Trehalase in the spermatophore from the bean-shaped accessory gland of the male mealworm beetle,Tenebrio molitor: purification,kinetic properties and localization of the enzyme

Trehalase from the bean-shaped accessory glands of the male mealworm beetle, Tenebrio molitor, was purified by acid treatment, with subsequent chromatography on columns of DEAE-cellulofine and Sephacryl S-300. The molecular masses of the native and the denatured forms were estimated to be 43 and 62 kDa by gel filtration and SDS-PAGE, respectively, an indication that the trehalase may be composed of a single polypeptide. The optimum pH of the reaction catalyzed by trehalase was 5.6–5.8. The K _m for trehalose was 4.4 mmol·l^–1. Immunohistochemical experiments with trehalase-specific antiserum showed that the enzyme was localized in one specific type of secretory cell in the bean-shaped accessory gland epithelium and within the semisolid secretory mass that was a precursor to the wall of spermatophore. SDS-PAGE and immunoblotting analysis revealed the presence of a polypeptide of about 62 kDa in the spermatophore, Immunohistochemical observations showed that the trehalase was located at the outgrowth in the anterior portion of the spermatophore. When a fresh spermatophore was immersed in phosphate-buffered saline it discharged sperm in the same manner as in the bursa copulatrix of the female. Before the rupture of the expanded bulb of the spermatophore, almost all of the trehalase had dissolved in the phosphate-buffered saline. The addition of validoxylamine A to the saline, a specific inhibitor of trehalase, did not affect the expansion and evacuation of the spermatophore. These results demonstrate that trehalase, synthesized by a specific type of secretory cell in the bean-shaped accessory gland epithelium, is actively passed into the lumen of the bean-shaped accessory gland and then incorporated into the spermatophore. Trehalase appears to be one of the structural proteins of the spermatophore, although the possibility can not yet be completely ruled out that the trehalase-trehalose system functions for the nourishment and/or activation of the sperm in the bursa copulatrix of the female.Abbreviations BAG bean-shaped accessory gland(s) - DEAE diethylaminoethyl - Kpi buffer K₂HPO₄/KH₂PO₄ buffer (pH 7.0) - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - SDS sodium dodecy sulphate - Spph spermatophore(s) - TAG tubular accessory gland(s)     

N-Linked Glycosylation of the α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate(AMPA)-Selective Glutamate Receptor Channel α2 Subunit Is Essential for the Acquisition of Ligand-Binding Activity

Abstract: The N-linked glycosylation of the α2 subunit of the mouse α-amino-3-hydroxy-5-methylisoxazole-4-propionate(AMPA)-selective glutamate receptor (GluR) channel was characterized. The receptor subunit protein has five putative N -glycosylation sites. The recombinant receptor proteins were identified by [³⁵S]methionine/[³⁵S]cysteine metabolic labeling, western blot analysis, immunocytochemical detection, and [³H]AMPA binding experiments when expressed in insect Spodoptera frugiperda cells using a baculovirus system. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein species of ∼102 kDa and a minor species of ∼98 kDa, correspond to glycosylated and unglycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. Immunofluorescence staining of tunicamycin-treated cells expressing only the unglycosylated form differed little from that of tunicamycin-nontreated cells expressing both glycosylated and unglycosylated forms. The lack of AMPA-binding activity of the unglycosylated form expressed in the presence of tunicamycin suggested that N -glycosylation is required, directly or indirectly, for functional expression in insect cells for ligand binding. These results demonstrate that occupancy of at least one N -glycosylation site is required for the formation and maintenance of the GluRα2 subunit protein in an active conformation for ligand binding. Possible roles of N -glycosylation of GluRα2 subunit protein are discussed.     

