APOLLON Protein Promotes Early Mitotic CYCLIN A Degradation Independent of the Spindle Assembly Checkpoint

In the mammalian cell cycle, both CYCLIN A and CYCLIN B are required for entry into mitosis, and their elimination is also essential to complete the process. During mitosis, CYCLIN A and CYCLIN B are ubiquitylated by the anaphase-promoting complex/cyclosome (APC/C) and then subjected to proteasomal degradation. However, CYCLIN A, but not CYCLIN B, begins to be degraded in the prometaphase when APC/C is inactivated by the spindle assembly checkpoint (SAC). Here, we show that APOLLON (also known as BRUCE or BIRC6) plays a role in SAC-independent degradation of CYCLIN A in early mitosis. APPOLON interacts with CYCLIN A that is not associated with cyclin-dependent kinases. APPOLON also interacts with APC/C, and it facilitates CYCLIN A ubiquitylation. In APPOLON-deficient cells, mitotic degradation of CYCLIN A is delayed, and the total, but not the cyclin-dependent kinase-bound, CYCLIN A level was increased. We propose APPOLON to be a novel regulator of mitotic CYCLIN A degradation independent of SAC.     

Dysregulated Gene Expression in the Primary Osteoblasts and Osteocytes Isolated from Hypophosphatemic Hyp Mice

Osteocytes express multiple genes involved in mineral metabolism including PHEX, FGF23, DMP1 and FAM20C. In Hyp mice, a murine model for X-linked hypophosphatemia (XLH), Phex deficiency results in the overproduction of FGF23 in osteocytes, which leads to hypophosphatemia and impaired vitamin D metabolism. In this study, to further clarify the abnormality in osteocytes of Hyp mice, we obtained detailed gene expression profiles in osteoblasts and osteocytes isolated from the long bones of 20-week-old Hyp mice and wild-type (WT) control mice. The expression of Fgf23, Dmp1, and Fam20c was higher in osteocytic cells than in osteoblastic cells in both genotypes, and was up-regulated in Hyp cells. Interestingly, the up-regulation of these genes in Hyp bones began before birth. On the other hand, the expression of Slc20a1 encoding the sodium/phosphate (Na⁺/Pi) co-transporter Pit1 was increased in osteoblasts and osteocytes from adult Hyp mice, but not in Hyp fetal bones. The direct effects of extracellular Pi and 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on isolated osteoblastic and osteocytic cells were also investigated. Twenty-four-hour treatment with 10⁻⁸ M 1,25(OH)₂D₃ increased the expression of Fgf23 in WT osteoblastic cells but not in osteocytic cells. Dmp1 expression in osteocytic cells was increased due to the 24-hour treatment with 10 mM Pi and was suppressed by 10⁻⁸ M 1,25(OH)₂D₃ in WT osteocytic cells. We also found the up-regulation of the genes for FGF1, FGF2, their receptors, and Egr-1 which is a target of FGF signaling, in Hyp osteocytic cells, suggesting the activation of FGF/FGFR signaling. These results implicate the complex gene dysregulation in osteoblasts and osteocytes of Hyp mice, which might contribute to the pathogenesis.     

Systemic corticosteroids and early administration of antiviral agents for pneumonia with acute wheezing due to influenza A(H1N1)pdm09 in Japan

Background

Pneumonia patients with wheezing due to influenza A(H1N1)pdm09 were frequently treated with systemic corticosteroids in Japan although systemic corticosteroid for critically ill patients with pneumonia caused by influenza A(H1N1)pdm09 has been controversial. Applicability of systemic corticosteroid treatment needs to be evaluated.

