Some Characteristics of Protein Kinases in Lemna paucicostata

The three protein kinases of Lemna paucicostata that are separableby DEAE-Sephacel chromatography have been designated PI, PIIand PIII [Kato et al. (1983) Plant & Cell Physiol. 24: 841].The optimum pH for the PI and PII enzymes was 7.5 and for thePHI enzyme 7.0. The activities of these enzymes were stimulatedby divalent cations, the maximum stimulation being producedby 5 nw Mg^{2 $} for PI, by 3 mM Co^{2 $} for PII and by 1 mM Mn^2$ for PIII. The cytokinins; benzyladenine, kinetin and zeatin,inhibited the activity of the PIII enzyme. The molecular weightsof the PI and PII enzymes did not change after incubation withcAMP even though their activities were regulated by this compound. (Received October 17, 1983; )     

Isolation and Chemical Characterization of a Mating Pheromone Produced by Saccharomyces kluyveri

The pullulanase gene (pul) of Klebsiella aerogenes was transferred in vivo to Escherichia coli by using RP4:: Mu cts. The pul gene was expressed in E. coli, although the level of pullulanase activity in E. coli was lower than that in K. aerogenes, and the Pul⁺ transconjugants were relatively unstable in an unselective medium. Production of pullulanase, which is used to make maltose from starch, was induced in E. coli by pullulan, waxy maize amylopectin, soluble starch and maltose. When the transconjugant cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Retransfer of the pul_k gene from E. coli to K. aerogenes by conjugation resulted in an increase of the production of extracellular pullulanase.     

Effects of activin A and somatostatin on intact FSH secretion and intracellular Ca2+ concentration in human FSH-secreting pituitary adenoma cells.

Activin A stimulated synthesis and secretion of intact FSH in dispersed human FSH-secreting adenoma cells. Significant stimulation was observed after 24 hr. Activin A caused an increase in Ca2+ concentration ([Ca2+]i). This response occurred soon after the activin A action. These effects were blocked in Ca(2+)-deficient medium and by nitrendipine (5 microM). Somatostatin inhibited the activin A-induced intact FSH secretion and the [Ca2+]i response. These findings indicated that Ca2+ influx through voltage-gated Ca2+ channel was involved in the activin A induced synthesis and secretion of intact FSH.     

Stabilization of G-quadruplex structure on vascular endothelial growth factor gene promoter depends on CpG methylation site and cation type

Background

DNA methylation at the 5-position of cytosine is an epigenetic modification of CpG dinucleotides. In addition to CpG methylation, the G-quadruplex (G4) structure has been reported as a regulator of gene expression. The identification of G4 forming sequences in CpG islands suggests an involvement of CpG-methylated G4 structures in biological processes; however, few reports have addressed the effects of CpG methylation on G4 structure.

Simple SNP typing assay using a base-discriminating fluorescent probe

We have developed a new concept involving a single-step homogeneous method for single-nucleotide polymorphism (SNP) typing. In this method, a probe containing base-discriminating fluorescent (BDF) bases is added to a sample solution. BDF base-containing DNA usually shows only a weak fluorescence, but emits a strong blue fluorescence when it recognizes a target base at a specific site in a hybridized strand. By utilizing this feature, a simple mix-and-read SNP typing assay was achieved without any tedious probe-designing or washing processes for exclusion of hybridization error or any addition of DNA-modifying enzymes. This is very different from conventional methods. We simultaneously analyzed a number of samples with ease, with a high accuracy, using our BDF assay.     

Fibril formation of hsp10 homologue proteins and determination of fibril core regions: differences in fibril core regions dependent on subtle differences in amino acid sequence

Heat shock protein 10 (hsp10) is a member of the molecular chaperones and works with hsp60 in mediating various protein folding reactions. GroES is a representative protein of hsp10 from Escherichia coli. Recently, we found that GroES formed a typical amyloid fibril from a guanidine hydrochloride (Gdn-HCl) unfolded state at neutral pH. Here, we report that other hsp10 homologues, such as human hsp10 (Hhsp10), rat mitochondrial hsp10 (Rhsp10), Gp31 from T4 phage, and hsp10 from the hyperthermophilic bacteria Thermotoga maritima, also form amyloid fibrils from an unfolded state. Interestingly, whereas GroES formed fibrils from either the Gdn-HCl unfolded state (at neutral pH) or the acidic unfolded state (at pH 2.0-3.0), Hhsp10, Rhsp10, and Gp31 formed fibrils from only the acidic unfolded state. Core peptide regions of these protein fibrils were determined by proteolysis treatment followed by a combination of Edman degradation and mass spectroscopy analyses of the protease-resistant peptides. The core peptides of GroES fibrils were identical for fibrils formed from the Gdn-HCl unfolded state and those formed from the acidic unfolded state. However, a peptide with a different sequence was isolated from fibrils of Hhsp10 and Rhsp10. With the use of synthesized peptides of the determined core regions, it was also confirmed that the identified regions were capable of fibril formation. These findings suggested that GroES homologues formed typical amyloid fibrils under acidic unfolding conditions but that the fibril core structures were different, perhaps owing to differences in local amino acid sequences.     

The effects of aggregation-inducing motifs on amyloid formation of model proteins related to neurodegenerative diseases

To examine the effects of aggregation-inducing motifs related to neurodegenerative diseases on amyloid formation of host protein, we prepared several chimera myoglobins, in which various aggregation-inducing motifs were inserted. The focused aggregation-inducing motifs included five (R5) or two (R2) oligopeptide repeats in yeast Sup35p, five octapeptide repeats (OPR) in the human prion protein, a nonamyloid beta component (NAC) in alpha-synuclein, and tandem repeats of 50 glutamines (Q50). Circular dichroism and infrared spectroscopies suggested that the OPR, R5, and Q50 motifs formed an antiparallel beta sheet as well as a random coil, whereas the R2 and NAC motifs mainly formed random coils. The OPR, R5, and Q50 mutants, but not the R2 and NAC mutants, readily formed the SDS-resistant aggregates under physiological condition, and electron microscopy revealed that the aggregates contained amyloid fibrils. The destabilization and increase in gyration radius of the OPR, R5, and Q50 mutants correlated with the tendency to form amyloid fibrils. A control mutant bearing a nonamyloidgenic sequence was also moderately destabilized but did not form amyloid fibrils. Therefore, we concluded that the OPR, R5, and Q50 motifs, even in a quite stable protein such as myoglobin, led the host protein to formation of amyloid fibrils under physiological condition.     

The mitochondrial Ca2+ uptake regulator,MICU1, is involved in cold stress‐induced ferroptosis

Ferroptosis has recently attracted much interest because of its relevance to human diseases such as cancer and ischemia‐reperfusion injury. We have reported that prolonged severe cold stress induces lipid peroxidation‐dependent ferroptosis, but the upstream mechanism remains unknown. Here, using genome‐wide CRISPR screening, we found that a mitochondrial Ca²⁺ uptake regulator, mitochondrial calcium uptake 1 (MICU1), is required for generating lipid peroxide and subsequent ferroptosis under cold stress. Furthermore, the gatekeeping activity of MICU1 through mitochondrial calcium uniporter (MCU) is suggested to be indispensable for cold stress‐induced ferroptosis. MICU1 is required for mitochondrial Ca²⁺ increase, hyperpolarization of the mitochondrial membrane potential (MMP), and subsequent lipid peroxidation under cold stress. Collectively, these findings suggest that the MICU1‐dependent mitochondrial Ca²⁺ homeostasis‐MMP hyperpolarization axis is involved in cold stress‐induced lipid peroxidation and ferroptosis.