Takahiko Fujikawa

Structures of mollusc shell framework proteins

Mollusc shells consist of the nacreous mother-of-pearl layer and the prismatic layer. Both layers are microlaminate composites of CaCO3 crystals (aragonite in the nacre and calcite in the prismatic layer) and biopolymers. The main biopolymers are structural proteins, insoluble in water and methanoic acid, which determine the framework of each shell layer and bind soluble polyanionic proteins which determine the type of CaCO3 crystal that grows. Here we report the sequences and structures of the framework proteins for the nacreous and prismatic layers of the pearl oyster, Pinctada fucata.

Essential role for ERK2 mitogen-activated protein kinase in placental development

Background:  Extracellular signal‐regulated kinase 2 (ERK2) has been implicated in cell proliferation, differentiation, and survival. However, its role in vivo remains to be determined.

Critical role of p38 MAPK for regeneration of the sciatic nerve following crush injury in vivo

BackgroundThe physiological function of p38α, which is an isoform of p38 MAPK, has been investigated previously in several studies using pharmacological inhibitors. However, the results regarding whether p38α promotes or inhibits nerve regeneration in vivo have been controversial.MethodsWe generated novel p38α mutant mice (sem mice) with a point mutation in the region encoding the p38α substrate-docking-site, which serves as a limited loss-of-function model of p38α. In the present study, we utilized sem mice and wild-type littermates (wt mice) to investigate the physiological role of p38α in nerve regeneration following crush injuries.ResultsAt four weeks after crush injury, the average axon diameter and the average axon area in sem mice were significantly smaller than those in wt mice. The average myelin sheath thickness in sem mice was reduced compared to wt mice, but no significant difference was observed in the G-ratio between the two groups. The sciatic functional index value demonstrated that functional nerve recovery in sem mice following crush injury was delayed, which is consistent with the histological findings. To investigate the underlying mechanisms of these findings, we examined inflammatory responses of the sciatic nerve by immunohistochemistry and western blotting. At an early phase following crush injury, sem mice showed remarkably lower expression of inflammatory cytokines, such as TNF-α and IL-1β, than wt mice. The expression of Caspase-3 and Tenascin-C were also lower in sem mice. Conversely, at a late phase of the response, sem mice showed considerably higher expression of TNF-α and of IL-1β with lower expression of S-100 than wt mice.ConclusionsThis is the first study of the physiological role of p38 MAPK in nerve regeneration that does not rely on the use of pharmacological inhibitors. Our results indicate that p38α insufficiency may cause an inflammatory disorder, resulting in a delay of histological and functional nerve recovery following crush injury. We conclude that p38 MAPK has an important physiological role in nerve regeneration and may be important for controlling both initiation of inflammation and recovery from nerve injury.

Threshold-like pattern of neuronal activation in the hypothalamus during treadmill running: establishment of a minimum running stress (MRS) rat model.

Despite the indication that the hypothalamo-pituitary-adrenal (HPA) axis is activated during treadmill running, there have not been any studies focusing on the relationship between exercise intensity and region-specific neural activities in hypothalamus. To address this, rats were subjected to 30 min of running, either at middle (supra-LT, 25 m min(-1)) or low speeds (sub-LT, 15 m min(-1)), and c-Fos-(+) cells were counted and compared with control rats. Significant increases in blood glucose and lactate levels, and plasma ACTH and osmolality levels were observed during supra-LT running. Only supra-LT running significantly increased c-Fos induction in various hypothalamic regions, namely, the medial preoptic area (MPO), periventricular nucleus (Pe), suprachiasmatic nucleus (SCN), supraoptic nucleus (SON), parvocellular division of the paraventricular nucleus (pPVN), anterior hypothalamic area (AH), arcuate nucleus (ARC) and posterior hypothalamic nucleus (PH). However, sub-LT caused no effect on c-Fos accumulation. This indicates that the hypothalamus responds uniquely to running in a threshold-like pattern distinct from the speed-dependent pattern previously reported for the medulla oblongata [Ohiwa et al., 2006a,b]. In addition, these results showed a physiologic basis for mild exercise useful for establishing our minimum running stress (MRS) rat model, or the running conditions that minimize the activation of the HPA axis.

Brain glycogen supercompensation following exhaustive exercise

Non‐technical summary  Exercise training elicits an increase in the basal level of muscular glycogen. This happens when glycogen recovers to above its basal level (supercompensation) after it decreases with acute exercise. Although untested, it is hypothesized that, similar to that of skeletal muscle, brain glycogen supercompensation occurs after acute exhaustive exercise. We provide evidence that exhaustive exercise induces glycogen supercompensation not only in skeletal muscles, but also in the brain. Furthermore, we observed exercise training‐induced increases in basal glycogen levels in the cortex and hippocampus, which are involved in motor control and cognitive function. This suggests that, like skeletal muscles, the brain adapts metabolically, probably to meet the increased energy demands of exercise training.

A biphasic regulation of receptor mRNA expressions for growth hormone, glucocorticoid and mineralocorticoid in the rat dentate gyrus during acute stress.

Acute stress increases circulating ACTH and glucocorticoid levels. The hippocampus (HIP) is a target of such stress hormones as glucocorticoid and it also expresses receptors for growth hormone (GH), particularly in the dentate gyms (DG). In order to understand the interactions between glucocorticoids and functions of GH in HIP during acute stress, the mRNA levels for GH receptor (GHR), glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) were investigated in DG in rats exposed to restraint stress in the water (RSW). Using in situ hybridization histochemistry (ISHH), high level expressions of GHR mRNA were detected in DG. These were down-regulated by 14% after 0.5 h of RSW and then up-regulated by 38% over the initial level after 4 h of RSW. This biphasic enhancement of GHR mRNA expression in DG followed the elevation of plasma glucocorticoid levels and paralleled with biphasic expressions of mRNAs for GR and MR in DG. Although circulating GH levels did not show any correlation with the hippocampal GHR mRNA expression, adrenalectomy (ADX) decreased GHR mRNA expression in DG, and the dexamethasone treatment (DEX; 20 microg/100 microl, i.p.) of ADX rats rapidly increased the GHR mRNA expression in DG. These results have suggested that the GHR mRNA expression in the DG is regulated, at least in part, by glucocorticoids and that GH may be involved in responses of the DG to acute stress.