Plasminogen Binds Specifically to α-Enolase on Rat Neuronal Plasma Membrane

Abstract: Plasminogen (PGn) that we identified in microglial-conditioned medium has a neurotrophic factor-like effect on cultured neurons. We have also shown that PGn binds specifically to a protein with a molecular mass of 45 kDa in the neuronal plasma membrane. As a candidate PGn receptor-like molecule on the neuronal surface, this 45-kDa protein was purified from the plasma membrane of embryonic rat brain. Amino acid sequence analysis of polypeptides derived from the cleavage of the protein with cyanogen bromide and V8 protease revealed that the 45-kDa protein is identical to rat α-enolase. In fact, PGn was found to bind to purified rat α-enolase and also to a synthetic peptide (30 residues) that corresponds to the carboxyl terminal region of rat α-enolase. Physical properties of the 45-kDa protein, such as molecular mass, isoelectric point, and the ability to form dimers, are quite similar to those of α-enolase. The 45-kDa PGn-binding protein in the plasma membrane was also recognized by anti-rat α-enolase antibody, and pretreatment with α-enolase antibody markedly diminished the PGn-binding to the plasma membrane. In addition, immunocytochemical staining of the cultured cells under the nonpermeable condition showed that α-enolase is present on the cell surface of a certain population of neurons. These results suggest that α-enolase may function as a PGn-binding molecule on the neuronal cell surface.     

Monoclonal anti-Fc epsilon receptor antibodies with different specificities and studies on the expression of Fc epsilon receptors on human B and T cells

Three monoclonal antibodies, 1-7 (gamma 2b), 3-5 (gamma 1), and 8-30 (mu), specific to Fc epsilon receptors (Fc epsilon R) on human B cells were established. The two monoclonals (1-7 and 8-30) could inhibit the binding of IgE to Fc epsilon R in rosette formation assays, as well as FACS analysis, and were shown to recognize the same epitope of Fc epsilon R. The other monoclonal antibody (3-5) recognized the same molecule but a different epitope, and marginally inhibited the IgE binding. The molecules on RPMI 8866 cells recognized by these monoclonal antibodies had Mr of 46,000 and 25,000 to 30,000 daltons as determined by immunoprecipitation and SDS-PAGE analysis. By employing these monoclonal antibodies, the expression of Fc epsilon R on circulating lymphocytes was studied. Approximately 50% of B cells from normal, nonatopic individuals were found to express Fc epsilon R, and a remarkable increase in the expression of Fc epsilon R was observed in B cells of atopic patients. The expression of Fc epsilon R was not detected in T cells from atopic patients (including hyper IgE syndrome) as well as normal individuals. Incubation of B cells with PHA-conditioned medium plus IgE augmented the expression of Fc epsilon R in the Fc epsilon R+ B cell population but not in Fc epsilon R- population. PHA-conditioned medium plus IgE did not induce Fc epsilon R expression on T cells.     

Amplification of epidermal growth factor receptor (EGFR) gene and oncogenes in human gastric carcinomas

DNAs from 37 human gastric carcinomas and seven lymph node metastases were analyzed for alterations of the epidermal growth factor receptor (EGFR) gene and oncogenes by the Southern blot hybridization method. The probes used were EGFR gene, c-Ha-ras, v-Ki-ras, N-ras, c-myc, v-myb, v-fos, c-erbB-2, v-erbA, v-abl and v-fes. Amplification of the EGFR gene was detected in only one poorly differentiated adenocarcinoma. Amplifications of c-myc gene and c-erbB-2 gene were each observed in two well differentiated adenocarcinomas. One of these tumors had coamplification of c-erbB-2 and c-erbA genes but there were no amplifications nor rearrangements of other oncogenes. The poorly differentiated adenocarcinom with amplified EGFR gene also showed enhanced expression of EGFR gene by Northern blot analysis and additionally had strong synchronous immunoreactivity for EGFR and EGF. Supported in part by Grants-in Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan and for Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan     

TaqI polymorphism in the LDL receptor gene and a TaqI 1.5-kb band associated with familial hypercholesterolemia

