PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin

Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG_2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.     

Establishment of a monoclonal antibody PMab-225 against alpaca podoplanin for immunohistochemical analyses

Podoplanin (PDPN) is known as a lymphatic endothelial cell marker. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse PDPN have been established in our previous studies. However, mAbs against alpaca PDPN (aPDPN), required for immunohistochemical analysis, remain to be developed. In the present study, we employed the Cell-Based Immunization and Screening (CBIS) method for producing anti-aPDPN mAbs. We immunized mice with aPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/aPDPN), and hybridomas producing mAbs against aPDPN were screened using flow cytometry. One of the mAbs, PMab-225 (IgG_2b, kappa), specifically detected CHO/aPDPN cells via flow cytometry and recognized the aPDPN protein on Western blotting. Further, PMab-225 strongly stained lung type I alveolar cells, colon lymphatic endothelial cells, and kidney podocytes via immunohistochemistry. These findings demonstrate that PMab-225 antibody is useful to investigate the function of aPDPN via different techniques.     

Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil podoplanin

Monoclonal antibodies (mAbs) against not only human, mouse, and rat but also rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. PDPN is used as a lymphatic endothelial cell marker in pathological diagnoses. However, mAbs against Tasmanian devil PDPN (tasPDPN), which are useful for immunohistochemical analysis, remain to be developed. Herein, mice were immunized with tasPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/tasPDPN) cells, and hybridomas producing mAbs against tasPDPN were screened using flow cytometry. One of the mAbs, PMab-233 (IgG₁, kappa), specifically detected CHO/tasPDPN cells by flow cytometry and recognized tasPDPN protein by western blotting. Furthermore, PMab-233 strongly detected CHO/tasPDPN cells by immunohistochemistry. These findings suggest that PMab-233 may be useful as a lymphatic endothelial cell marker of the Tasmanian devil.     

Development of a novel tocopheryl ester for suppression of lipid accumulation without cytotoxicity by optimization of dicarboxylic ester moiety

Tocopheryl succinate (Tsuc) is a succinic acid ester of the well-known antioxidant α-tocopherol (T). Tsuc exhibits various biological activities, including tumor growth suppression via activation of cell signaling and prevention of lipid accumulation in mouse adipocyte 3T3-L1 cells. The latter findings suggest that Tsuc may be a drug candidate for the treatment of obesity. However, Tsuc was found to induce apoptosis of normal cells (in addition to cancer cells), demonstrating the need to reduce the cytotoxicity of Tsuc without losing the suppression effect on lipid accumulation. Based on our previous findings, we focused on the ester structure of Tsuc for controlling cytotoxicity. Herein, we examined the cytotoxicity and lipid accumulation suppression effect of various T ester derivatives. We found that the terminal carboxylic group is necessary for suppression of lipid accumulation. We synthesized tocopheryl glutarate (Tglu) and tocopheryl adipate (Tadi) by elongation of carbon atoms 1 and 2 of the dicarboxylic moiety, respectively. Tglu and Tadi did not show any cytotoxicity, and both esters suppressed lipid accumulation, although their suppression activities were weaker than that of Tsuc. Tadi showed a more potent lipid accumulation inhibitory effect than Tglu. Although Tadi inhibited lipogenesis and promoted lipolysis, lipolysis was induced at lower concentrations than inhibition of lipogenesis, suggesting that Tadi mainly affects lipolysis. Taken together, we succeeded in the reduction of cytotoxicity, without loss of the suppression effect on lipid accumulation, by elongation of the dicarboxylic moiety of Tsuc. Tadi may be a promising candidate as an anti-obesity drug.     

Inheritance of two M type mitochondrial DNA from sperm and unfertilized eggs to offspring in Mytilus galloprovincialis

In Mytilus mussels, paternal mitochondrial DNA (mtDNA) from sperm is known to be transmitted to offspring. This phenomenon is called doubly uniparental inheritance (DUI). Under DUI, sperm mtDNA (M type) is inherited only by males. Female mussels receive maternal mtDNA (F type). However, in our previous study, we showed female and unfertilized eggs have both F and M types. We hypothesized that the two M types both from sperm and unfertilized eggs were transmitted to offspring. To test the hypothesis, we examined the number of M type haplotypes in mature M. galloprovincialis. The M type in larvae was compared with those of the parents. Cross experiments were carried out to test the inheritance of M type. In six of 20 mature mussels, two M types were detected by sequence analysis and polymerase chain reaction-restriction fragment length polymorphism. In cross experiments of larval samples from five of 12 crosses, double peak wave was observed by single nucleotide polymorphisms analysis. In these larval samples, the higher peak wave was identical to the parental M type. Larvae received much more paternal M type than the maternal ones. We demonstrated that two M types from sperm and unfertilized eggs were transmitted to offspring in M. galloprovincialis.     

Properties of blocking and non-blocking monoclonal antibodies specific for human macrophage galactose-type C-type lectin (MGL/ClecSF10A/CD301)

Monoclonal antibodies (mAbs) specific for the human macrophage galactose-type calcium-type lectin (MGL) were established. The recombinant extracellular domain of MGL was used to immunize a mouse, and 10 hybridoma clones were obtained. Binding of recombinant MGL to asialo-bovine submaxillary mucin was shown to be blocked by mAbs MLD-1, 4 and 6. Immunoprecipitation of MGL from lysates of COS-1 cells transfected with MGL cDNA (form 6A) was achieved with mAbs MLD-1, 4, 7, 8 and 16. Chimeric recombinant proteins between human MGL and mouse MGL1 were used to determine the location of the epitopes for these mAbs. mAbs MLD-8, 13, 15 and 16 interacted with the amino terminal side of the conserved WVDGTD sequence immediately upstream of QPD, whereas mAbs MLD-7, 12 and 17 interacted with the other side. mAbs MLD-1, 4, and 6 apparently required both sides of this boundary. mAbs MLD-15 and 16 were shown to recognize the protein products of alternatively spliced mRNA 6A/8A and 6C/8A, having deletions at the boundary of exons 7 and 8, in addition to full length and other spliced forms of MGL (6A, 6B and 6C), whereas the other mAbs bound only full length and forms 6A, 6B and 6C.     

