Fermented milk retains beneficial effects on skeletal muscle protein anabolism after processing by centrifugation and supernatant removal.

Lactobacillus-fermented milk can stimulate anabolic effects in skeletal muscle. Fermented milk containing Lactobacillus produces aqueous molecules, such as free AA and lactate. This study aimed to investigate how processing fermented milk by centrifugation and removal of supernatant affects AA absorption and postprandial skeletal muscle protein synthesis (MPS) when mice are fed fermented milk. We gavaged male Sprague-Dawley rats with skim milk (S), fermented milk (F), or processed fermented milk (P), and examined the total AA content in portal vein blood (reflecting AA absorption) and plantaris muscle MPS at 30, 60, and 90 min following administration. Relative to fasted rats, at 30 min the total AA concentration in portal vein blood from rats in the P groups was significantly higher, followed by F and S, respectively. The MPS rates were higher for the F or P groups compared with the S group. Phosphorylation levels of p70S6 kinase in the P and F groups were significantly higher than those for the S group 30 min after administration, although the level of Akt phosphorylation was similar among the groups. These results suggested that fermentation improves AA absorption that in turn enhances postprandial MPS via Akt-independent mechanisms, and that processed fermented milk retains these favorable effects on MPS.

Towards a unified model of neutrino-nucleus reactions for neutrino oscillation experiments.

A precise description of neutrino-nucleus reactions will play a key role in addressing fundamental questions such as the leptonic CP violation and the neutrino mass hierarchy through analyzing data from next-generation neutrino oscillation experiments. The neutrino energy relevant to the neutrino-nucleus reactions spans a broad range and, accordingly, the dominant reaction mechanism varies across the energy region from quasi-elastic scattering through nucleon resonance excitations to deep inelastic scattering. This corresponds to transitions of the effective degree of freedom for theoretical description from nucleons through meson-baryon to quarks. The main purpose of this review is to report our recent efforts towards a unified description of the neutrino-nucleus reactions over the wide energy range; recent overall progress in the field is also sketched. Starting with an overview of the current status of neutrino-nucleus scattering experiments, we formulate the cross section to be commonly used for the reactions over all the energy regions. A description of the neutrino-nucleon reactions follows and, in particular, a dynamical coupled-channels model for meson productions in and beyond the [Formula: see text](1232) region is discussed in detail. We then discuss the neutrino-nucleus reactions, putting emphasis on our theoretical approaches. We start the discussion with electroweak processes in few-nucleon systems studied with the correlated Gaussian method. Then we describe quasi-elastic scattering with nuclear spectral functions, and meson productions with a [Formula: see text]-hole model. Nuclear modifications of the parton distribution functions determined through a global analysis are also discussed. Finally, we discuss issues to be addressed for future developments.

Separation of pygmy dipole and M1 resonances in 90Zr by a high-resolution inelastic proton scattering near 0°.

A high-resolution measurement of inelastic proton scattering off (90)Zr near 0° was performed at 295 MeV with a focus on a pronounced strength previously reported in the low-energy tail of giant dipole resonance. A forest of fine structure was observed in the excitation energy region 7-12 MeV. A multipole decomposition analysis of the angular distribution for the forest was carried out using the ECIS95 distorted-wave Born approximation code with the Hartree-Fock plus random-phase approximation model of E1 and M1 transition densities and inclusion of E1 Coulomb excitation. The analysis separated pygmy dipole and M1 resonances in the forest at E(PDR)=9.15±0.18 MeV with Γ(PDR)=2.91±0.64 MeV and at E(M1)=9.53±0.06 MeV with Γ(M1)=2.70±0.17 MeV in the Lorentzian function, respectively. The B(E1)↑ value for pygmy dipole resonance over 7-11 MeV is 0.75±0.08 e(2)fm(2), which corresponds to 2.1±0.2% of the Thomas-Reiche-Kuhn sum rule.

Altered growth, differentiation, and responsiveness to epidermal growth factor of human embryonic mesenchymal cells of palate by persistent rubella virus infection.

