Dielectric properties of polyelectrolytes. V. Dielectric dispersion of heavy meromyosin

Dielectric constant and dielectric loss of heavy meromyosin (HMM) were measured with varying pH. HMM showed a broader dispersion pattern than that with a single relaxation time especially on the high-frequencey side. The dielectric increment increased sharply with pH, above p^{H 6}, whereas the mean relaxation time and whole dispersion pattern were unchanged in the same region. The values of the increment and the mean relaxation time were much larger than those of usual globular proteins. The dispersion profile, pH dependence, and values of the increment are well explained by Oosawa's counterion fluctuation theory. Other mechanisms are more or less inadequate to our results. In the low pH region below the isoelectric precipitation region, both the increment and the mean relaxation time decreased; this is probably due to partial denaturation and suppression of the dissociation of carboxyl groups. An experiment on a urea-denatured sample supports this assumption. The biological significance of the pH dependence is discussed.     

Pacemaker Potentials for the Periodic Burst Discharge in the Heart Ganglion of a Stomatopod, Squilla oratoria

From somata of the pacemaker neurons in the Squilla heart ganglion, pacemaker potentials for the spontaneous periodic burst discharge are recorded with intracellular electrodes. The electrical activity is composed of slow potentials and superimposed spikes, and is divided into four types, which are: (a) "mammalian heart" type, (b) "slow generator" type, (c) "slow grower" type, and (d) "slow deficient" type. Since axons which are far from the somata do not produce slow potentials, the soma and dendrites must be where the slow potentials are generated. Hyperpolarization impedes generation of the slow potential, showing that it is an electrically excitable response. Membrane impedance increases on depolarization. Brief hyperpolarizing current can abolish the plateau but brief tetanic inhibitory fiber stimulation is more effective for the abolition. A single stimulus to the axon evokes the slow potential when the stimulus is applied some time after a previous burst. Repetitive stimuli to the axon are more effective in eliciting the slow potential, but the depolarization is not maintained on continuous stimulation. Synchronization of the slow potential among neurons is achieved by: (a) the electrotonic connections, with periodic change in resistance of the soma membrane, (b) active spread of the slow potential, and (c) synchronization through spikes.     

Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.     

Close linkage of MEN2A with RBP3 locus in Japanese kindreds

Summary The gene responsible for multiple endocrine neoplasia type 2A (MEN2A) has recently been assigned to the pericentromeric region of chromsome 10 in European Caucasian kindreds by linkage analysis using a DNA marker, interstitial retinol-binding protein 3 (RBP3). We have found tight linkage between the MEN2A and RBP3 loci in Japanese MEN2A kindreds. The maximum lod score is 5.19 at a recombination fraction of 0.00. This result suggests that mutation of a certain gene close to RBP3 is responsible for MEN2A irrespective of ethnic backgrounds.     

Effects of a New Plant Growth Regulator Prohexadione Calcium (BX-112) on Shoot Elongation Caused by Exogenously Applied Gibberellins in Rice (Oryza sativa L.) Seedlings

The effects of a novel plant growth regulator (PGR) prohexadionecalcium (BX-112; calcium 3,5-dioxo-4-propionylcyclohexanecarboxylate)on shoot elongation caused by exogenously applied GA₁, GA₃,GA₄₎ GA₁₉ and GA₂₀ were investigated in rice (Oryza sativa L.cv. Nihonbare and cv. Tan-ginbozu) seedling test. Dependingon the dose, BX-112 reduced shoot elongation in both cultivarscaused by GA₁₉ and GA₂₀, but not by GA₁. When a high dose ofBX-112 (e.g. 250 ng/plant and over) was applied with GA₁, orGA₄, shoot elongation was even promoted. This promotive effect,however, was not observed with GA₃. These results suggest thatBX-112 inhibits gibberellin (GA) biosynthesis in the rice plantat the 3ß- and 2ß-hydroxylation of GAs,namely steps of activation and inactivation, respectively. (Received September 6, 1989; Accepted November 27, 1989)     

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.     

Prosaposin Facilitates Sciatic Nerve Regeneration In Vivo

Abstract: Prosaposin, a multifunctional protein, is the precursor of saposins, which activate sphingolipid hydrolases. In addition to acting as a precursor for saposins, prosaposin has been shown to rescue hippocampal CA1 neurons from lethal ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Here we show that prosaposin, when added to a collagen-filled nerve guide after sciatic nerve transection in guinea pigs, increased dramatically the number of regenerating nerve fibers within the guide. To identify the target neurons of prosaposin during peripheral nerve regeneration, we determined the degree of atrophy and chromatolysis of neurons in the spinal anterior horn and dorsal root ganglia on the prosaposin-treated and untreated side. The effect of prosaposin on large spinal neurons and small neurons of the dorsal root ganglion was more conspicuous. Subsequent immunohistochemistry demonstrated that the atrophy of cholinergic large neurons in the anterior horn is prevented to significant extent by prosaposin treatment. These findings suggest that prosaposin promotes peripheral nerve regeneration by acting on α-motor neurons in the anterior horn and on small sensory neurons in the dorsal root ganglion. The present study raises the possibility of using prosaposin as a tool for the treatment of peripheral nerve injuries.     

Mutational analysis of the RNase-like domain in subunit 2 of fission yeast RNA polymerase II

Local sequence similarity exists between the subunit 2 of eukaryotic RNA polymerases II and the barnase-type bacterial RNases. The RNase-like domain from the Rpb2 ofSchizosaccharomyces pombe was expressed inEscherichia coli as a GST fusion protein and examined for its RNase activity. When the GST fusion protein was incubated in vitro with³²P-labeled RNA, the RNA degradation activity was less than 0.1%, if any, of the level of synthetic barnase. In order to check the in vivo function of this region, we constructed two mutantrpb2 alleles,rpb2 ^E357A andrpb2 ^H3a6L , each carrying a single amino acid substitution at the site correponding to one of the three essential amino acid residues forming the catalytic site in barnase (mutation of barnase at the corresponding sites results in complete loss of RNase activity) and five other mutantrpb2 alleles, each carrying a single mutation at various positions within the RNase-like domain but outside the putative catalytic site for RNase activity. When these mutantrpb2 alleles were expressed in anrpb2-disruptedS. pombe strain, all the mutants grew as well as the wild-type parent and did not show any clear defective phenotypes. These results suggest either that the RNase-like domain in Rpb2 does not function as an RNase in vivo or that the RNase activity of this domain, if present at all, is not essential for cell growth.