Purification of a K(+)-channel protein of sarcoplasmic reticulum by assaying the channel activity in the planar lipid bilayer system

A K(+)-channel protein of the sarcoplasmic reticulum (SR) was purified by assaying the channel activity in a planar lipid bilayer system. The light fraction of SR vesicles was solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and fractionated by an anion-exchange chromatography and followed by gel filtration chromatography and affinity chromatography with concanavalin A. All fractions in each steps were mixed with asolectin solubilized in CHAPS and reconstituted into vesicles by dialysis. The channel activity of each fraction was assayed after the reconstituted vesicles had been fused into a planar lipid bilayer. The final fraction which showed the K(+)-channel activity contained only 100 kDa protein in a silver-stained gel after SDS-PAGE and an anti-Ca(2+)-ATPase antibody did not recognize the protein. The characteristics of the K(+)-channel were identical to those observed in native SR vesicles when using the same method. The channel showed a single-channel conductance of 120 pS in 0.1 M KCl and marked voltage dependence. The channel did not permeate Ca2+ and Cl- and was blocked by neomycin B.     

Distribution of retinoids in the chick limb bud: analysis with monoclonal antibody

Retinoic acid induces anteroposterior duplicate formation in developing chick limb bud, and it may be a natural morphogen involved in limb pattern formation. Retinoic acid is produced from retinol locally in the limb bud via retinal, and thus, to elucidate the distribution of these retinoids in the limb bud seems to be important for the understanding of the morphogen formation. We produced a monoclonal antibody against the retinoids with BSA-RA (bovine serum albumin-retinoic acid) conjugate for antigen, and investigated the distribution of retinoids in the chick limb bud. The antibody predominantly bound to retinoic acid, but weakly to retinol and retinal. Retinoids appeared in the limb bud at stage 18 and were distributed through stages 20-24, when the pattern formation in distal mesoderm was in progress. Initially they were found evenly in the whole mesoderm, but disappeared gradually from core mesoderm and remained only in the region of peripheral mesoderm at stage 24. At stage 26, retinoids were detected only in ectoderm. These results support the idea that the retinoids actually play roles in limb pattern formation and suggest that the retinoids in the peripheral mesoderm are important for pattern formation. Further, the role of retinoids in epidermis development at later limb bud stages is also suggested.     

A protein essential for recovering oxygen evolution in cholate-treated chloroplasts

A novel method for the reconstitution of oxygen evolution in cholate-extracted spinach thylakoid membranes was established and a protein essential for the reconstitution was purified from cholate extracts. Purification of the protein was accomplished by chromatography on a DEAE-Sephacel column. This protein (M_r 17 000) was reinserted into vesicular membranes reconstituted from cholate-extracted thylakoids in the presence of 25% glycerol to reactivate oxygen evolution.     

Sesquiterpenoids of Riccardia and Pallavicinia species

Riccardia species (Metzgeriales) contain various types of sesquiterpenes. R. jackii produces ent-selinane-, ent-aromadendrane-and ent-bicyclogermacrane-type sesquiterpenes together with (R)-cuparene and α-barbatene. Aneura pinguis (= Riccardia pinguis) is chemically quite different from R. multifida and R. jackii. The former produces a large amount of pinguisone. R. multifida contains 6-(3-methyl-2-butenyl)-indole and (+)-β-elemene as the major components. Pallavicinia longispina (Dilaenaceae; Metzgeriales) produces mainly spathulenol. The chiral properties of the sesquiterpenes isolated from R. jackii are quite similar to those of red algae, Laurencia species.     

Bibenzyls from Radula tokiensis and R. Japonica

A new bibenzyl having a dihydrooxepin ring was isolated from the acetone extract of the liverwort Radula tokiensis, together with the previously known 5 bibenzyls and 3 sesquiterpenes, trans-β-farnesene, cuparene and (Jcuparenol. Two known bibenzyls were isolated from R. japonica. The bibenzyl derivatives are significant chemosystematic markers of the Radulaceae.     

Soluble and bound forms of intracellular acid carboxypeptidase inAspergillus saitoi

The enzymological, physical, and immunological properties of soluble and bound forms of intracellular acid carboxypeptidase isolated from fresh mycelia ofAspergillus saitoi are reported. In the broken mycelia, about 60% of the total activity was found in the 2,000×g precipitate, with most of the remainder in the 100,000×g supernantant. The highly purified enzymes, I_a and I_b, from the 100,000×g supernatant were found to be homogeneous by such criteria as disc gel electrophoresis at pH 9.4 The bound enzyme, II, was solubilized from the 2,000×g precipitate by self-digestion at pH 6.4 and was highly purified by chromotography. The two forms of intracellular enzymes, the soluble enzymes (I_a and I_b) from the 100,00×g supernatant and the solubilized enzyme (II) from the 2,000×g precipitate, were closely related to, but not completely identical with, the extracellular acid carboxypeptidase.     

Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and γ-hydroxyphenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene—cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a μBondapak C₁₅ column using a mobile phase of methanol—0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5%/min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultra-violet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for γ-hydroxyphenylbutazone was 0.05 μg/ml.A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.     

On “intercolonial” cannibalism in Japanese paper wasps,Polistes chinensis antennalis Pérez andP. Jadwigae dalla torre (hymenoptera: Vespidae)

Summary Foundresses of two species of Japanese paper wasps,Polistes chinensis antennalis andP. jadwigae, attacked other colonies of the same species. A foundress ofP. chinensis antennalis visited two nests of the same species, and ate larvae from them, while two foundresses ofP. jadwigae each visited a nest of the same species, eating larvae and pupae even when the foundress of the attacked nest was on her nest. In addition, a foundress ofP. jadwigae distributed flesh balls thus obtained among their larvae. Discussion was made on the adaptive significance of the inter-colonial cannibalism. It was considered that, at first, it increases the foraging efficiency and secondly it plays a role in regulating population density.     

Control of pattern duplication in the retinotectal system of Xenopus. Suppression of duplication by eye-fragment interactions.

Recombining right nasal half-eyes with left temporal half-eyes at embryonic stage 32 in Xenopus produces a double or “twinned” pattern of functional connections between retina and midbrain optic tectum. The left temporal half-eye is reprogrammed such that it projects to the tectum as a mirror-image duplicate pattern of the nasal right half-eye; both half-eyes project across the entire tectum. However, recombining a right nasal half-eye (in situ) with a right dorsal half-eye (grafted into the temporal position; N_RD_R eye) produces a single normal retinotectal projection. Where interactions between N_R and T_L involve both axial reprogramming and duplication of N_R positional values in T_L, N_RD_R interactions involve axial reprogramming in D_R without duplication of N_R values. In a second approach to interactions which suppress pattern duplication, both nasal and temporal one-third-sized eye fragments form approximate “NN” or “TT” duplicate pattern maps, respectively, when either is allowed to round up and form a whole eye. Allowing nasal and temporal thirds to permanently fuse (after removal of a one-third-sized vertical center strip of retina at stage 32) produces a normal projection; allowing the nasal and temporal thirds to interact (fuse) for 35–40 hr, followed by removal of one or the other third, suppresses pattern duplication (produces normal maps) in the remaining third in a majority of cases. Allowing the thirds to interact for 18–30 hr before removal of the temporal third produces a majority result of partial duplication in the remaining nasal third. Partial duplicates are apparent spatial intermediates with regard to interactions which suppress duplication in either fragment type.     

Circular dichroism of squid rhodopsin and its intermediates

Circular dichroism (CD) and absorption spectra of squid (Todarodes pacificus) rhodopsin, isorhodopsin and the intermediates were measured at low temperatures. Squid rhodopsin has positive CD bands at wavelengths corresponding the - and β-absorption bands at liquid nitrogen temperature (CD maxima: 485 nm at -band and 348 nm at β-band) as well as at room temperature (CD maxima: 474 nm at -band and 347 nm at β-band). The rotational strength of the -band has a molecular ellipticity about twice that of cattle rhodopsin. The CD spectrum of bathorhodopsin displays a negative peak at 532 nm, the rotational strength of which has an absolute value slightly larger than that of rhodopsin. The reversal in sign at -band of the CD spectrum may indicate that the isomerization of retinal chromophore from twisted 11-cis form to twisted 11-trans form has occurred in the process of conversion from rhodopsin to bathorhodopsin. Lumirhodopsin has a small negative CD band at 490 nm, the maximum of which lies at 25 nm shorter wavelengths than the absorption maximum (515 nm), and a large positive CD band near 290 nm, which is not observed in rhodopsin and the other intermediates. This band may be derived from a conformational change of the opsin. In the process of changing from lumirhodopsin to LM-rhodopsin, the CD bands at visible and near ultraviolet regions disappear. Both alkaline and acid metarhodopsins have no CD bands at visible and near ultraviolet regions.