemal dynein light chain proteins are localized to sperm flagella and are known to be involved in sperm motility. Here, we focused on the gene axonemal dynein light chain domain containing 1 (Axdnd1) with the aim to determine the function of its protein product AXDND1. Methods: To elucidate the role of AXDND1 in spermatogenesis, we generated Axdnd1 knockout (KO) mice using the CRISPR/Cas9 system. The generated mice were subjected to fertility tests and analyzed by immunohistochemistry. Result: The Axdnd1 KO mouse exhibited sterility caused by impaired spermiogenesis during the elongation step as well as abnormal nuclear shaping and manchette, which are essential for spermiogenesis. Moreover, AXDND1 showed enriched testicular expression and was localized from the mid-pachytene spermatocytes to the early spermatids. Conclusion: Axdnd1 is essential for spermatogenesis in the mouse testes. These findings improve our understanding of spermiogenesis and related defects. According to a recent report, deleterious heterozygous mutations in AXDND1 were found in non-obstructive azoospermia (NOA) patients. Therefore, Axdnd1 KO mice could be used as a model system for NOA, which will greatly contribute to future NOA treatment studies.

Phosphopeptide enrichment using Phos-tag technology reveals functional phosphorylation of the nucleocapsid protein of SARS-CoV-2.

Phosphorylation of viral proteins serves as a regulatory mechanism during the intracellular life cycle of infected viruses. There is therefore a pressing need to develop a method to efficiently purify and enrich phosphopeptides derived from viral particles in biological samples. In this study, we utilized Phos-tag technology to analyze the functional phosphorylation of the nucleocapsid protein (N protein; NP) of severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral particles were collected from culture supernatants of SARS-CoV-2-infected VeroE6/TMPRSS2 cells by ultracentrifugation, and phosphopeptides were purified by Phos-tag magnetic beads for LC-MS/MS analysis. Analysis revealed that NP was reproducibly phosphorylated at serine 79 (Ser79). Multiple sequence alignment and phylogenetic analysis showed that the Ser79 was a distinct phospho-acceptor site in SARS-CoV-2 but not in other beta-coronaviruses. We also found that the prolyl-isomerase Pin1 bound to the phosphorylated Ser79 in NP and positively regulated the production of viral particles. These results suggest that SARS-CoV-2 may have acquired the potent virus-host interaction during its evolution mediated by viral protein phosphorylation. Moreover, Phos-tag technology can provide a useful means for analyzing the functional phosphorylation of viral proteins. SIGNIFICANCE: In this study, we aimed to investigate the functional phosphorylation of SARS-CoV-2 NP. For this purpose, we used Phos-tag technology to purify and enrich virus-derived phosphopeptides with high selectivity and reproducibility. This method can be particularly useful in analyzing viral phosphopeptides from cell culture supernatants that often contain high concentrations of fetal bovine serum and supplements. We newly identified an NP phosphorylation site at Ser79, which is important for Pin1 binding. Furthermore, we showed that the interaction between Pin1 and phosphorylated NP could enhance viral replication in a cell culture model.

Evaluation of four phosphopeptide enrichment strategies for mass spectrometry-based proteomic analysis.

Information about phosphorylation status can be used to prioritize and characterize biological processes in the cell. Various analytical strategies have been proposed to address the complexity of phosphorylation status and comprehensively identify phosphopeptides. In this study, we evaluated four strategies for phosphopeptide enrichment, using titanium dioxide (TiO2 ) and Phos-tag ligand particles from in-gel or in-solution digests prior to mass spectrometry-based analysis. Using TiO2 and Phos-tag magnetic beads, it was possible to enrich phosphopeptides from in-gel digests of phosphorylated ovalbumin separated by Phos-tag SDS-PAGE or in-solution serum digests, while minimizing non-specific adsorption. The tip-column strategy with TiO2 particles enabled enrichment of phosphopeptides from in-solution digests of whole-cell lysates with high efficiency and selectivity. However, the tip-column strategy with Phos-tag agarose beads yielded the greatest number of identified phosphopeptides. The strategies using both types of tip columns had a high degree of overlap, although there were differences in selectivity between the identified phosphopeptides. Together, our results indicate that multi-enrichment strategies using TiO2 particles and Phos-tag agarose beads are useful for comprehensive phosphoproteomic analysis.

A novel homozygous variant of the thrombomodulin gene causes a hereditary bleeding disorder.

