Effect on epidermal cell of soybean protein-degraded products and structural determination of the root hair promoting peptide

Peptide(s) produced from degraded soybean protein by an alkaline protease from Bacillus circulans HA12 (degraded soybean-meal products; DSP) increased the number of both the root hair cells (trichoblasts) and hairless cells (atrichoblasts) of Brassica rapa by about 4.4 times and 1.9 times, respectively. To identify the root hair-promoting peptide(s) in DSP, the origin protein of the root hair-promoting peptide(s) was identified as Kunitz trypsin inhibitor (KTI). The root hair-promoting peptide in the degraded products of KTI was purified and produced a signal of 1,198.2 Da with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) analysis. A search of the amino acid sequence of KTI located the peptide GGIRAAPTGNER, which had a molecular weight identical to 1,198.2 Da. The peptide GGIRAAPTGNER was chemically synthesized, and the synthetic peptide possessed root hair-promoting activity. Thus, it is concluded that this peptide in DSP is the foreign bioactive peptide promoting the differentiation of root hairs.     

Synchrony in the hatching of eggs in the desert locust <Emphasis Type="Italic">Schistocerca gregaria</Emphasis> (Orthoptera: Acrididae): egg condition influences hatching time in the laboratory and under simulated field temperatures

This study examined the time of hatching of the desert locust Schistocerca gregaria Forskål (Orthoptera: Acrididae) in the laboratory to test the effect of eggs within a pod versus individualized eggs. The pod organization of eggs is thought to play a role in controlling hatching time and to facilitate synchronous hatching at constant temperatures. In the present study, we examined the hatching times of eggs in a pod and individualized eggs under 24-h thermocycles and simulated field temperatures. We tested two patterns of thermocycles consisting of a 12-h thermoperiod (35 or 30 °C) and 12-h cryoperiod (low temperature period; 30 or 25 °C), and two patterns of field temperatures observed in a natural habitat, Mauritania, in May and September. The majority of eggs hatched during low temperature periods in all patterns tested. In addition, the variances of hatching times for individualized eggs were significantly greater than for egg pods in which a clear peak of time of hatching was observed. We show that egg condition influences hatching time under thermocycles of constant and fluctuating temperatures in the laboratory, and may play a role in the adaptive time of hatching.     

Unveiling cryptic species diversity of flowering plants: successful biological species identification of Asian Mitella using nuclear ribosomal DNA sequences

Background  

Although DNA sequence analysis is becoming a powerful tool for identifying species, it is not easy to assess whether the observed genetic disparity corresponds to reproductive isolation. Here, we compared the efficiency of biological species identification between nuclear ribosomal and chloroplast DNA sequences, focusing on an Asian endemic perennial lineage of Mitella (Asimitellaria; Saxifragaceae). We performed artificial cross experiments for 43 pairs of ten taxonomic species, and examined their F1 hybrid pollen fertility in vitro as a quantitative measure of postzygotic reproductive isolation.     

Maximal Voluntary Force Strengthened by the Enhancement of Motor System State through Barely Visible Priming Words with Reward

The topic of unconscious influences on behaviour has long been explored as a means of understanding human performance and the neurobiological correlates of intention, motivation, and action. However, what is relatively unknown is whether subconsciously delivered priming stimuli, with or without rewards, can affect individuals’ maximum level of force produced with their best effort. We demonstrated using transcranial magnetic stimulation that barely visible priming of an action concept, when combined with a reward in the form of a consciously visible positive stimulus, could alter the state of the motor system. In accordance with this neurophysiological alteration, the prime-plus-reward stimuli significantly increased the hand-grip force level of maximum voluntary contraction with little conscious awareness. This is the first objective evidence that the barely conscious presence of a behavioral goal can influence the state of the motor system and arouse latent ability for human force exertion.     

Digoxigenin-labeled RNA probes for untranslated regions enable the isoform-specific gene expression analysis of myosin heavy chains in whole-mount in situ hybridization

Myosin heavy chains (MyHCs), which are encoded by myosin heavy chain (Myh) genes, are the most abundant proteins in myofiber. Among the 11 sarcomeric Myh isoform genes in the mammalian genome, seven are mainly expressed in skeletal muscle. Myh genes/MyHC proteins share a common role as force producing units with highly conserved sequences, but have distinct spatio-temporal expression patterns. As such, the expression patterns of Myh genes/MyHC proteins are considered as molecular signatures of specific fiber types or the regenerative status of mammalian skeletal muscles. Immunohistochemistry is widely used for identifying MyHC expression patterns; however, this method is costly and is not ideal for whole-mount samples, such as embryos. In situ hybridization (ISH) is another versatile method for the analysis of gene expression, but is not commonly applied for Myh genes, partly because of the highly homologous sequences of Myh genes. Here we demonstrate that an ISH analysis with the untranslated region (UTR) sequence of Myh genes is cost-effective and specific method for analyzing the Myh gene expression in whole-mount samples. Digoxigenin (DIG)-labeled antisense probes for UTR sequences, but not for protein coding sequences, specifically detected the expression patterns of respective Myh isoform genes in both embryo and adult skeletal muscle tissues. UTR probes also revealed the isoform gene-specific polarized localization of Myh mRNAs in embryonic myofibers, which implied a novel mRNA distribution mechanism. Our data suggested that the DIG-labeled UTR probe is a cost-effective and versatile method to specifically detect skeletal muscle Myh genes in a whole-mount analysis.     

