Cloning and characterization of genes specifically expressed during infection stages in the rice blast fungus

We isolated promoters of 12 genes from the rice blast fungus based on the sequences of randomly selected expressed sequence tags (ESTs) (appressorium formation stage cDNA library of Magnaporthe available from GenBank). These promoters (and the 5' coding regions if any) were fused in frame with egfp, and their expression patterns were examined under the epifluorescence microscope. Among them, two turned out to be specifically active in structures necessary for infection, viz. a promoter of adenylate cyclase interacting protein 1-like gene expressed in conidia, germ tubes, and appressoria, and a promoter of putative membrane-associated or secreted protein gene specifically expressed in appressoria. Although targeted knockout mutants of either gene failed to show detectable phenotypic alterations under laboratory conditions, these ESTs should be useful for identification of genes expressed during infection stages.     

LATS1 and LATS2 Phosphorylate CDC26 to Modulate Assembly of the Tetratricopeptide Repeat Subcomplex of APC/C

In budding yeast, the Mitotic Exit Network (MEN) regulates anaphase promoting complex/cyclosome (APC/C) via the Dbf2-Cdc14 signaling cascade. Dbf2 kinase phosphorylates and activates Cdc14 phosphatase, which removes the inhibitory phosphorylation of the APC/C cofactor Cdh1. Although each component of the MEN was highly conserved during evolution, there is presently no evidence supporting direct phosphorylation of CDC14 by large tumor suppressor kinase 1 (LATS1), the human counterpart of Dbf2; hence, it is unclear how LATS1 regulates APC/C. Here, we demonstrate that LATS1 phosphorylates the Thr7 (T7) residue of the APC/C component CDC26 directly. Nocodazole-induced phosphorylation of T7 was reduced by knockdown of LATS1 and LATS2 in HeLa cells, indicating that both of these kinases contribute to the phosphorylation of CDC26 in vivo. The T7 residue of CDC26 is critical for its interaction with APC6, a tetratricopeptide repeat-containing subunit of APC/C, and mutation of this residue to Asp (T7D) reduced the interaction of CDC26 with APC6. Replacement of endogenous CDC26 in HeLa cells with exogenous phosphor-mimic T7D-mutated CDC26 increased the elution size of APC/C subunits in a gel filtration assay, implying a change in the APC/C assembly upon phosphorylation of CDC26. Furthermore, T7D-mutated CDC26 promoted the ubiquitination of polo-like kinase 1, a well-known substrate of APC/C. Overall, these results suggest that LATS1/2 are novel kinases involved in APC/C phosphorylation and indicate a direct regulatory link between LATS1/2 and APC/C.     

Ceramide kinase expression is altered during macrophage-like cell differentiation of the leukemia cell line HL-60

Ceramide kinase (CerK) has important roles in leukocyte functions, including the role in degranulation of mast cells and the phagocytosis of polymorphonuclear leukocytes, so its expression levels should be strictly regulated. Here, we report that the mRNA expression and enzyme activity of CerK were decreased during macrophage-like cell differentiation of the leukemia cell line HL-60, yet neither was altered during granulocytic differentiation of the same cells. Our findings demonstrate that HL-60 cells are useful for studying CerK functions in leukocyte differentiation, and they also suggest that CerK might have an important role in such differentiation.     

Cleavage of hexokinase II to two domains by trypsin without significant change in catalytic activity

Hexokinase II prepared from Ehrlich-Lettre hyperdiploid tumor cells (ELD cells) was subjected to a limited digestion by trypsin. After 60 min digestion, hexokinase II (100 kDa) was completely cleaved to two fragments with the molecular weight of about 60 kDa and 40 kDa as manifested in SDS-PAGE. It was noteworthy that the enzyme activity was observed even at the time when the native enzyme molecule was no more detectable. These fragments were separated by SDS-PAGE irrespective of the presence of a reducing agent, but neither by native PAGE nor by cellulose acetate membrane electrophoresis under the nondenaturing conditions. Neither kinetic parameters such as Km values for ATP and glucose nor an ability of binding to mitochondria were changed significantly by the tryptic digestion. These results indicate that an essential conformation of hexokinase II can be restored by the self-association of two fragments produced as a result of the cleavage by trypsin at the middle of the molecule. Affinity labeling with 2-3-dialdehyde ATP followed by the trypsin digestion showed that ATP binding site resided in the 40 kDa fragment. Furthermore, the mode of the response in the incorporation of this ATP analog to hexose phosphate, moreover, was similar to that in the catalytic activity.Abbreviations SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis - EDTA Ethylenediamine Tetraacetic Acid - CBB Coomassie Brilliant Blue - PMSF Phenylmethylsulfonyl Fluoride - TPCK N-tosyl-Lphenylalanyl Chloromethyl Ketone - ELD cells Ehrlich-Lettre Hyperdiploid Tumor Cells     