We report a 19-year-old Vietnamese woman who experienced several life-threatening bleeding events, including ovarian hemorrhage. Blood analysis revealed a decreased fibrinogen level with markedly elevated fibrinogen/fibrin degradation products and D-dimer levels. Despite hemostatic surgery and administration of several medications, such as nafamostat mesylate, tranexamic acid, and unfractionated heparin, the coagulation abnormalities were not corrected, and the patient experienced repeated hemorrhagic events. We found that administration of recombinant human thrombomodulin (rhTM) remarkably improved the patient's pathophysiology. Screening and sequencing of the TM gene (THBD) revealed a previously unreported homozygous variation: c.793T>A (p.Cys265Ser). Notably, the Cys265 residue forms 1 of 3 disulfide bonds in the epidermal growth factor (EGF)-like domain 1 of TM. Transient expression experiments using COS-1 cells demonstrated markedly reduced expression of TM-Cys265Ser on the plasma membrane relative to wild-type TM. The TM-Cys265Ser mutant was intracellularly degraded, probably because of EGF-like domain 1 misfolding. The reduced expression of TM on the endothelial cell membrane may be responsible for the disseminated intravascular-coagulation-like symptoms observed in the patient. In summary, we identified a novel TM variant, c.793T>A (p.Cys265Ser). Patients homozygous for this variant may present with severe bleeding events; rhTM should be considered a possible treatment option for these patients.

Gonadal somatic cell-specific transforming growth factor-ß superfamily member in the Yesso scallop reveals gonadal somatic cell distribution during the reproductive phase.

The objective of this study was to identify the gonadal somatic cells in the Yesso scallop using a novel molecular marker. This study is the first to identify the bone morphogenetic protein 2a (Bmp2a) gene as a gonadal somatic cell-specific gene in this bivalve. We performed a transcriptomic survey to identify the transforming growth factor-ß (TGFß) superfamily members that act in Yesso scallop gonad development. BLAST survey, phylogenetic tree, and RT-PCR analyses screened BMP molecules (i.e., bmp2a and bmp10a), which are members of the TGFß superfamily that show gonad-specific expression. Among the BMPs from the Yesso scallop, in situ hybridization accompanied by RNAscope assay identified that bmp2a mRNA was specifically expressed in the gonadal somatic cells localized in the interspace between germ cells. Real-time quantitative PCR (qPCR) analysis revealed that bmp2a mRNA expression increased during the reproductive phase. The relative expression of bmp2a mRNA was lowest at the beginning of the growing stage and peaked at the mature stage in both sexes. These observations indicate that bmp2a-positive gonadal somatic cells support germ cell growth and differentiation during the reproductive phase for both sexes. This study provides new insights into gonadal somatic cell biology in marine invertebrates and we propose that TGFß signaling is necessary for gonad development in bivalves.

Diversified Biomineralization Roles of Pteria penguin Pearl Shell Lectins as Matrix Proteins.

Previously, we isolated jacalin-related lectins termed PPL2, PPL3 (PPL3A, 3B and 3C) and PPL4 from the mantle secretory fluid of Pteria penguin (Mabe) pearl shell. They showed the sequence homology with the plant lectin family, jacalin-related ß-prism fold lectins (JRLs). While PPL3s and PPL4 shared only 35%-50% homology to PPL2A, respectively, they exhibited unique carbohydrate binding properties based on the multiple glycan-binding profiling data sets from frontal affinity chromatography analysis. In this paper, we investigated biomineralization properties of these lectins and compared their biomineral functions. It was found that these lectins showed different effects on CaCO3 crystalization, respectively, although PPL3 and PPL2A showed similar carbohydrate binding specificities. PPL3 suppressed the crystal growth of CaCO3 calcite, while PPL2A increased the number of contact polycrystalline calcite composed of more than one crystal with various orientations. Furthermore, PPL4 alone showed no effect on CaCO3 crystalization; however, PPL4 regulated the size of crystals collaborated with N-acetyl-D-glucosamine and chitin oligomer, which are specific in recognizing carbohydrates for PPL4. These observations highlight the unique functions and molecular evolution of this lectin family involved in the mollusk shell formation.

Phos-tag diagonal electrophoresis precisely detects the mobility change of phosphoproteins in Phos-tag SDS-PAGE.