Two Conserved Cysteine Residues Are Required for the Masculinizing Activity of the Silkworm Masc Protein

We have recently discovered that the Masculinizer (Masc) gene encodes a CCCH tandem zinc finger protein, which controls both masculinization and dosage compensation in the silkworm Bombyx mori. In this study, we attempted to identify functional regions or residues that are required for the masculinizing activity of the Masc protein. We constructed a series of plasmids that expressed the Masc derivatives and transfected them into a B. mori ovary-derived cell line, BmN-4. To assess the masculinizing activity of the Masc derivatives, we investigated the splicing patterns of B. mori doublesex (Bmdsx) and the expression levels of B. mori IGF-II mRNA-binding protein, a splicing regulator of Bmdsx, in Masc cDNA-transfected BmN-4 cells. We found that two zinc finger domains are not required for the masculinizing activity. We also identified that the C-terminal 288 amino acid residues are sufficient for the masculinizing activity of the Masc protein. Further detailed analyses revealed that two cysteine residues, Cys-301 and Cys-304, in the highly conserved region among lepidopteran Masc proteins are essential for the masculinizing activity in BmN-4 cells. Finally, we showed that Masc is a nuclear protein, but its nuclear localization is not tightly associated with the masculinizing activity.     

Requirement for C-mannosylation to be secreted and activated a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4)

BackgroundC-mannosylation is a unique type of glycosylation. A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a multidomain extracellular metalloproteinase that contains several potential C-mannosylation sites. Although some ADAMTS family proteins have been reported to be C-mannosylated proteins, whether C-mannosylation affects the activation and protease activity of these proteins is unclear.MethodsWe established wild-type and mutant ADAMTS4-overexpressing HT1080 cell lines. Recombinant ADAMTS4 was purified from the conditioned medium of the wild-type ADAMTS4-overexpressing cells, and the C-mannosylation sites of ADAMTS4 were identified by LC-MS/MS. The processing, secretion, and intracellular localization of ADAMTS4 were examined by immunoblot and immunofluorescence analyses. ADAMTS4 enzymatic activity was evaluated by assessing the cleavage of recombinant aggrecan.ResultsWe identified that ADAMTS4 is C-mannosylated at Trp⁴⁰⁴ in the metalloprotease domain and at Trp⁵²³, Trp⁵²⁶, and Trp⁵²⁹ in the thrombospondin type 1 repeat (TSR). The replacement of Trp⁴⁰⁴ with Phe affected ADAMTS4 processing, without affecting secretion and intracellular localization. In contrast, the substitution of Trp⁵²³, Trp⁵²⁶, and Trp⁵²⁹ with Phe residues suppressed ADAMTS4 secretion, processing, intracellular trafficking, and enzymatic activity.ConclusionsOur results demonstrated that the C-mannosylation of ADAMTS4 plays important roles in protein processing, intracellular trafficking, secretion, and enzymatic activity.General significanceBecause C-mannosylation appears to regulate many ADAMTS4 functions, C-mannosylation may also affect other members of the ADAMTS superfamily.     

High-performance liquid chromatographic determination of irinotecan (CPT-11) and its active metabolite (SN-38) in human plasma

A simplified method for the simultaneous determination of irinotecan (CPT-11, I) and its active metabolite (SN-38, II) in human plasma by high-performance liquid chromatography (HPLC) with fluorescence detection has been developed. Following the addition of the internal standard (I.S.) camptothecin, the drugs were extracted from plasma using methanol. The average extraction efficiencies were 87% for I, 90% for II and 90% for the I.S. Chromatography was performed using a TSK gel ODS-80Ts column, monitored at 556 nm (excitation wavelength, 380 nm) and the mobile phase was acetonitrile-50 mM disodium hydrogen phosphate (28:72) containing 5 mM heptanesulphonate (pH 3.0). The linear quantitation ranges for I and II were 30–2000 and 1–30 ng/ml, respectively.     

Induction of delayed type hypersensitivity-like skin reaction by peptidoglycans of bacterial cell walls

Water-soluble glycopeptides isolated from Lactobacillus plantarum and Staphylococcus epidermidis cell walls elicited a delayed type hypersensitivity (DTH)-like skin reaction in rats previously immunized with Mycobacterium tuberculosis cell walls, but not in unimmunized rats. Histological examination of the skin reaction sites in immunized animals revealed a close similarity of this skin reaction to a typical DTH reacton with respect to the time course of development and the types of cells that infiltrated into the skin reaction sites, which were characterized by a predominant infiltration of mononuclear cells at 48 hr. This DTH-like reaction was also demonstrated by immunizing the rats with the cell wall peptidoglycans of L. plantarum or S. epidermidis and skin testing them with homologous as well as heterologous peptidoglycans. The DTH-like reaction appeared to be caused by peptidoglycans that exist in common in the cell walls of phylogenetically distant bacterial species. Furthermore, it was also suggested that the putative antigenic determinants(s) might include both the glycan chain and part of the peptide moieties of the cell wall peptidoglycan rather than either of the single moieties.