Locally controlled release of basic fibroblast growth factor from multilayered capsules

Biodegradable multilayered capsules encapsulating basic fibroblast growth factor (bFGF) were developed as a cytokine release carrier for drug delivery systems. The multilayered hollow capsules were fabricated via the layer-by-layer (LbL) assembly of chitosan (CT) and dextran sulfate (Dex). The bFGF was encapsulated into the CT/Dex multilayered capsules by controlling the membrane permeability, and the local and sustained release of bFGF from the capsules was examined. At pH < 8.0, the capsule membrane tightened, and FITC-dextran ( Mw = 4000) could not enter the capsules. However, FITC-dextran ( M w = 250000) easily entered the capsules at pH > 8.0, which can be attributed to the electrostatic repulsion of Dex caused by the deprotonation of the amine group in CT. After treatment with acetic acid buffer (pH 5.6), FITC-dextran or bFGF was successfully encapsulated into the capsules. The amount of encapsulated bFGF was approximately 34 microg/1 mg of capsule. Initially, about 30% of the encapsulated bFGF was released in serum-free medium within a few hours, however, the release was sustained over 70 h. When the bFGF encapsulating capsules were added to cell culture medium (serum-free), the mouse L929 fibroblast cells proliferated well for 2 weeks as compared to cultures, where bFGF was added to the medium or where bFGF and empty hollow capsules were added separately. The proliferation is due to the local and sustained release of bFGF from the adsorbent capsule to the cell surface.     

The Molting Biomarker Molecule Exists as 2‐Acetamido‐2‐deoxy‐gluconic Acid in Urine of Blue Crabs and Helmet Crabs

N‐Acetyl‐d ‐glucosamino‐1,5‐lactone 1 has been reported as a candidate component of the sex pheromone mixture of female blue crabs, Callinectes sapidus, since it is present in the urine of reproductive females and males detect it. Theoretically, 1 can convert to a 1,4‐lactone isomer 2 or to the corresponding carboxylic acid, 2‐acetamido‐2‐deoxygluconic acid 3 by hydrolysis in aqueous solution. In this study, we examined the biologically relevant state of equilibrium mixture of 1 , 2 , and 3 in crab urine using ESI‐MS and NMR analyses. The ESI‐MS analysis showed that the dominant form of solubilized synthetic 1 is lactone 1 and/or 2 , immediately after solubilization in deuterated water, seawater, and phosphate buffer and gradually changing to carboxylic acid 3 which becomes most predominant in phosphate buffer. The NMR analysis showed that synthetic 1 converts to other forms in deuterated water and seawater, and reaches an equilibrium mixture of at least three forms within 24 h. In contrast, 1 converts to a single state of another form in deuterated water with 35 mm phosphate buffer pH 7.6 within 24 h, which is identical to the state in urine with or without phosphate buffer. Thus, we conclude that the molting biomarker sensed by male crabs is 3 .     

Molecular characterization of a cold-induced plasma membrane protein gene from wheat

As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Δpmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat. The nucleotide sequence data for wpi6 have been recorded in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AB030210 (cDNA) and AB221353 (genomic DNA).     

Sexual Maturation, Annual Reproductive Cycle, and Spawning Periodicity of the Shore Scorpionfish, Scorpaenodes littoralis

The ovarian structure, sexual maturation, annual reproductive cycle, and spawning periodicity of the shore scorpionfish, Scorpaenodes littoralis, in Uchiura Bay, central Japan, were examined using specimens collected between May 1995 and March 1998 and fishes reared in laboratory. The ovarian stroma and blood vessels run longitudinally through the center of each ovarian lobe. The ovarian peduncles radiate from the central stroma. During the spawning season, gelatinous material is secreted from the epithelia of both the ovarian peduncle and ovarian wall, and the epithelia show morphological changes accompanying the ovarian maturation cycle. The minimum standard length at maturity was 55.2mm for males and 40.2mm for females. Males with mature testes were collected from March to November. Females in the mature or post spawning stages were collected between May and October, when the mean gonadosomatic indices were also high. This indicates that the spawning season of this species occurs between May and October. Four successive types of oocytes were grouped in the mature ovary, comprised of mature, late and early vitellogenic and previtellogenic oocytes respectively, suggesting that this species is a multiple spawner. Four captive females spawned repeatedly at intervals of 2–8 days over a prolonged period (4–8 months); a 2-day spawning interval was the most common for all females. This suggests that female S. littoralis have a 48-h spawning cycle in captivity.     

