cDNA cloning of the Cry1Aa receptor variants from Bombyx mori and their expression in mammalian cells

We cloned cDNA of three variants of BtR175, a putative Bombyx mori receptor for Bacillus thuringiensis Cry1Aa delta-endotoxin by PCR. These variants were likely to be allelic to BtR175. cDNA of BtR175b, the most distant variant from BtR175, was introduced into mammalian cells. BtR175b protein was expressed in the plasma membrane of the cells and showed binding activity to Cry1Aa.     

Augmentation of antitumor resistance by a strain of unicellular green algae,Chlorella vulgaris

Summary Growth of Meth-A tumor in CDF1 mice was inhibited significantly by injection of a hot water extract of a strain of Chlorella vulgaris (CE) into the tumor or into the subcutaneous tissue near the tumor. The augmentation of resistance by CE may require the participation of T cells and macrophages, since it was abolished or reduced in athymic nude mice or mice treated with carrageenan, a macrophage blocker. Mice treated with CE exhibited antigen-specific augmented resistance against rechallenge with tumor. Moreover, the antitumor effect of CE was comparable with that of Corynebacterium parvum, but its mechanism of effect might be different.     

Immunocytochemical study of the surrounding envelope of autophagic vacuoles in cultured rat hepatocytes

By the use of electron immunoperoxidase cytochemistry at the ultrastructural level, the relationship of the surrounding sac of the autophagic vacuoles to the different cytomembranes was studied. When the endoplasmic reticulum was completely stained for microsomal carboxyesterase E1, the enzyme was not found to be labeled in the developed envelopes forming autophagic vacuoles. The autophagic envelope at the formative stages was also devoid of albumin which intensely stained Golgi cisternae. However, although it was rare, the endoplasmic reticulum showed an electron-lucent region like an early autophagic envelope in its cisternae which was lacking in carboxyesterase E1. In addition, deeply curving swelled cisternae where carboxyesterase E1 was found at the edges were occasionally encountered. These observations suggest that the segregating membranes arise from an endoplasmic reticulum and the structural characteristics of the endoplasmic membranes change at very early stages of formation of autophagic vacuoles. Acid phosphatase, a lysosomal marker enzyme, began to be localized on sections of the double membranes of newly created autophagic vacuoles. The enzyme spread all along the limiting membranes of the autophagic vacuoles, while, at the same time, the double membranes were converted into a single membrane. A lysosomal membrane glycoprotein (LGP107) was also localized on the surrounding envelope of autophagic vacuoles in a fashion similar to that of acid phosphatase. Lysosomal hydrolases seem to play some role in the conversion of double limiting membranes into a single limiting membrane.     

Biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase in rat hepatocytes.

The biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase was studied using an in vitro cell-free translation system, pulse-chase experiments with primary cultured rat hepatocytes and subcellular fractionation techniques of rat liver after pulse-labeling with [35S]methionine in vivo. The single polypeptide of 45 kDa translated in the cell-free system from membrane-bound polysomal RNAs was converted to the 64 kDa form when the translation was carried out in the presence of microsomal vesicles. Pulse-chase experiments using cultured rat hepatocytes showed that acid phosphatase is initially synthesized as an endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form of 64 kDa, and processed via an Endo H-sensitive intermediate form of 62 kDa to an Endo H-resistant form with a 67 kDa mass. Phase separation with Triton X-114 showed that both the 64 and 67 kDa forms have hydrophobic properties. Treatment of the cells with chloroquine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphatase to lysosomes. Acid phosphatase did not contain the phosphorylated high mannose type of oligosaccharide chains observed in cathepsin D. Subcellular fractionation experiments in conjunction with pulse-labeling in vivo showed that the acid phosphatase of the 67 kDa form was present in the Golgi heavy fraction (GF3) and the Golgi light fraction (GF1+2) enriched in cis and trans Golgi elements, respectively, at 30 min after the administration of [35S]methionine. Simultaneously, this polypeptide was also found in the lysosomal membrane fraction, thereby indicating that acid phosphatase is delivered to lysosomes in a membrane-bound form, immediately after reaching the trans-Golgi region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Performance of a time-resolved IR facility for assessment of protonation states and polarity changes in carboxyl groups in a large membrane protein,mammalian cytochrome c oxidase,under turnover conditions in a sub-millisecond time resolution

Time-resolved IR analyses for the protonation and polarity changes of carboxyl groups involved in proton pump enzymes under turnover conditions are indispensable for elucidation of their proton-pump mechanisms. We have developed a new time-resolved infrared facility by introducing a flow system for transferring highly concentrated and thus viscous protein solution to a thin (50?μm) flow cell equipped in a highly sensitive IR spectrometer constructed with the femtosecond mid-IR pulse laser with spectral width of 350?cm^?1 as an IR white light source equipped with multi-channel MCT detector. This facility equipped with O₂ supply system enables the sub-millisecond time scale infrared measurements of the O₂ reduction coupled with proton pumping by bovine cytochrome c oxidase (CcO) initiated by CO-flash photolysis in the COOH (1725–1770?cm^?1) region with the accuracy of about 10?μO.D. under the background O.D. of 1. The facility identifies a band intensity change at ~1744?cm^?1 assignable to protonation of a carboxyl group coupled with a single electron transfer to the O₂ reduction center within 1?ms after initiation of the reaction. The results suggest that the facility detects protonation of a single carboxyl group included in large proteins like as CcO (210?kDa). The present facility sensitively identifies also polarity changes in COOH group by detecting shifts of the bands near 1750?cm^?1 and 1760?cm^?1, without significant intensity changes. These findings show the performance of this facility sufficiently high for providing crucial information for understanding the proton transferring mechanisms of protein carboxyl groups.     

