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.     

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induces caspase-dependent apoptosis in mononuclear cells

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), one of the tryptophan pyrolysates, is a dietary carcinogen and is formed in cooked meat and fish in our daily diet. Trp-P-1 will affect the cells in the blood circulation system before it causes carcinogenicity in target organs such as the liver. In this study, the cytotoxicity of Trp-P-1 was investigated in mononuclear cells (MNCs) from blood. Trp-P-1 (10-15 microM) decreased cell viability and induced apoptosis characterized both by morphological changes and by DNA fragmentation 4 h after treatment. DNA fragmentation was also observed following treatment at 1 nM after 24 h in culture. This result suggested that apoptosis would occur in the body following unexpected intake of foods containing Trp-P-1. To determine the mechanism of apoptosis, we investigated the activation of the caspase cascade in MNCs. Trp-P-1 (10-15 microM) activated the caspase cascade, i.e. the activity of caspase-3, -6, -7, -8 and -9 increased dose-dependently using peptide substrates, the active forms of caspase-3, -8 and -9 were detected by immunoblotting, and cleavage of poly(ADP-ribose) polymerase and protein kinase C-delta as the intracellular substrates for caspases was observed. A peptide inhibitor of caspase-8 completely suppressed activation of all other caspases, while an inhibitor of caspase-9 did not. These results indicated that caspase-8 may act as an apical caspase in the Trp-P-1-activated cascade.     

Detoxification of cadmium in tobacco plants: formation and active excretion of crystals containing cadmium and calcium through trichomes

In tobacco (Nicotiana tabacum L.), long and short trichomes can be distinguished morphologically. The established function of long trichomes is to exude a sticky gum containing diterpenes, whereas that of short trichomes is not known. When tobacco seedlings were exposed to toxic levels of cadmium (Cd), growth was retarded, but trichome number was increased up to 2-fold in comparison with untreated samples. Observation by variable-pressure scanning electron microscopy (VP-SEM) indicated that large crystals of 150 μm in size were formed on head cells of both short and long trichomes. An energy-dispersive X-ray analysis system fitted with VP-SEM revealed the crystals to contain amounts of Cd and calcium (Ca) at much higher concentrations than in the head cells themselves. Transmission electron microscopy demonstrated crystal formation in amorphous osmiophilic deposits in vacuoles. When seedlings were treated with Cd in the presence of Ca, tolerance was increased in proportion to the increase in Ca concentration. These results indicate that tobacco plants actively exclude toxic Cd by forming and excreting Cd/Ca-containing crystals through the head cells of trichomes. Received: 26 June 2000 / Accepted: 20 September 2000     

Tyrosine phosphorylation and association of BIT with SHP-2 induced by neurotrophins

BIT (brain immunoglobulin-like molecule with tyrosine-based activation motifs) is a membrane glycoprotein that has two cytoplasmic TAMs (tyrosine-based activation motifs). We previously reported that tyrosine-phosphorylated TAMs of BIT interact with the Src homology 2 domain-containing protein tyrosine phosphatase SHP-2 both in vitro and in transfected cells, and this association results in a potent stimulation of the phosphatase activity of SHP-2. Both BIT and SHP-2 are highly expressed in the mammalian brain, and they may play important roles in the regulation of synaptic function. In this study, we found that nerve growth factor (NGF) treatment of PC12 cells leads to the tyrosine phosphorylation of BIT and a subsequent complex formation between BIT and SHP-2. Furthermore, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) also induced the tyrosine phosphorylation of BIT and the association with SHP-2 in primary cultured rat neurons. Our results suggest that the BIT-SHP-2 signaling pathway is a novel signal transduction mechanism of neurons that acts in response to neurotrophic factors such as NGF, BDNF, and NT-3.