Angiotensin II AT1 receptors regulate ACE2 and angiotensin-(1-7) expression in the aorta of spontaneously hypertensive rats

When increased in vascular tissues, angiotensin-converting enzyme 2 (ACE2), a carboxypeptidase that hydrolyzes angiotensin II to angiotensin-(1-7), may augment the growth inhibitory and vasodilatory effects of the heptapeptide. We investigated the regulation of ACE2 and angiotensin-(1-7) expression in aortas and carotid arteries of 12-wk-old male spontaneously hypertensive rats (SHR) by determining the effect of sustained angiotensin type 1 (AT(1)) receptor blockade with olmesartan (10 mg.kg(-1).day(-1), n = 13) compared with those that received atenolol (30 mg.kg(-1).day(-1), n = 13), hydralazine (10 mg.kg(-1).day(-1), n = 13), or vehicle (n = 21). Systolic blood pressures were approximately 30% lower (P < 0.05) in rats treated for 2 wk with olmesartan compared with vehicle-treated rats. Both atenolol and hydralazine produced similar decreases in systolic blood pressure. ACE2 mRNA in the thoracic aorta of olmesartan-treated rats (n = 8) was fivefold greater (P < 0.05) than that in vehicle-treated rats (n = 16), whereas atenolol (n = 8) or hydralazine (n = 8) had no effect. Immunostaining intensities in rats treated with olmesartan (n = 5) were also associated with increased (P < 0.05) ACE2 and angiotensin-(1-7) in thoracic aorta media compared with vehicle-treated rats. In contrast, immunostaining intensities for both ACE2 and angiotensin-(1-7) were not different from vehicle (n = 5) in carotid arteries of SHR medicated with either atenolol (n = 5) or hydralazine (n = 5). A comparison of vessel wall dimensions showed that olmesartan selectively reduced the thoracic aorta media-to-lumen ratio (P < 0.05) and media thickness (P < 0.05) without an effect on carotid artery morphometry. Compared with vehicle-treated SHR, vascular hypertrophy determined from media and lumen measurements was not changed in SHR given either atenolol or hydralazine. These data represent the first report of ACE2 and angiotensin-(1-7) expression in the aorta and carotid arteries of SHR. Increased ACE2 and angiotensin-(1-7) in association with altered dimensions of the thoracic aorta but not carotid arteries in response to olmesartan treatment provides evidence that this pathway is regulated by AT(1) receptors and may be important in mediating the pressure-independent vascular remodeling effects of angiotensin peptides.     

Periostin, discovered by nano-flow liquid chromatography and mass spectrometry, is a novel marker of diabetic retinopathy

Diabetes can lead to serious microvascular complications including proliferative diabetic retinopathy (PDR), the leading cause of blindness in adults. Recent studies using gene array technology have attempted to apply a hypothesis-generating approach to elucidate the pathogenesis of PDR, but these studies rely on mRNA differences, which may or may not be related to significant biological processes. To better understand the basic mechanisms of PDR and to identify potential new biomarkers, we performed shotgun liquid chromatography (LC)/tandem mass spectrometry (MS/MS) analysis on pooled protein extracts from neovascular membranes obtained from PDR specimens and compared the results with those from non-vascular epiretinal membrane (ERM) specimens. We detected 226 distinct proteins in neovascular membranes and 154 in ERM. Among these proteins, 102 were specific to neovascular membranes and 30 were specific to ERM. We identified a candidate marker, periostin, as well as several known PDR markers such as pigment epithelium-derived factor (PEDF). We then performed RT-PCR using these markers. The expression of periostin was significantly up-regulated in proliferative membrane specimens. Periostin induces cell attachment and spreading and plays a role in cell adhesion. Proteomic analysis by LC/MS/MS, which permits accurate quantitative comparison, was useful in identifying new candidates such as periostin potentially involved in the pathogenesis of PDR.     

Oral supplementation with carbohydrate- and branched-chain amino acid-enriched nutrients improves postoperative quality of life in patients undergoing hepatic resection

The long-term outcomes of branched-chain amino acid (BCAA) administration in patients undergoing hepatic resection remain unclear. The aim of this study is to assess the impact of oral supplementation with BCAA-enriched nutrients on postoperative quality of life (QOL) in patients undergoing liver resection. A prospective randomized clinical trial was conducted in 96 patients undergoing hepatic resection. Patients were randomly assigned to receive BCAA supplementation (AEN group, n = 48) or a conventional diet (control group, n = 48). Postoperative QOL and short-term outcomes were regularly and continuously evaluated in all patients using a short-form 36 (SF-36) health questionnaire and by measuring various clinical parameters. This study demonstrated a significant improvement in QOL after hepatectomy for liver neoplasm in the AEN group based on the same patients’ preoperative SF-36 scores (P < 0.05). Perioperative BCAA supplementation preserved liver function and general patient health in the short term for AEN group patients compared to those not receiving the nutritional supplement. BCAA supplementation improved postoperative QOL after hepatic resection over the long term by restoring and maintaining nutritional status and whole-body kinetics. This study was registered at (registration number: NCT00945568).     