Identification of proteins that bind to the neuroprotective agent neoechinulin A

Neoechinulin A is an indole alkaloid with several biological activities. We previously reported that this compound protects neuronal PC12 cells from cytotoxicity induced by the peroxynitrite generator 3-morpholinosydnonimine (SIN-1), but the target proteins and precise mechanism of action of neoechinulin A were unclear. Here, we employed a phage display screen to identify proteins that bind directly with neoechinulin A. Our findings identified two proteins, chromogranin B and glutaredoxin 3, as candidate target binding partners for the alkaloid. QCM analyses revealed that neoechinulin A displays high affinity for both chromogranin B and glutaredoxin 3. RNA interference-mediated depletion of chromogranin B decreased the sensitivity of PC12 cells against SIN-1. Our results suggested chromogranin B is a plausible target of neoechinulin A.     

Nucleotide Sequence and Gene Organization of the Starfish Asterina Pectinifera Mitochondrial Genome

The 16,260-bp mitochondrial DNA (mtDNA) from the starfish Asterina pectinifera has been sequenced. The genes for 13 proteins, two rRNAs and 22 tRNAs are organized in an extremely economical fashion, similar to those of other animal mtDNAs, with some of the genes overlapping each other. The gene organization is the same as that for another echinoderm, sea urchin, except for the inversion of a 4.6-kb segment that contains genes for two proteins, 13 tRNAs and the 16S rRNA. Judging from the organization of the protein coding genes, mammalian mtDNAs resemble the sea urchin mtDNA more than that of the starfish. The region around the 3' end of the 12S rRNA gene of the starfish shows a high similarity with those for vertebrates. This region encodes a possible stem and loop structure; similar potential structures occur in this region of vertebrate mtDNAs and also in nonmitochondrial small subunit rRNA. A similar stem and loop structure is also found at the 3' end of the 16S rRNA genes in A. pectinifera, in another starfish Pisaster ochraceus, in vertebrates and in Drosophila, but not in sea urchins. The full sequence data confirm the presumption that AGA/AGG, AUA and AAA codons, respectively, code for serine, isoleucine, and asparagine in the starfish mitochondria, and that AGA/AGG codons are read by tRNA(GCU)(Ser), which possesses a truncated dihydrouridine arm, that was previously suggested from a partial mtDNA sequence. The structural characteristics of tRNAs and possible mechanisms for the change in the mitochondrial genetic code are also discussed.     

Endothelin receptor in microvessels isolated from human meningiomas: Quantification with radioluminography

Summary 1. We characterized specific¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in microvessels isolated from human meningiomas, using anin vitro quantitative receptor autoradiographic technique coupled to a radioluminographic imaging plate system.2. This newly developed and highly sensitive method revealed high-affinity ET receptors present in pellet sections of the microvessels from all the meningiomas studied, regardless of histological subtypes (dissociation constant, 1.2 ± 0.3 nM; maximum binding capacity, 185 ± 56 fmol/mg; means ± SE for nine tumors).3. In five cases of meningiomas, ET-3 competed for¹²⁵I-ET-1 binding to microvessels from those tumors with a low affinity [50% inhibiting concentration (IC₅₀) of 1.6 ± 0.4 × 10^–6 M], and a selective ET_B receptor agonist, sarafotoxin S6c, up to 10^–6 M, did not displace ET binding from the sections.4. In the sections of microvessels from four other tumors, biphasic competition curves were obtained in the case of incubation in the presence of increasing concentrations of ET-3, with an IC₅₀ of 1.1 ± 0.2 × 10^–9 M for the high-affinity component and 1.6 ± 0.3 × 10^–6 M for the low-affinity component, respectively. In addition, S6c competed for ET binding to those sections (IC₅₀=2.3 ± 0.2 × 10^–10 M) and 10^–6 M S6c displaced 30% of the control, corresponding to the high-affinity component of competition curves obtained in the presence of ET-3.5. Our results suggest that (a) capillaries in human meningiomas express a large number of high-affinity ET_A (non-ET_B) receptors with a small proportion of ET_B receptors, and (b) ET may have a role in neovascularization, tumor blood flow, and/or function of the blood-tumor barrier in meningioma tissues by interacting with specific receptors present on the surface of the endothelium.     

The Endothelin ETA Receptor Exists in the Caudal Solitary Tract Nucleus of the Rat Brain

1. The receptor autoradiographic method done on the rat lower brain stem and cerebellum plus ¹²⁵I-endothelin-1, BQ-123, an antagonist for the endothelin ET_A receptor, and sarafotoxin S6c, an agonist for the ET_B receptor, revealed minute amounts of the ET_A receptor coexisting with the ET_B receptor in the caudal solitary tract nucleus of the rat lower brain stem.2. The ET_B receptor is present predominantly in other parts of the lower brain stem.3. Knowledge of the heterogeneous distribution of the central endothelin receptor subtypes aids in understanding the neurophysiology of endothelins.