We previously demonstrated that human embryonic mesenchymal cells derived from the palate (HEMP cells) retain alkaline phosphatase (ALP) content and capacity for collagen synthesis after long-term culture, and their growth is markedly stimulated by epidermal growth factor (EGF). There was a dramatic decrease in ALP content and capacity to synthesize collagen in HEMP cells (HEMP-RV cells) persistently infected with rubella virus (RV). EGF increased ALP activity and decreased collagen synthesis in HEMP cells, whereas EGF showed no effect on these activities in HEMP-RV cells. Growth of HEMP-RV cells was slightly reduced compared with that of HEMP cells. EGF stimulated growth of HEMP cells and to a lesser extent of HEMP-RV cells. Binding of 125I-EGF to cell-surface receptors in HEMP-RV cells was, to our surprise, twice as much as that in HEMP cells. However, internalization of bound 125I-EGF in HEMP-RV cells was profoundly diminished. Thus, persistent RV infection causes not only changes in HEMP cell growth and differentiation but a decrease in or loss of HEMP cell responsiveness to EGF. The effects of persistent RV infection on palatal cell differentiation as well as growth may be responsible for the pathogenesis of congenital rubella. Furthermore, since HEMP cells appear to be closely related to osteoblasts, these results suggest a mechanism for RV-induced osseous abnormalities manifested in congenital rubella patients.

Control of VX2 carcinoma cell growth in culture by calcium, calmodulin, and prostaglandins.

Based on our in vivo observation that growth of VX2 carcinoma transplanted in rabbits paralleled development of hypercalcemia, we studied the regulation of VX2 tumor growth using a clonal cell line isolated from VX2 tumor (VX2-L). VX2-L cell growth was dependent on prostaglandins released by the cultured cells into the medium, since indomethacin suppressed VX2-L growth, and prostaglandins A2, E1, E2, F1 alpha, and F2 alpha stimulated VX2-L proliferation. In contrast, prostaglandins D2 and I2 inhibited VX2-L proliferation. In contrast to previous reports, increases in extracellular calcium concentration promoted VX2-L growth not only directly but indirectly through augmentation of prostaglandin E synthesis. Antagonists of the intracellular calcium binding protein calmodulin inhibited cell replication. Increases in extracellular calcium also stimulated production of a nonprostaglandin macromolecular bone-resorbing factor. This factor may account for the hypercalcemia which we were unable to block by indomethacin. These results suggest a close relationship between VX2-L growth, prostaglandin production, and hypercalcemia. It is proposed that calcium blockers and anticalmodulin drugs might be powerful anticancer and/or antihypercalcemic agents for malignant cells such as VX2-L.

Occurrence of hypercalcemia and leukocytosis with cachexia in a human squamous cell carcinoma of the maxilla in athymic nude mice: a novel experimental model of three concomitant paraneoplastic syndromes.

Hypercalcemia and leukocytosis may occur in conjunction as paraneoplastic syndromes associated with malignant disease. Here we describe a human squamous cell carcinoma of the maxilla that was associated with hypercalcemia and leukocytosis, and also cachexia. The primary tumor was surgically removed and established in permanent cell culture. When either primary tumors or cultured tumor cells were inoculated into nude mice, the nude mice developed the same paraneoplastic syndromes as those which occurred in the patient from whom the tumor was originally derived. The plasma calcium was increased two and one-half-fold and the WBC count 30-fold, and the body weight was decreased by 45% in tumor-bearing animals. Each of these paraneoplastic syndromes was alleviated by surgical excision of the tumor, indicating that the paraneoplastic syndromes were due to a factor or factors produced by the primary tumor. The development of each of these paraneoplastic syndromes in nude mice correlated positively with the other two syndromes. We examined the organs of tumor-bearing mice and found striking histopathologic abnormalities in the bones, spleen, and liver, but no infiltration with tumor cells. The bones showed marked evidence of osteoclastic bone resorption. This model of a human tumor associated with the hypercalcemia-leukocytosis paraneoplastic syndrome, together with cachexia, should make it possible to determine the mechanisms responsible for these paraneoplastic syndromes and their relationship to each other.

