The Ndc80 loop region facilitates formation of kinetochore attachment to the dynamic microtubule plus end

Proper chromosome segregation in mitosis relies on correct kinetochore-microtubule (KT-MT) interactions. The KT initially interacts with the lateral surface of a single MT (lateral attachment) extending from a spindle pole and is subsequently anchored at the plus end of the MT (end-on attachment). The conversion from lateral to end-on attachment is crucial because end-on attachment is more robust and thought to be necessary to sustain KT-MT attachment when tension is applied across sister KTs upon their biorientation. The mechanism for this conversion is still elusive. The Ndc80 complex is an essential component of the KT-MT interface, and here we studied a role of the Ndc80 loop region, a distinct motif looping out from the coiled-coil shaft of the complex, in Saccharomyces cerevisiae. With deletions or mutations of the loop region, the lateral KT-MT attachment occurred normally; however, subsequent conversion to end-on attachment was defective, leading to failure in sister KT biorientation. The Ndc80 loop region was required for Ndc80-Dam1 interaction and KT loading of the Dam1 complex, which in turn supported KT tethering to the dynamic MT plus end. The Ndc80 loop region, therefore, has an important role in the conversion from lateral to end-on attachment, a crucial maturation step of KT-MT interaction.     

PLK4 overexpression and its effect on centrosome regulation and chromosome stability in human gastric cancer

Polo-like kinase 4 (PLK4) is a centrosomal protein that is involved in the regulation of centrosome duplication. This study aimed to determine whether the genetic abnormality of PLK4 is involved in human gastric cancer. First, we examined the status of PLK4 mRNA expression in 7 gastric cancer cell lines and 48 primary gastric cancers using an RT-PCR analysis. The upregulation of PLK4 mRNA expression was detected in 57.1 % (4/7) of the gastric cancer cell lines, and a novel PLK4 variant with exon 4, but without exon 5, was identified. In the primary gastric cancers, the upregulation of PLK4 mRNA expression in the cancerous cells was detected in 50.0 % (24/48) of the cases, and this upregulation was statistically significant (P value = 0.0139). Next, we established AGS gastric cancer cells capable of inducibly expressing PLK4 using the piggyBac transposon vector system and showed that PLK4 overexpression induced centrosome amplification and chromosome instability using immunofluorescence and FISH analyses, respectively. Furthermore, PLK4 overexpression suppressed primary cilia formation. Our current findings suggested that PLK4 is upregulated in a subset of primary gastric cancers and that PLK4 overexpression induces centrosome amplification and chromosome instability and causes the suppression of primary cilia formation.     

NARCOBIT: a newly developed inhalational anesthesia system for mice.

NARCOBIT is the first anesthetic system for mice and rats to incorporate a ventilator. Therefore, it is expected to improve the reliability of mice and rat experiments by accurately controlling and maintaining the depth of anesthesia. In this study, we used NARCOBIT for inducing inhalational anesthesia in mice and evaluated the changes in their hemodynamic parameters. ICR mice were anesthetized with 5% isoflurane and room air, followed by endotracheal intubation. Subsequently, they were mechanically ventilated, and anesthesia was maintained by 2% isoflurane for a 60-min period (maintenance state) using NARCOBIT. In study 1, the heart rate (HR) and mean arterial blood pressure (MAP) were measured. The skin blood flow (SBF) from the hind legs was continuously measured during the maintenance state. Subsequently, the concentration-dependent effects of isoflurane on MAP were examined. In study 2, blood samples were obtained from the abdominal aorta for blood gas analysis. The HR and MAP decreased after anesthesia but were stable during the maintenance state. Decreased MAP and concentration-dependent effects of isoflurane were observed. The SBF increased slightly during the maintenance state but this increase was insignificant. The blood gas analysis showed neither hypoxia nor hypercapnia. Since the use of NARCOBIT enables the anesthetic concentration of isoflurane to be easily changed, a suitable anesthesia depth can be obtained for experimental purposes. Therefore, we conclude that NARCOBIT can be used for providing inhalational anesthesia to mice.     

Inhibition of PKCbeta improves glucocorticoid-induced insulin resistance in rat adipocytes

Pretreatment with glucocorticoids for 60 min depressed insulin-stimulated uptake of 2-[3H] deoxyglucose (2-DOG), an effect that neither cycloheximide, an inhibitor of protein synthesis, nor RU38486, a glucocorticoid receptor antagonist, could restore. Preincubation with conventional PKC inhibitors restored dexamethasone-induced insulin resistance. We also examined the dexamethasone-mediated inhibitory effect on insulin-induced 2-DOG uptake in adipocytes overexpressed with wild-type and dominant negative forms of PKCbeta. The dexamethasone-mediated inhibitory effect on insulin-induced 2-DOG uptake was abrogated in adipocytes overexpressed with dominant-negative PKCbeta. These results indicate that PKCbeta may play an important role in glucocorticoid-induced insulin resistance.     

Regional distribution of glucose in mouse brain

After rapid inactivation of the enzymes responsible for glucose metabolism by microwave irradiation, concentrations of glucose in 20 regions of the mouse brain were estimated with combined gas chromatography-mass spectrometry (GC-MS). The highest concentrations of glucose were found in the periventricular nuclei of the hypothalamus and nucleus preopticus (P<0.05). The septum and nucleus amygdaloideus showed significantly higher glucose concentration compared with the cerebral neocortex, olfactory bulb, corpus striatum, cingulum, fornix, colliculus inferior, cerebellar cortex, corpus geniculatum laterale, substantia nigra, and nucleus ruber (P<0.05). The glucose concentration in the substantia nigra and nucleus ruber was significantly lower than in the other regions (P<0.01).     

Detection and Identification of Amanitins in the Wood-Rotting Fungi Galerina fasciculata and Galerina helvoliceps

More than 600 strains of wood-rotting fungi were screened for the detection of amanitins. Three strains of Galerina fasciculata and 18 strains of Galerina helvoliceps contained amanitins. These strains contained mainly α- and β-amanitins in the native fruit bodies, while α- and γ-amanitins were found in liquid-cultured mycelia. Purified amanitins were confirmed by their chromatographic profiles, spectra (UV, Fourier transform infrared, and atmospheric ionization mass), cytotoxicity for mammalian cell lines (3T3 and SiHa), and inhibitory effects on RNA polymerase II. The results revealed that the purified amanitin fractions from these species are identical to authentic amanitins and suggest that these two species must be handled as poisonous mushrooms.     

Sex-specific differences in rat soleus muscle signaling pathway responses to a bout of horizontal and downhill running

Journal of Physiology and Biochemistry - Males and females of many species, including humans, exhibit different muscle responses and adaptations to exercise stress; however, the molecular...