Methods/Principal Findings

We retrospectively reviewed 89 subjects who were diagnosed with influenza A(H1N1)pdm09 and admitted to a national hospital, Tokyo during the pandemic period. The median age of subjects (45 males) was 8 years (range, 0–71). All subjects were treated with antiviral agents and the median time from symptom onset to initiation of antiviral agents was 2 days (range, 0–7). Subjects were classified into four groups: upper respiratory tract infection, wheezing illness, pneumonia with wheezing, and pneumonia without wheezing. The characteristics of each group was evaluated. A history of asthma was found more frequently in the wheezing illness (55.6%) and pneumonia with wheezing (43.3%) groups than in the other two groups (p = 0.017). Corticosteroid treatment was assessed among subjects with pneumonia. Oxygen saturation was lower in subjects receiving corticosteroids (steroid group) than in subjects not receiving corticosteroids (no-steroid group) (p<0.001). The steroid group required greater oxygen supply than the no-steroid group (p<0.001). No significant difference was found by the Kaplan-Meier method between the steroid and the no-steroid groups in hours to fever alleviation from the initiation of antiviral agents and hospitalization days. In logistic regression analysis, wheezing, pneumonia and oxygen saturation were independent factors associated with using systemic corticosteroids.

Conclusion

Patients with wheezing and a history of asthma were frequently found in the study subjects. Systemic corticosteroids together with early administration of antiviral agents to pneumonia with wheezing and possibly without wheezing did not result in negative clinical outcomes and may prevent progression to severe pneumonia in this study population.     

Validity of the top-down approach of inverse dynamics analysis in fast and large rotational trunk movements

This study investigated the validity of the top-down approach of inverse dynamics analysis in fast and large rotational movements of the trunk about three orthogonal axes of the pelvis for nine male collegiate students. The maximum angles of the upper trunk relative to the pelvis were approximately 47°, 49°, 32°, and 55° for lateral bending, flexion, extension, and axial rotation, respectively, with maximum angular velocities of 209°/s, 201°/s, 145°/s, and 288°/s, respectively. The pelvic moments about the axes during the movements were determined using the top-down and bottom-up approaches of inverse dynamics and compared between the two approaches. Three body segment inertial parameter sets were estimated using anthropometric data sets (Ae et al., Biomechanism 11, 1992; De Leva, J Biomech, 1996; Dumas et al., J Biomech, 2007). The root-mean-square errors of the moments and the absolute errors of the peaks of the moments were generally smaller than 10 N·m. The results suggest that the pelvic moment in motions involving fast and large trunk movements can be determined with a certain level of validity using the top-down approach in which the trunk is modeled as two or three rigid-link segments.     

A mechanism of Ca2+ release from Ca2+ stores coupling to the Na+/Ca2+ exchanger in cultured smooth muscle cells.

We previously observed Ca2+ release from intracellular Ca2+ stores caused by reduction in extracellular Na+ concentration ([Na+]o). The purpose of this study was to determine whether lowering [Na+]o can elicit Ca2+ release from Ca2+ stores via the Na+/Ca2+ exchanger and to elucidate the mechanisms related to the Ca2+ release pathway in cultured longitudinal smooth muscle cells obtained from guinea pig ileum. Low [Na+]o-induced Ca2+ release was inhibited by antisense oligodeoxynucleotides for Na+/Ca2+ exchanger type 1 (anti-NCX). Application of anti-NCX to cells attenuated both the number of Ca2+ responding cells and the expression of the exchanger. Moreover, microinjection of heparin, a blocker of inositol 1,4,5-trisphosphate (IP3) receptors, into the cells inhibited low [Na+]o-induced Ca2+ release. These findings suggest that low [Na+]o-induced Ca2+ release occurs through an IP3-induced Ca2+ release mechanism due to changes in the Ca2+ flux regulated by the Na+/Ca2+ exchanger.     

Spontaneous and flow-induced Ca2+ transients in retracted regions in endothelial cells

Changes in intracellular calcium concentration ([Ca2+]i) and focal adhesion sites of cultured bovine aortic endothelial cells (BAECs) were simultaneously visualized in real time. Local [Ca2+]i transients were observed at the rear edges of spontaneously migrating BAECs. Furthermore, the majority of starting regions of [Ca2+]i transients retracted continuously. Frequency of [Ca2+]i transients increased with the application of fluid flow. The majority of starting regions of flow-induced [Ca2+]i transients retracted following the occurrence of [Ca2+]i transients. In addition, retracted areas were distributed in the upstream regions of the cell. Application of GdCl3, a mechanosensitive cation channel blocker, resulted in a clear reduction of [Ca2+]i transients and rear retractions in cases of spontaneous and flow-induced BAEC migration. Flow-induced directional rear retractions were also inhibited. Consequently, we conclude that local [Ca2+]i transients play an important role in the migration of BAECs with respect to rear retraction. Furthermore, flow-induced [Ca2+]i transients regulate directional rear retraction under flow conditions.     