Ameloblast-lineage cells of rat tooth germs proliferate and scatter in response to hepatocyte growth factor in culture.

Hepatocyte growth factor (HGF) is considered to be one of the mediators of epithelial-mesenchymal interactions during early organogenesis and to be involved in the development of murine molars. In this study, the immunohistochemical localization of HGF and of its receptor, c-Met, revealed that HGF was distributed in the proliferating mesenchymal cells in the dental papillae and that c-Met was continuously expressed in the epithelial cells during the development of rat incisors. These observations confirmed the involvement of HGF in the development of rat incisors, as demonstrated previously in molars. We then used a primary culture of ameloblast-lineage cells, prepared from mandibular incisors of young rats, to examine the direct effects of HGF on the growth and differentiation of ameloblasts. We found that HGF at 2-20 ng/ml induced a marked increase in the number of ameloblast-lineage cells and in the scattering of such cells. Our results suggest that HGF promotes the proliferation and scattering of ameloblast-lineage cells simultaneously.

Induction of teratocarcinoma F9 cell differentiation with cis-diammine dichloroplatinum(II) (CDDP).

cis-Diammine dichloroplatinum(II) (CDDP) is the salt of a platinum compound which has been noted to have a wide spectrum of activity against malignant disorders. We have studied the effects of CDDP on embryonal carcinoma F9 cell differentiation. In the presence of this agent in vitro, the cells showed rapid morphological changes, a marked increase in the mRNA expression of various differentiation markers accompanied by a loss of tumorigenicity. These results indicate that the differentiation of F9 cells is induced with CDDP.

The development of peritumoral stroma required for IL-12 induced tumor regression depends on the T cell/IFN-gamma-involving host-tumor interaction.

T cell migration into tumor masses is a critical process in the scenario of IL-12-induced tumor regression. Our previous study showed that this depends on the development of peritumoral stroma prior to IL-12 therapy. The present study investigated the regulation of the development of peritumoral stroma in comparison with tumor-parenchymal stroma. In the OV-HM and CSA1M tumor models, tumor regression associated with T cell migration was induced following IL-12 treatment. Both OV-HM and CSA1M tumor masses growing in syngeneic mice developed peritumoral stroma before IL-12 treatment. However, peritumoral stroma was not observed in these two types of tumor masses generated in nude mice, T cell-depleted syngeneic mice, anti-IFN-gamma mAb-treated mice or IFN-gamma-deficient mice. In contrast, parenchymal stroma formation did not appear to be affected because tumors generated in these groups of mice exhibited rather higher growth rates than those of tumors in normal syngeneic mice. Importantly, the lack of peritumoral stroma in tumor masses was associated with the failure of T cells to migrate to these tumor masses: splenic T cells prepared from IL-12-treated tumor-bearing mice migrated into the corresponding tumor mass growing in untreated syngeneic recipient mice, whereas portions of the same donor cells failed to migrate into the above stroma-negative tumor masses. These results indicate that the development of peritumoral and parenchymal stroma is differentially regulated; there exist functional differences in the two types of stroma; and the formation of peritumoral stroma requires components of the host's immune system such as IFN-gamma and T cells.

Insolubilized properties of UV-irradiated CO3 apatite-collagen composites.

To examine the response to biological hard tissues, a carbonate-containing hydroxyapatite with chemical composition and crystallinity similar to those of bone was synthesized at pH 7.4 and 60 degrees C. The apatite powder was mixed with collagen solution, whose antigenicity had been removed by enzymatic treatment, and formed into apatite-collagen pellets. After insolubilization by UV-irradiation for 4 h, the composites showed remarkably reduced disintegration and maintained their shape under 3.6 MPa of stress after 1 wk incubation in 0.9% NaCl solution. They showed good biocompatibility when implanted beneath the periosteum cranii of rats. The UV-irradiated sample kept its features well and was packed with newly created material 3 wk after implantation.

Regeneration of periodontal primary afferents of the rat incisor following injury of the inferior alveolar nerve with special reference to neuropeptide Y-like immunoreactive primary afferents.