Purification and identification of neuromedin U as an endogenous ligand for an orphan receptor GPR66 (FM3)

GPR66 is an orphan G-protein-coupled receptor (GPCR) whose structure is similar to the ghrelin and motilin receptors. We have tried to purify a natural ligand for GPR66 in rat tissues and identified a 23-amino-acid peptide as the endogenous ligand. Sequence analysis revealed the peptide as neuromedin U (NMU), a smooth-muscle-contracting peptide that was first purified from porcine spinal cord by our group. NMU binds to GPR66-expressing cells with high specificity to induce intracellular calcium mobilization. When NMU was injected intracerebroventricularly (ICV) into rats, it potently suppressed food intake. In contrast, ICV injection of NMU-antibody increased food intake. These results suggest that NMU is a potent endogenous anorexic peptide.     

Acoustic Features Used in Song Discrimination by the Veery

To investigate which song features are used for discrimination by the veery (Catharus fuscescens), we synthesized songs by computer and manipulated or deleted various features. These songs were then played to territorial males in their natural habitat. Differences in response to these stimuli indicated the relative importance of the manipulated song components. We found that certain features must be maintained in order to elicit a territorial response similar to that elicited by the natural song. These features were the frequency changes between and within syllables, the dominant higher frequency band (or voice), the intra-syllable syntax, and the rapid repetitive amplitude and frequency modulations. Manipulations of inter-syllable syntax independent of frequency changes between syllables, broad changes in amplitude, and the lower frequency band, did not have a significant effect on response. We thus concluded that they are not essential as cues for discrimination or species identification. Re-recordings of songs broadcast along transects indicate that song components used in encoding species identity are those which transmit well across veery territories.     

Intermittent pulsed electromagnetic field stimulation activates the mTOR pathway and stimulates the proliferation of osteoblast‐like cells

Pulsed electromagnetic fields (PEMFs) have been shown to be a noninvasive physical stimulant for bone fracture healing. However, PEMF stimulation requires a relatively long period of time and its mechanism of action has not yet been fully clarified. Recently, the mammalian target of rapamycin (mTOR) pathway has been shown to be involved in bone formation. This study aimed to investigate the effects of PEMFs on osteoblastic MC3T3‐E1 cells by examining various cellular responses including changes in the mTOR pathway. Continuous PEMF stimulation induced a transient phosphorylation of the mTOR pathway, whereas intermittent PEMF stimulation (1 cycle of 10 min stimulation followed by 20 min of stimulation pause) revitalized the reduced phosphorylation. Moreover, PEMF stimulation stimulated cell proliferation (bromodeoxyuridine incorporation) rather than differentiation (alkaline phosphatase activity), with a more notable effect in the intermittently stimulated cells. These results suggest that intermittent PEMF stimulation may be effective in promoting bone fracture healing by accelerating cell proliferation, and in shortening stimulation time. Bioelectromagnetics. 2019;40:412–421. © 2019 Bioelectromagnetics Society.     

Tópicos y reflexiones sobre la reducción de ingresos hospitalarios: de la evidencia a la práctica

Demographic changes and the economic situation of the recent years have conditioned a turning point in health policies, which have decided to progressively prioritize chronicity care programs. Given that hospital costs were concentrated in attention to patients with chronic diseases, reduction on admissions is now a priority target.     

Secretion of Active-Form Streptoverticillium mobaraense Transglutaminase by Corynebacterium glutamicum: Processing of the Pro-Transglutaminase by a Cosecreted Subtilisin-Like Protease from Streptomyces albogriseolus

The transglutaminase secreted by Streptoverticillium mobaraense is a useful enzyme in the food industry. A fragment of transglutaminase was secreted by Corynebacterium glutamicum when it was coupled on a plasmid to the promoter and signal peptide of a cell surface protein from C. glutamicum. We analyzed the signal peptide and the pro-domain of the transglutaminase gene and found that the signal peptide consists of 31 amino acid residues and the pro-domain consists of 45 residues. When the pro-domain of the transglutaminase was used, the pro-transglutaminase was secreted efficiently by C. glutamicum but had no enzymatic activity. However, when the plasmid carrying the S. mobaraense transglutaminase also encoded SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus, the peptide bond to the C side of 41-Ser of the pro-transglutaminase was hydrolyzed, and the pro-transglutaminase was converted to an active form. Our findings suggest that C. glutamicum has potential as a host for industrial-scale protein production.