The facilitative influence of experiential factors on the parental behavior of male mice (Mus musculus): (i) The effects of the experience of copulation and cohabitation with a pregnant female

Effects of the experience of copulation and cohabitation with a pregnant female on male parental behavior were examined in ICR-strain male mice. The experience of copulation and/or cohabitation with a pregnant female promoted parental behavior, although the cohabitation with a pregnant female per se failed to inhibit infanticide in male mice. Therefore, it is possible that the facilitation of parental behavior is caused by a motivation independent from that of the inhibition of infanticide in male mice. As to the facilitation of parental behavior, the effect of copulation was stronger than that of cohabitation with a pregnant female, and these 2 effects seem to be additive. It is possible that there are 2 independent mechanisms for the facilitation of parental behavior in male mice.     

Serratia marcescens S-layer protein is secreted extracellularly via an ATP-binding cassette exporter, the Lip system

The Serratia marcescens Lip exporter belonging to the ATP-binding cassette (ABC) exporter is known to be involved in signal peptide-independent extracellular secretion of a lipase and a metalloprotease. Although the genes of secretory proteins and their ABC exporters are usually all reported to be linked in several Gram-negative bacteria, neither the lipase nor the protease gene is located close to the Lip exporter genes, lipBCD . A gene ( slaA ) located upstream of the lipBCD genes was cloned, revealing that it encodes a polypeptide of 100 kDa and is partially similar to the Caulobacter crescentus paracrystalline cell surface layer (S-layer) protein. The Lip exporter-deficient mutants of S . marcescens failed to secrete the SlaA protein. Electron micrography demonstrated the cell surface layer of S . marcescens . The S-layer protein was secreted to the cultured media in Escherichia coli cells carrying the Lip exporter. Three ABC exporters, Prt, Has and Hly systems, could not allow the S-layer secretion, indicating that the S . marcescens S-layer protein is strictly recognized by the Lip system. This is the first report concerning secretion of an S-layer protein via its own secretion system.     

Asymmetric amination of 4-methoxyphenylacetone and its related compounds with microorganisms

Summary Asymmetric amination of 4-methoxyphenylacetone and its related compounds by microorganisms was investigated. Among 630 type culture strains, 4-methoxyphenylacetone-aminating ability was found inBrevibacterium, Chromobacterium, Flavobacterium, Mycobacterium, Pseudomonas, andSarcina spp. 4-Methoxyamphetamine produced by these microorganisms was the (S)-(+)-enantiomer.B. linens IFO 12141 was selected as the best strain. The optimum pH of amination was about 7.0, andl-alanine was the most effective aminov donor for the amination. By using this strain, 37.6 mM (S)-(+)-4-methoxyamphetamine was formed with a 94% conversion yield from 4-methoxyphenylacetone. As for substrate specificity,B. linens IFO 12141 catalysed amination of 3,4-dimethoxyphenylacetone and 4-(4-methoxyphenyl)-2-butanone, and formed the corresponding optically active amines.     

Production of Coenzyme A by Sarcina lutea

To develop an efficient method for the production of coenzyme A (CoA), optimal conditions for its formation from pantothenic acid, cysteine, and adenine were studied. A number of microorganisms were screened for production of CoA. Strains belonging to the genera Sarcina, Bacillus, Microbacterium, Micrococcus, and Serratia accumulated CoA. Among these, Sarcina lutea was selected as the best organism, and the culture conditions for the production of CoA were investigated with this organism. Under optimal conditions, 600 mug of CoA per ml was accumulated in the culture broth. CoA was readily isolated in high purity by the use of charcoal, diethylaminoethyl-cellulose, Sephadex G-25, and Dowex-50. Yields of isolated CoA were over 33% from culture broth.