cDNA cloning of an mRNA encoding a sulfur-rich 10 kDa prolamin polypeptide in rice seeds

Using a rice maturing seed pUC9 expression library, we isolated a cDNA clone corresponding to 10 kDa sulfurrich prolamin by immunoscreening. A longer cDNA clone was obtained from a gtll library by plaque hybridization using this ³²P-labeled cDNA as a probe. A polypeptide sequence composed of 134 amino acids was deduced from the nucleotide sequence. A 24 amino acid signal peptide was assigned by computer calculation for the membrane spanning region and Edman sequencing of the purified mature polypeptide. Remarkably, 20% of methionine and 10% of cysteine were found in the mature polypeptide as well as high contents of glutamine, and hydrophobic amino acids. Part of the amino acid sequence was homologous with a conserved cysteine-rich region found in other plant prolamins. Two repeats of amino acid sequence were found in the polypeptide.     

Fertilization Potential of the Medaka Egg

Fertilization of the medaka egg in 10% Ringer's solution generates a depolarization of 4 mV just before the appearance of a characteristically longer hyperpolarization (25). The depolarization appears to be the result of a nonspecific leak triggered by sperm stimulation and the amplitude of the depolarization is thought to be independent of [Ca²⁺]_o (25). We have investigated the ionic dependence of this depolarization. An initial small depolarization (3–4 mV; duration, 5–8 sec) is followed by a rising phase of a spike-like depolarization in the range of 10–60 mV. The amplitude of this spike-like depolarization is propodional to log [Ca²⁺], ranging from 0.33–18 mM. Calcium antagonists, e.g. 10 mM cobalt or 10 μg/ml verapamil in 10% Ringer do not block the depolarization in the presence of 1.1 mM CaCl₂.     

Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) shows sugar-binding specificity for L-fucose. A λgt11 expression library was constructed from A. aurantia poly(A) RNA and screened with a polyclonal antiserum directed against AAL. An immunopositive clone carrying 1.3-kb EcoRI fragment was obtained. The fragment encoded AAL, but lacked a nucleotide sequence corresponding to the two amino-terminal amino acids. The 5′-terminal part of the fragment was replaced with a chemically synthesized DNA fragment and inserted into an expression vector to yield a plasmid pKA-1. Escherichia coli carrying pKA-1 expressed functional AAL and the recombinant AAL showed the same immunological properties as those of natural AAL.     

Tissue-specific expression of three types of beta-protein precursor mRNA: enhancement of protease inhibitor-harboring types in Alzheimer's disease brain

Expression of three types of mRNA encoding amyloid beta-protein precursor (APP) in various tissues was analysed, using a ribonuclease protection assay, with special reference to Alzheimer's disease (AD). The total content and the proportion of APP mRNAs were specific to each tissue. Among eight tissues examined, the brain was distinct in that the expression level was highest and APP695 mRNA was expressed in abundance. The ratio of APP770/APP751/APP695 mRNAs was approximately 1:10:20 in the cerebral cortex of control brain. The proportions of APP770 mRNA and APP770-plus-APP751 mRNAs increased up to 2.6- and 1.4-fold, respectively, in various regions of AD brain compared with control. The enhanced expression of protease inhibitor-harboring types (APP770 and APP751) may disturb the balance between biosynthesis and degradation of APPs and ultimately lead to accumulation of beta-protein as amyloid.     

Endothelin induces two types of contractions of rat uterus: phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels

Effects of endothelin on nonvascular smooth muscle have been examined using rat uterine horns and two modes of endothelin action have been revealed. Endothelin (0.3 nM) caused rhythmic contractions of isolated uterus in the presence of extracellular calcium. The rhythmic contractions were completely inhibited by calcium channel antagonists. These characteristics of endothelin-induced contractions were very similar to those induced by oxytocin. Binding assays using 125I-endothelin showed that endothelin and the calcium channel blockers did not compete for the binding sites. However, endothelin was unique in that it caused, in addition to rhythmic contractions, a slowly developing monophasic contraction that was insensitive to calcium channel blockers. This developing contraction became dominant at higher concentrations of endothelin and was also calcium dependent.     

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study

1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP.     

Osmoelastic coupling in biological structures: a comprehensive thermodynamic analysis of the osmotic response of phospholipid vesicles and a reevaluation of the "dehydration force" theory

A comprehensive thermodynamic analysis of the osmotic response of phospholipid vesicles is presented, using the Gibbs free energy of a vesicle suspension including the elastic contribution of the bilayer membrane. The results indicate that, in addition to the hydrostatic pressure difference across the membrane and the interbilayer pressure due to electrostatic repulsion, the elastic pressure arising from the coupling between the osmotic stress and the elasticity of the membrane (osmoelastic coupling) should participate in the osmotic response of phospholipid vesicles. The data of Cowley et al. [Cowley, A. C., Fuller, N. L., Rand, R. P., & Parsegian, V. A. (1978) Biochemistry 17, 3163-3168] and of Parsegian et al. [Parsegian, V. A., Fuller, N., & Rand, R. P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2750-2754] on the osmotic shrinkage of multilayer vesicles are discussed in terms of the elastic pressure and the interbilayer pressure, and the proposed "dehydration force" theory is reevaluated from the viewpoint of the present analysis.