Age-based demography and reproductive biology of three <Emphasis Type="Italic">Epinephelus</Emphasis> groupers, <Emphasis Type="Italic">E. polyphekadion,E. tauvina,</Emphasis> and <Emphasis Type="Italic">E. howlandi</Emphasis> (Serranidae), inhabiting coral reefs in Okinawa

The demography and reproductive biology of three Epinephelus groupers (Serranidae), namely E. polyphekadion, E. tauvina, and E. howlandi in the Yaeyama Islands, Okinawa, were examined based on age assessment using otoliths and gonadal histology. The maximum ages for these three species were 26 year, 23 year, and 17 year. The von Bertalanffy growth functions were also determined for each species. The size and age at 50% female maturity were estimated to be 358 mm in total length (TL) and 6.0 year for E. polyphekadion, 371 mm TL and 6.7 year for E. tauvina, and 327 mm TL and 4.1 year for E. howlandi, respectively. Significant differences between the sexes in size and age frequencies were found in all three species, with males being larger and older than females, or transitional individuals. These results strongly indicated that the population of these three grouper species showed monandric protogynous hermaphroditism. The sex ratios of E. polyphekadion and E. tauvina were biased in favor of females, but that of E. howlandi was equivalent between sexes. The relative sizes of ripe testes indicated that the intensity of sperm competition varied among species suggesting different mating system of each species. Reproductive seasonality was similar among species, with active vitellogenesis coinciding with the annual rise in water temperature. The active spawning period was determined to be between April and May for E polyphekadion, in May for E. howlandi, and from March to June for E. tauvina.     

Factors associated with severity of daytime sleepiness and indications for initiating treatment in patients with periodic limb movements during sleep

Obstructive sleep apnea syndrome (OSAS) is closely associated with hypertension. Activity of angiotensin II (Ang II) and non-dipping nocturnal blood pressure (BP) variability are implicated in hypertension-related target organ damage. We examined the correlation between OSAS with serum Ang II levels and evaluated the risk of non-dipping BP variability in 180 patients with essential hypertension (EHT). Eligible patients were divided into three subgroups based on their apnea-hypopnea index (AHI) evaluated by polysomnography. EHT alone, EHT with mild OSAS, and EHT with moderate/severe OSAS. Ambulatory BP monitoring was used to calculate mean BP over 24 h, as well as diurnal and nocturnal BP variability. Serum Ang II was determined with enzyme-linked immun-osorbent assay. EHT patients with OSAS had significantly higher systolic BP calculated either over 24 h, or by diurnal or nocturnal monitoring (P < 0.05). More EHT patients with OSAS showed non-dipping BP profiles than did EHT patients alone (P < 0.05). The number of patients with non-dipping BP increased with increasing OSAS severity. Surgical treatment alleviated OSAS and reduced AHI (P < 0.05). Preoperative serum Ang II in EHT patients with OSAS was significantly higher than that in those without OSAS (P < 0.05), and showed a rising trend with OSAS severity (P < 0.05). Postoperative serum Ang II, BP and the incidence of non-dipping BP were reduced by surgery to levels lower than preoperative values in patients with OSAS. We therefore conclude that OSAS leads to increased serum Ang II and increased risk of non-dipping BP in patients with EHT.

     

Crystal structure of highly thermostable glycerol kinase from a hyperthermophilic archaeon in a dimeric form

The crystal structure of glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-GK) in a dimeric form was determined at a resolution of 2.4 A. This is the first crystal structure of a hyperthermophilic glycerol kinase. The overall structure of the Tk-GK dimer is very similar to that of the Escherichia coli glycerol kinase (Ec-GK) dimer. However, two dimers of Ec-GK can associate into a tetramer with a twofold axis, whereas those of Tk-GK cannot. This may be the reason why Tk-GK is not inhibited by fructose 1,6-bisphosphate, because the fructose 1,6-bisphosphate binding site is produced only when a tetrameric structure is formed. Differential scanning calorimetry analyses indicate that Tk-GK is a highly thermostable protein with a melting temperature (T(m)) of 105.4 degrees C for the major transition. This value is higher than that of Ec-GK by 34.1 degrees C. Comparison of the crystal structures of Tk-GK and Ec-GK indicate that there is a marked difference in the number of ion pairs in the alpha16 helix. Four ion pairs, termed IP1-IP4, are formed in this helix in the Tk-GK structure. To examine whether these ion pairs contribute to the stabilization of Tk-GK, four Tk-GK and four Ec-GK derivatives with reciprocal mutations at the IP1-IP4 sites were constructed. The determination of their stabilities indicates that the removal of each ion pair does not affect the stability of Tk-GK significantly, whereas the mutations designed to introduce one of these ion pairs stabilize or destabilize Ec-GK considerably. These results suggest that the ion pairs in the alpha16 helix contribute to the stabilization of Tk-GK in a cooperative manner.     

Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

Objectives

Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2.

Materials and methods

Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR.

Results

BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and nuclear translocation were up-regulated when MG63 cells were cultured with both BMP-2 and HA. Western blot analysis revealed that phosphorylation of ERK protein was diminished by HA. Furthermore, the mRNA expressions of noggin and follistatin induced by BMP-2 were preferentially blocked by HA.

Conclusions

These results indicate that HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation.     

Activation of the JNK pathway by nanosecond pulsed electric fields

Nanosecond pulsed electric fields (nsPEFs) are increasingly recognized as a novel and unique tool in various life science fields, including electroporation and cancer therapy, although their mode of action in cells remains largely unclear. Here, we show that nsPEFs induce strong and transient activation of a signaling pathway involving c-Jun N-terminal kinase (JNK). Application of nsPEFs to HeLa S3 cells rapidly induced phosphorylation of JNK1 and MKK4, which is located immediately upstream of JNK in this signaling pathway. nsPEF application also elicited increased phosphorylation of c-Jun protein and dramatically elevated c-jun and c-fos mRNA levels. nsPEF-inducible events downstream of JNK were markedly suppressed by the JNK inhibitor SP600125, which confirmed JNK-dependency of these events in this pathway. Our results provide novel mechanistic insights into the mode of nsPEF action in human cells.     

Accelerating Effect of Soy Peptides Containing Collagen Peptides on Type I and III Collagen Levels in Rat Skin

Two weeks of feeding soy peptides containing 2% collagen peptides increased the levels of type I and III tropocollagen and their mRNAs. In contrast, the diet did not increase the mRNA levels of rat hyaluronan synthases, serine palmitoyltransferase (the rate-limiting enzyme of ceramide synthesis), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (the key enzyme of cholesterol synthesis). These results suggest that feeding of soy peptides with collagen peptides specifically enhanced the tropocollagen level in the skin.     

Skeletal FGFR1 signaling is necessary for regulation of serum phosphate level by FGF23 and normal life span

Fibroblast growth factor (FGF) 23 produced by the bone is the principal hormone to regulate serum phosphate level. Serum FGF23 needs to be tightly regulated to maintain serum phosphate in a narrow range. Thus, we hypothesized that the bone has some phosphate-sensing mechanism to regulate the production of FGF23. Previously we showed that extracellular phosphate induces the phosphorylation of FGF receptor 1 (FGFR1) and FGFR1 signaling regulates the expression of Galnt3, whose product works to increase FGF23 production in vitro. In this study, we show the significance of FGFR1 in the regulated FGF23 production and serum phosphate level in vivo. We generated late-osteoblast/osteocyte-specific Fgfr1-knockout mice (Fgfr1^fl/fl; Ocn^Cre/+) by crossing the Ocn-Cre and the floxed Fgfr1 mouse lines. We evaluated serum phosphate and FGF23 levels, the expression of Galnt3 in the bone, the body weight and life span. A selective ablation of Fgfr1 aborted the increase of serum active full-length FGF23 and the enhanced expression of Galnt3 in the bone by a high phosphate diet. These mice showed more pronounced hyperphosphatemia compared with control mice. In addition, these mice fed with a control diet showed body weight loss after 23 weeks of age and shorter life span. These results reveal a novel significance of FGFR1 signaling in the phosphate metabolism and normal life span.     

Prevalence of antibodies against Simkania negevensis in a healthy Japanese population determined by the microimmunofluorescence test

Simkania negevensis has been associated with bronchiolitis in infants and community-acquired pneumonia in adults. Reports of exposure to this microorganism are only available from Israel, North America and Western Europe. Currently, no standard method for diagnosis of S. negevensis infection has been established nor have prevalence rates been shown in Japan. For the first time we demonstrated the ability of the microimmunofluorescence (MIF) test to detect S. negevensis-specific immunoglobulin G and exposure to S. negevensis in Japan. The positive rate in healthy volunteers was 4.3% (25/588), with rates increasing with age. Results indicate the usefulness of the MIF test as a serological method for detecting S. negevensis-specific antibodies. A standard serological test for infection with S. negevensis is needed.     

Antitumor activity of Nocardia cell wall skeleton preparations in transplantable tumors in syngeneic mice and patients with malignant pleurisy

Summary The antitumor activity of the cell wall skeleton preparations of four species of Nocardia, N. brasiliensis strain 146, N. coeliaca strain 122, N. polychromogenes strain 6, and N. rubra, which showed potent adjuvant activity on the induction of cell-mediated cytotoxicity in allogeneic mice, was examined with the aid of EL-4 leukemia, melanoma B16, and MH-134 hepatoma in syngeneic mice. Preliminary clinical trials were performed and the results suggest that the cell wall skeleton of N. rubra, upon intrapleural injection, may be useful as an immunotherapeutic agent for patients with malignant pleurisy. The chemical properties of these cell wall skeleton preparations are described.