A novel role of group VIB calcium-independent phospholipase A2 (iPLA2gamma) in the inducible expression of group IIA secretory PLA2 in rat fibroblastic cells

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.     

Possibility of SARS-CoV-2 Infection in the Metastatic Microenvironment of Cancer

According to a report from the World Health Organization (WHO), the mortality and disease severity induced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are significantly higher in cancer patients than those of individuals with no known condition. Common and cancer-specific risk factors might be involved in the mortality and severity rates observed in the coronavirus disease 2019 (COVID-19). Similarly, various factors might contribute to the aggravation of COVID-19 in patients with cancer. However, the factors involved in the aggravation of COVID-19 in cancer patients have not been fully investigated so far. The formation of metastases in other organs is common in cancer patients. Therefore, the present study investigated the association between lung metastatic lesion formation and SARS-CoV-2 infectivity. In the pulmonary micrometastatic niche of patients with ovarian cancer, alveolar epithelial stem-like cells were found adjacent to ovarian cancer. Moreover, angiotensin-converting enzyme 2, a host-side receptor for SARS-CoV-2, was expressed in these alveolar epithelial stem-like cells. Furthermore, the spike glycoprotein receptor-binding domain (RBD) of SARS-CoV-2 was bound to alveolar epithelial stem-like cells. Altogether, these data suggested that patients with cancer and pulmonary micrometastases are more susceptible to SARS-CoV-2. The prevention of de novo niche formation in metastatic diseases might constitute a new strategy for the clinical treatment of COVID-19 for patients with cancer.     

Downregulation of the voltage-dependent calcium channel (VDCC) beta-subunit mRNAs in pancreatic islets of type 2 diabetic rats

The present study was undertaken to determine whether altered expression of the VDCC beta-subunits in pancreatic beta-cells could play a role in the changes in beta-cell sensitivity to glucose that occur with diabetes. Application of competitive RT-PCR procedure revealed that in normal Wistar rats, LETO and prediabetic OLETF rats, the beta(2)-subunit mRNA levels were 60-200-fold greater than the levels for the beta(3)-subunit. These findings suggest that the beta(2)-subunit as well as the beta-cell type VDCC1 alpha(1)-subunit may be the predominant form of the VDCC expressed in pancreatic beta-cells. The levels of mRNA encoding the beta-subunits and the beta-cell type alpha(1)-subunit as well as insulin were significantly reduced in diabetic rats. Perfusion experiments revealed that diabetic rats showed the higher basal insulin secretion and profoundly impaired insulin secretory responses to glucose compared with non-diabetic rats. Alternatively, impaired insulin secretory responses to glucose in high dose glucose-infused rats were recovered partly with the elevation of mRNA levels of the VDCC beta(2)- and beta(3)-subunits as well as the alpha(1)-subunit by the treatment with diazoxide. Thus, considering the possibility that the most striking effect of the VDCC alpha(1) beta-subunit coexpression in pancreatic beta-cells might occur on activation kinetics like the skeletal muscle, the impairment of further activation of the VDCCs to acute glucose challenge caused by the reduced expressions of the alpha(1) beta-subunits mRNAs in type 2 diabetic animals might be at least partly associated with the alterations in beta-cell sensitivity to glucose.     

Extracellular Toll-like receptor 2 region containing Ser40-Ile64 but not Cys30-Ser39 is critical for the recognition of Staphylococcus aureus peptidoglycan

Toll-like receptor 2 (TLR2) and CD14 function as pattern recognition receptors for bacterial peptidoglycan (PGN). TLRs and CD14 possess repeats of the leucine-rich motif. To address the role of the extracellular domain of TLR2 in PGN signaling, we constructed CD14/TLR2 chimeras, in which residues 1-356 or 1-323 of CD14 were substituted for the extracellular domain of TLR2, and five deletion mutants of TLR2, in which the progressively longer regions of extracellular TLR2 regions were deleted. PGN induced NF-kappaB activation in HEK293 cells expressing TLR2 but not in cells expressing CD14/TLR2 chimeras. The cells transfected with a deletion mutant TLR2(DeltaCys30-Ile64) as well as TLR2(DeltaCys30-Asp160) and TLR2(DeltaCys30-Asp305) failed to respond to PGN, indicating the importance of the TLR2 region Cys(30)-Ile(64). Although TLR2(DeltaCys30-Ser39) conferred cell responsiveness to PGN, the cells expressing TLR2(DeltaSer40-Ile64) failed to induce NF-kappaB activation. In addition, NF-kappaB activity elicited by PGN was significantly attenuated in the presence of synthetic peptide corresponding to the TLR2 region Ser(40)-Ile(64). From these results, we conclude that; 1) CD14 cannot functionally replace the extracellular domain of TLR2 in PGN signaling; 2) the TLR2 region Cys(30)-Ser(39) is not required for PGN recognition; 3) the TLR2 region containing Ser(40)-Ile(64) is critical for PGN recognition.