Expression and purification of a mutant human growth hormone that is resistant to proteolytic cleavage by thrombin, plasmin and human plasma in vitro

The region having a sequence from amino acid 134 to 150 in human growth hormone (hGH) is known to be cleaved by proteases in human plasma, plasmin and thrombin. In this study, oligonucleotide primer-directed mutagenesis was used to produce recombinant mutant hGHs resistant to limited proteolysis by these proteases. Substitution of Arg134 and Thr135 of hGH with Asp134 and Pro135 yielded a thrombin-resistant hGH mutant, and substitution of Arg134, Thr135 and Lys140 with Asp134, Pro135 and Ala140 yielded a plasmin-resistant hGH mutant. The latter mutant hGH was also insensitive to in vitro proteolysis by human plasma incubated for 7 days. These alterations in amino acid residues of hGH did not disrupt its biological conformation and retained full growth promoting activities on rat Nb2 cells and human T-47D breast cancer cells.

Anti-obesity compounds in green leaves of Eucommia ulmoides

The anti-hypertensive effect of Eucommia leaves has been confirmed clinically, and the study of their anti-obesity properties has advanced. However, the compounds involved in their anti-obesity effect have not been fully elucidated. In this Letter, we examined the anti-obesity effect of Eucommia green leaf extract (EGLE) divided into five fractions with high porous polystyrene gel and of the compounds isolated, geniposidic acid, asperuloside and chlorogenic acid, respectively. A metabolic syndrome-like clinical model in mice was generated by feeding a 40% high-fat diet to examine the anti-obesity effects of chronic administration of test substance. After 4 weeks, body weight, white adipose tissue weight, plasma triglyceride levels and total cholesterol levels in the model mice were significantly inhibited by the 30% MeOH fraction (containing much higher levels of asperuloside than the other fractions), and these effects were similar to those of EGLE. Chronic administration of isolated asperuloside in Eucommia leaves suppressed increases in model mouse body weight, white adipose tissue weight, plasma triglyceride levels and free fatty acids levels. These results suggest that asperuloside in Eucommia leaves has important anti-obesity effects.

Induction of Brain Prolactin Receptor Long-Form mRNA Expression and Maternal Behavior in Pup-Contacted Male Rats: Promotion by Prolactin Administration and Suppression by Female Contact

Prolactin (PRL) is considered to induce maternal behavior toward foster young in female rats. In the present study, we studied the relationship between pup contact-induced maternal behavior and serum PRL concentrations and brain PRL receptor (PRL-R) mRNA expression in male rats. Both intact and castrated male rats exposed to foster pups gradually developed caretaking behavior such as crouching and licking, but their exhibitions of other maternal behavior components, retrieval/grouping and nest building, were incomplete. However, in the male rats displaying crouching and licking, the concomitant increases in serum PRL concentration and brain mRNA expression for long-form PRL-R were observed. The expression of short-form PRL-R mRNA in the brain was not stimulated by pup contact. Administration of PRL remarkably promoted the onset of those maternal responses in male rats. On the other hand, when an intact male rat was housed in a cage where a lactating female rat and her pups were living, his scores in maternal behavior tests toward pups were lowered. And, concomitantly, increases in serum PRL concentration and brain expression of long-form PRL-R mRNA were reduced. In castrated male rats, however, the ratings of maternal behavior toward foster young, serum PRL concentration increase, or brain long-form PRL-R mRNA expression were not reduced at all by cohabitation with a female and her pups. These findings indicated that maternal behavior was triggered and maintained in pup-contacted male rats through elevated serum PRL levels and induced brain long-form PRL-R.

Eucommia Leaf Extract (ELE) Prevents OVX-Induced Osteoporosis and Obesity in Rats

The cortex of Eucommia ulmoides Oliver is widely used to treat kidney deficiency in traditional Chinese medicine. Its leaves have recently been reported to have anti-obesity properties in metabolic syndrome-like rat models. Due to a sharp decline in estrogen production, obesity, together with osteoporosis, are common problems in postmenopausal women. In this study, we examined the potential effect of Eucommia leaf extract (ELE) in preventing osteoporosis and obesity induced by ovariectomy (OVX). Forty-six female Wistar rats were divided into six groups: Sham-Cont, OVX-Cont, and four OVX groups administered estradiol and different concentrations of ELE 1.25%, ELE 2.5%, and ELE 5%. Treatments were administered after ovariectomy at six weeks of age and continued for 12 weeks. OVX induced a significant decrease in the bone mineral density (BMD) of the lumbar, femora, and tibiae, together with a marked increase in body mass index (BMI). The administration of 5% ELE led to a significant increase in tibial and femoral BMD, as well as significantly increased bone-strength parameters when compared with OVX-Cont rats. According to the suppressed Dpd and increased osteocalcin concentrations in ELE 5% rats, we suggest that varying proportions of bone formation and bone absorption contributed to the enhanced BMD in the femora and tibiae. In addition, significant decreases in body weight, BMI and fat tissue in 5% ELE rats were also observed. These results suggest that ELE may have curative properties for BMD and BMI in OVX rats, and could provide an alternative therapy for the prevention of both postmenopausal osteoporosis and obesity.