Phos-tag diagonal electrophoresis was developed to identify precisely a change in electrophoretic mobility of phosphoproteins in Phos-tag SDS-PAGE. Previously, if a single protein band was detected, it was impossible to determine whether mobility of the protein altered by Mn2+ Phos-tag in Phos-tag SDS-PAGE gels because SDS-PAGE and Phos-tag SDS-PAGE were performed on different gels. Moreover, when multiple protein bands were detected, it was difficult to determine whether the band with the highest mobility was altered mobility by Mn2+ Phos-tag. However, these problems were resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns were provided on a single gel. Using this technique we identified phosphorylation states of various proteins such as α-lactalbumin, α- and ß-casein, ovalbumin, basic 7S globulin, and 26S proteasome subunits. In the analyses of 26S proteasome subunits from humans and yeast, we could confirm that all subunits are phosphorylated, and find that the number of major proteins with different phosphorylation states is a few in each of the subunits despite having many phosphorylation sites. SIGNIFICANCE: Previously, Phos-tag SDS-PAGE has been developed to identify a change in electrophoretic mobility of phosphoproteins. However, we had a problem in this technique; it was often difficult to recognize the mobility shift by Mn2+ Phos-tag when we used separately SDS-PAGE and Phos-tag SDS-PAGE. Such a problem was resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns are provided on a single gel. This technique was useful to identify phosphorylation states of various proteins. : Phos-tag diagonal electrophoresis, mass spectrometry, phosphoproteins, basic 7S globulin, proteasome.

Glycan Binding Profiling of Jacalin-Related Lectins from the Pteria Penguin Pearl Shell.

We determined the primary structures of jacalin-related lectins termed PPL3s (PPL3A, 3B, and 3C, which are dimers consisting of sequence variants α + α, α + ß, ß + ß, respectively) and PPL4, which is heterodimer consisting of α + ß subunits, isolated from mantle secretory fluid of Pteria penguin (Mabe) pearl shell. Their carbohydrate-binding properties were analyzed, in addition to that of PPL2A, which was previously reported as a matrix protein. PPL3s and PPL4 shared only 35-50% homology to PPL2A, respectively; they exhibited significantly different carbohydrate-binding specificities based on the multiple glycan binding profiling data sets from frontal affinity chromatography analysis. The carbohydrate-binding specificity of PPL3s was similar to that of PPL2A, except only for Man3Fuc1Xyl1GlcNAc2 oligosaccharide, while PPL4 showed different carbohydrate-binding specificity compared with PPL2A and PPL3s. PPL2A and PPL3s mainly recognize agalactosylated- and galactosylated-type glycans. On the other hand, PPL4 binds to high-mannose-and hybrid-type N-linked glycans but not agalactosylated- and galactosylated-type glycans.

Erratum: Nagasawa, K. et al. Phenotypic Stability of Sex and Expression of Sex Identification Markers in the Adult Yesso Scallop Mizuhopecten yessoensis throughout the Reproductive Cycle. Animals 2019, 9(5), 277.

In the published article, "Phenotypic Stability of Sex and Expression of Sex Identification Markers in the Adult Yesso Scallop Mizuhopecten yessoensis throughout the Reproductive Cycle. [...].

Soluble CLEC-2 is generated independently of ADAM10 and is increased in plasma in acute coronary syndrome: comparison with soluble GPVI.

Soluble forms of platelet membrane proteins are released upon platelet activation. We previously reported that soluble C-type lectin-like receptor 2 (sCLEC-2) is released as a shed fragment (Shed CLEC-2) or as a whole molecule associated with platelet microparticles (MP-CLEC-2). In contrast, soluble glycoprotein VI (sGPVI) is released as a shed fragment (Shed GPVI), but not as a microparticle-associated form (MP-GPVI). However, mechanism of sCLEC-2 generation or plasma sCLEC-2 has not been fully elucidated. Experiments using metalloproteinase inhibitors/stimulators revealed that ADAM10/17 induce GPVI shedding, but not CLEC-2 shedding, and that shed CLEC-2 was partially generated by MMP-2. Although MP-GPVI was not generated, it was generated in the presence of the ADAM10 inhibitor. Moreover, antibodies against the cytoplasmic or extracellular domain of GPVI revealed the presence of the GPVI cytoplasmic domain, but not the extracellular domain, in the microparticles. These findings suggest that most of the GPVI on microparticles are induced to shed by ADAM10; MP-GPVI is thus undetected. Plasma sCLEC-2 level was 1/32 of plasma sGPVI level in normal subjects, but both soluble proteins significantly increased in plasma of patients with acute coronary syndrome. Thus, sCLEC-2 and sGPVI are released by different mechanisms and released in vivo upon platelet activation.