Leptin in sarcopenic visceral obesity: possible link between adipocytes and myocytes

The combination of sarcopenia, age-related loss of muscle strength and mass, and obesity has been recognized as a new category of obesity among the elderly. Given that leptin has been hypothesized to be involved in the pathogenesis of sarcopenic obesity, we investigated the relationship between plasma leptin levels and thigh muscle sarcopenia and visceral obesity. Thigh muscle cross-sectional area (CSA) and visceral fat area were measured using computed tomography as indices for muscle mass and visceral fat, respectively, in 782 middle-aged to elderly subjects (303 men and 479 women), participating in a medical check-up program. Visceral obesity was defined as visceral fat area >100 cm², and sarcopenia was defined as < (one standard deviation − mean of thigh muscle CSA/body weight of young subjects [aged <50 years]).Thigh muscle CSA was significantly and negatively associated with plasma levels of leptin in both men (β = -0.28, p<0.0001) and women (β = -0.20, p<0.0001), even after correcting for other confounding parameters, including age, body weight, body height, visceral fat area, blood pressure, homeostatic model assessment index, and high sensitive C reactive protein. Subjects were divided into four groups based on presence or absence of sarcopenia or visceral obesity. Plasma levels of leptin were higher in subjects with sarcopenic visceral obesity than in those with either sarcopenia or visceral obesity alone. These findings indicate that sarcopenic visceral obesity is a more advanced, and suggest that leptin may link visceral obesity and sarcopenia.     

Immunoreactivity of gonadotrophs (FSH and LH Cells) and gonadotropin subunit gene expression in the male chub mackerel Scomber japonicus pituitary during the reproductive cycle

The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are heterodimers composed of a common α subunit (GPα) and a unique β subunit (FSHβ or LHβ); they are synthesized in and secreted from gonadotrophs (FSH and LH cells) in the pituitary. Little is known about the roles of FSH and LH during spermatogenesis in perciform fishes. In this study, we examined immunoreactive changes in FSH and LH cells, and changes in the gene expression of the three gonadotropin subunits in the pituitary of male chub mackerel Scomber japonicus during testicular development. FSHβ-immunoreactive (ir) and LHβ-ir cell area were measured immuno-histochemically based on the FSH and LH cell-occupying area in the proximal pars distalis. The FSHβ-ir cell area increased significantly during spermiation, while FSHβ mRNA levels, already high at the beginning of spermatogenesis, increased further, peaking during spermiation. In contrast, LHβ-ir cell area and LHβ mRNA levels, which were low at the beginning of spermatogenesis, increased significantly during late spermatogenesis, peaking during spermiation. For both FSH and LH, GtHβ-ir cell area and GtHβ mRNA levels decreased until gonadal resting. GPα mRNA levels showed similar changes to LHβ mRNA levels. These results suggest that in the chub mackerel, FSH may play an important role in the early and late phases of spermatogenesis, and that LH may play a role during late spermatogenesis and spermiation. Moreover, our results demonstrate that changes in GtHβ-ir cell area were accompanied by similar changes in the expression of the FSHβ and LHβ genes, both of which increased during testicular development.     

Steroidogenic and maturation-inducing potency of native gonadotropic hormones in female chub mackerel, Scomber japonicus

ABSTRACT: BACKGROUND: The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are produced in the pituitary gland and regulates gametogenesis through production of gonadal steroids. However, respective roles of two GtHs in the teleosts are still incompletely characterized due to technical difficulties in the purification of native GtHs. METHODS: Native FSH and LH were purified from the pituitaries of adult chub mackerel, Scomber japonicus by anion-exchange chromatography and immunoblotting using specific antisera. The steroidogenic potency of the intact chub mackerel FSH (cmFSH) and LH (cmLH) were evaluated in mid- and late-vitellogenic stage follicles by measuring the level of gonadal steroids, estradiol-17beta (Epsilon2) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In addition, we evaluated the maturation-inducing potency of the GtHs on same stage follicles. RESULTS: Both cmFSH and cmLH significantly stimulated E2 production in mid-vitellogenic stage follicles. In contrast, only LH significantly stimulated the production of 17,20beta-P in late-vitellogenic stage follicles. Similarly, cmLH induced final oocyte maturation (FOM) in late-vitellogenic stage follicles. CONCLUSIONS: Present results indicate that both FSH and LH may regulate vitellogenic processes, whereas only LH initiates FOM in chub mackerel.