Isolation and characterization of a calcium-binding protein derived from mRNA termed p9Ka, pEL-98, 18A2, or 42A by the newly synthesized vasorelaxant W-66 affinity chromatography.

W-66 (N-(2-aminoethyl)-N-[2-(4-chlorocinnamylamino) ethyl]-5-isoquinolinesulfonamide), a newly synthesized isoquinolinesulfonamide, was shown to have a potent vasodilatory action and calmodulin (CaM)-antagonizing action. Using the W-66 affinity chromatographic technique, we purified two Ca(2+)-binding proteins from the EGTA-soluble fraction of bovine aorta. One was CaM and the other was an acidic protein with a molecular mass of 11 kDa. It was tentatively named "calvasculin." Calvasculin was a dimeric protein. Equilibrium dialysis showed that 1 mol of calvasculin (dimer) bound to 1.98 +/- 0.30 mol of Ca2+ in the presence of 10(-3) M Ca2+. Calvasculin failed to activate Ca2+/CaM-dependent enzymes such as myosin light chain kinase, Ca2+/CaM-dependent phosphodiesterase, or Ca2+/CaM-dependent protein kinase II and to inhibit the CaM stimulation of these enzymes. The partial amino acid sequence of calvasculin revealed a high homology to the predicted protein derived from mRNA, named pEL-98, 18A2, 42A, or p9Ka. We also examined the physicochemical and biochemical properties of calvasculin. Using the antibody specific for calvasculin, we obtained evidence that calvasculin is present in abundance in bovine aorta but not in brain, lung, heart, or testis.     

cDNA cloning and structure of mouse putative Ah receptor.

Mouse cDNA clones for a putative Ah receptor have been isolated from a cDNA library of mRNA from Hepa-1 cells by an oligonucleotide probe produced by PCR with a pair of primers which was synthesized according to the reported N-terminal sequence of 26 amino acids. The cDNA clones encode a polypeptide of 805 amino acids with a helix-loop-helix motif and with some similarity to a certain region designated PAS of Drosophila Per and Sim, and human Arnt protein. Cotransfection of an expression vector of the Ah receptor with a reporter plasmid pMC6.3k consisting of CYP1A1 promoter and CAT structural gene into CV-1 cells enhanced the CAT expression in response to added 3-methylcholanthrene.     

Intergeneric hybridization betweenMonascus anka andAspergillus oryzae by protoplast fusion

Summary To breed industrially useful strains of a slow-growing, red-pigment-producing strain ofMonascus anka, protoplasts ofM. anka MAK1 (arg) andAspergillus oryzae AOK1 (met, thr) were fused. A mixture of protoplasts prepared from mycelia ofM. anka MAK1 treated with 2% Usukizyme and ofA. oryzae AOK1 treated with 2% Usukizyme and 0.2% NovoZym 234 was incubated with 30% (w/v) polyethylene glycol no. 6000. Heterokaryon fusants complementing the auxotrophies of both mutants were isolated on minimal medium, but segregated into red (MAK1) and white (AOK1) sectors after being cultured on a complete medium. After irradiation with UV light, the fusants gave stable heterozygous diploids that formed long white hyphae. These diploids, which had twice as much DNA in the nucleus as their parents, grew more rapidly than the parent strain YZT1, and produced ethanol earlier than the parents. Production of amylase, protease, and kojic acid by the fusants was intermediate in amount between that of the two parents.     

Effect of penicillin G on the electroporation of Rhodococcus rhodochrous CF222

M. SUNAIRI, N. IWABUCHI, K. MURAKAMI, F. WATANABE, Y. OGAWA, H. MUROOKA AND M. NAKAJIMA. 1996. Suitable conditions for the introduction of bacteriophage DNA into cells of Rhodococcus rhodochrous CF222 by electroporation were established, and penicillin G was found to enhance the transfection frequency. When conditions optimal for the parental strain were applied to its colony-morphological mutants, different transfection frequencies were observed. Penicillin G enhanced the transfection frequency of smooth and mucoidal mutants but not of rough mutants.     

Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain

The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highests levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide -II (422–434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the -II tubulin fragment, but it showed interaction with the Affigel-conjugated -I (431–444) tubulin peptide. The different MAPs componentes were characterized by western blots using specific monoclonal antibodies. A salient feature of neonatal rat brain MAP-3 was its interactions with site-directed antibodies that recognize binding epitopes on the repetitive sequences of tau and MAP-2. However, these site-specific antibodies did not interact with MAP-1B from the neonatal rat brain tissue.Abbreviations PAA poly (L-aspartic acid) - HMW-MAPs high molecular weight microtubule associated proteins     

Characterization of Low pH-induced Catecholamine Secretion in the Rat Adrenal Medulla

Abstract: Catecholamine (CA) secretion was evoked when the isolated rat adrenal gland was perfused with HEPES-buffered Krebs solution acidified by the addition of HCI or by gassing with 95% O₂/5% CO₂. The secretion was detectable at pH 7.0 and increased with decreasing pH until at ～6.4. The low pH-induced CA secretion consisted of two phases, an initial transient response followed by a sustained phase. An intracellular Ca²⁺ antagonist, 3,4,5-trimethoxybenzoic acid 8-(N,N-diethylamino)octyl ester, selectively inhibited the initial phase of secretion. Both of the responses were resistant to nifedipine, a blocker of voltage-gated Ca²⁺ channel, but were completely inhibited in Ca²⁺-free (1 mM EGTA containing) solution. Adrenaline was an exclusive component in CAs released by low pH. The time course and extent of intracellular acidification caused either by low pH in the external medium or by the offset of a transitory NH₄CI application had no correlation with those of the secretory responses in the corresponding period. These results suggest that extracellular acidification preferentially activates adrenaline secretive cells to evoke CA secretion and that this low pH-induced CA secretion may be mediated by dihydropyridine-insensitive Ca²⁺ influx. Furthermore, the initial transient phase of the low pH-induced CA secretion might be caused by a Ca²⁺ release from intracellular stores, which is also induced by the Ca²⁺ influx.     

Purification and Characterization of Membrane Protein (90 kDa) from Mycoplasma salivarium,Which Binds Immunoglobulin (Ig) G Fc Fragment

A 90 kDa protein of Mycoplasma salivarium was released from cell membranes of the organism with Triton X-100 and purified by ion-exchange chromatography and chromatofocusing. The protein was eluted at pH 5.5 by chromatofocusing. The protein was shown to react with the Fc fragments of IgG from human and nine different animal species and did not distinguish between IgG from different species, while protein A, tested for comparative purposes, displayed a strong specificity for human and swine IgG. Furthermore, the protein reacted with antigen specific goat IgG (specific for gamma chains of human IgG), sheep red blood cells (SRBC) sensitized with rabbit antiserum to SRBC, that is, the Fc part of rabbit IgG, and concanavalin A as well. These findings may suggest that the protein is a lectin which binds the carbohydrate moiety of the Fc part of IgG.     

Effects of body and head positions on bilateral difference in tympanic temperatures

We have examined the nonparallel changes in tampanic membrane temperatures (T _ty) from the two ears in response to various changes in body and head positions. Upon assuming a lateral recumbent position, the T _ty on the lower side increased while that on the upper side decreased. Pressure application over a wide area of the lateral chest only caused inconsistent and obscure asymmetric changes in T _ty. A lateral flexion of the head with the subject sitting upright and a rotation of the head to the side in a supine position induced an increase in the T _ty on the lower side compared to that on the upper side. The temperature and blood flow of the forehead often decreased on the lower side and increased on the upper side, although such responses were not always concomitant with the asymmetric changes in T _ty. A dorsal flexion of the head with the subject in a reclining position caused a slight increase in the T _ty, whereas raising the head upright induced a slight decrease in them. Two additional experiments were carried out with single photon emission computed tomography using ^99mTc-hexamethylpropyleneamine oxime as tracer, and a slight, relative decrease in counts was noted in the right hemisphere during rotation of the head to the right. These results would strongly suggest that unilateral increases and decreases in T _ty could have been caused by one-sided decreases and increases, respectively, in blood flow to the brain and/or the tympanic membrane, induced by a vasomotor reflex involving vestibular stimulation.