Summary The low density lipoprotein (LDL) receptor gene was analyzed in 67 unrelated healthy Japanese and 38 members of six consecutive families with familial hypercholesterolemia (FH) by Southern blot hybridization with TaqI, an LDL receptor cDNA fragment containing exons 1 to 8 being used as a probe. A new TaqI RFLP at the LDL receptor locus was detected with allele frequencies of 0.67 and 0.33. The data obtained with smaller cDNA subfragment probes revealed that the TaqI RFLP site is located within 1.1 kb of the 5 side of the EcoRI site of exon 5. The TaqI RFLP was in linkage disequilibrium with the PstI RFLP but showed no significant linkage disequilibrium with the RFLPs for AvaII, ApaLI/I15, PvuII, NcoI, and ApaLI/3. Among the seven RFLPs at the LDL receptor locus, the TaqI RFLP was the only useful genetic marker in one of the six families with FH. Furthermore, the association of an additional TaqI 1.5-kb band with a mutant LDL receptor gene was observed in another family with FH in which the proband was homozygous for all of the seven RFLPs. The data obtained with various restriction enzymes and smaller cDNA subfragments probes suggested that a minor change in nucleotide sequences in the region including exons 5 to 8 is present in the mutant gene. These data suggest that the TaqI RFLP is a useful genetic marker at the LDL receptor locus and that TaqI serves for the analysis of some mutant LDL receptor genes, when used with small LDL receptor cDNA probes.     

Determination of beta-phenylethylamine in catfish (Parasilurus asotus) tissues by gas chromatography/mass spectrometry

1. beta-Phenylethylamine (PEA) was detected and quantitated in tissues of the catfish, Parasilurus asotus, by very specific and sensitive gas chromatography/mass spectrometry. 2. The selected ion monitoring was made with a strong quasi-molecular ion of the pentafluoropropionic derivative of PEA in the positive chemical ionization mode. 3. PEA was found in all tissues tested ranging from 2.8 to 38.2 ng/g wet wt tissue. It was highest in the spinal cord, followed by the skin, brain and intestine.     

Detection of γH2AX foci in mouse normal brain and brain tumor after boron neutron capture therapy

Aim

In this study, we investigated γH2AX foci as markers of DSBs in normal brain and brain tumor tissue in mouse after BNCT.

Background

Boron neutron capture therapy (BNCT) is a particle radiation therapy in combination of thermal neutron irradiation and boron compound that specifically accumulates in the tumor. ¹⁰B captures neutrons and produces an alpha (⁴He) particle and a recoiled lithium nucleus (⁷Li). These particles have the characteristics of extremely high linear energy transfer (LET) radiation and therefore have marked biological effects. High LET radiation causes severe DNA damage, DNA DSBs. As the high LET radiation induces complex DNA double strand breaks (DSBs), large proportions of DSBs are considered to remain unrepaired in comparison with exposure to sparsely ionizing radiation.

Materials and methods

We analyzed the number of γH2AX foci by immunohistochemistry 30 min or 24 h after neutron irradiation.

Results

In both normal brain and brain tumor, γH2AX foci induced by ¹⁰B(n,α)⁷Li reaction remained 24 h after neutron beam irradiation. In contrast, γH2AX foci produced by γ-ray irradiation at contaminated dose in BNCT disappeared 24 h after irradiation in these tissues.

Conclusion

DSBs produced by ¹⁰B(n,α)⁷Li reaction are supposed to be too complex to repair for cells in normal brain and brain tumor tissue within 24 h. These DSBs would be more difficult to repair than those by γ-ray. Excellent anti-tumor effect of BNCT may result from these unrepaired DSBs induced by ¹⁰B(n,α)⁷Li reaction.     

Hemoglobin Decrease with Iron Deficiency Induced by Daclatasvir plus Asunaprevir Combination Therapy for Chronic Hepatitis C Virus Genotype 1b

Background

Decreased hemoglobin (Hb) level has been supposed to be a relatively rare side effect of a combination therapy against hepatitis C virus that consists of the NS5A inhibitor daclatasvir (DCV) and the NS3/4A protease inhibitor asunaprevir (ASV).

Methods

The study was conducted in 75 patients with genotype 1b chronic hepatitis C virus infection who had started combination therapy with DCV and ASV at St. Marianna University School of Medicine Hospital between September 2014 and December 2014.

Results

Among the patients examined, decreased Hb level by ≥1.5 g/dL from the values at treatment initiation was observed in 11 individuals. This was accompanied by decreased mean corpuscular volume, and iron and ferritin levels.

Conclusions

These findings suggest that the mechanism of the phenomenon is caused by iron deficiency. The underlying mechanism and clinical impacts will need to be further examined.