Regeneration of primary afferents and the expression of neuropeptide Y (NPY) in the lingual periodontal ligament of the rat incisor were examined following different types of injury (resection or crush) of the inferior alveolar nerve (IAN) combined with superior cervical ganglionectomy. In normal animals, protein gene product 9.5 (PGP 9.5)-like immunoreactivity (-LI) was localized in the middle areas of the alveolus-related part of lingual periodontal ligament; some of these nerve fibers showed terminal ramification and morphologies resembling those of the periodontal Ruffini endings, and very few thin varicose NPY-like immunoreactive (-IR) nerve fibers were detected around the blood vessels. Three days following crush injury of the IAN, the number of PGP 9.5-IR nerve fibers decreased, then increased to the normal levels around 10-15 days following injury. NPY-IR primary afferents first appeared around 5 days following crush injury, increased in number gradually, reaching a peak around 14 days, and then decreased. No NPY-IR primary afferents were detected 56 days following crush injury of the IAN. The terminal morphology of NPY-IR primary afferents observed around 10-14 days following injury was similar to that of PGP 9.5-IR nerve fibers in the normal animals, but less expanded. The changes in distribution of PGP 9.5-IR and NPY-IR nerve fibers following resection were similar to those observed following crush injury but regeneration was slightly delayed. The present results suggest that injury-evoked NPY is closely associated with the regeneration process of mechanoreceptors in the periodontal ligament following injury of the IAN.

Altered distribution of Schwann cells in the periodontal ligament of the rat incisor following resection of the inferior alveolar nerve: an immunohistochemical study on S-100 proteins.

The present study employed immunohistochemistry for the detection of S-100 proteins to reveal the alteration in the distribution of Schwann cells in the periodontal ligament of the rat incisor following resection of the inferior alveolar nerve (IAN). In normal animals, S-100-immunostaining demonstrated the profiles of Ruffini endings, primary mechanoreceptors in the periodontal ligament, in the alveolus-related part of the ligament. Under the electron microscope, S-100-like immunoreactivity (-LI) was observed in the cytoplasm of the terminal Schwann cell elements and in some axon profiles of the Ruffini endings. During the regeneration, S-100-like immunoreactive (-IR) terminal Schwann cells in the alveolus-related part of the ligament gradually decreased in number. In contrast, S-100-LI was found in the spindle-shaped cells at the shear zone (the border between alveolus-related and tooth-related parts) and in the tooth-related part, where S-100-LI was rarely detected in normal animals. Immunoelectron microscopic observations revealed that some S-100-IR spindle-shaped cells contained fibrous long spacing (FLS) fibers, suggesting that they were Schwann cells. Some regenerating axons were observed at the shear zone, but were rarely found in the tooth-related part. With the progress of the regeneration of the periodontal Ruffini endings, S-100-IR terminal Schwann cells became rearranged in the alveolus-related part by 42-56 days post injury, whereas the S-100-IR spindle-shaped Schwann cells in the shear zone and tooth-related part disappeared when the regeneration was complete.

Effects of neonatal injury of the inferior alveolar nerve on the development and regeneration of periodontal nerve fibers in the rat incisor.

Our previous study showed that the migration of terminal Schwann cells occurred in the periodontal ligament of the rat lower incisor following transection of the inferior alveolar nerve (IAN) in the adult animals [Y. Atsumi, K. Matsumoto, M. Sakuda, T. Maeda, K. Kurisu, S. Wakisaka, Altered distribution of Schwann cells in the periodontal ligament of the rat incisor following resection of the inferior alveolar nerve: An immunohistochemical study on S-100 proteins, Brain Res. 849 (1999) 187-195]. The aim of the present study was to investigate the effects of neonatal transection of the IAN on the regeneration of axon elements and Schwann cells in the periodontal ligament of the rat lower incisor. Following transection of IAN at post-natal day 5 (PN 5d), when the numbers of both axon elements and the terminal Schwann cells were very small, regenerating nerve fibers appeared between post-injured days 7 (PO 7d) and PO 14d, and increased in number thereafter gradually. Although the terminal morphologies of regenerated Ruffini endings became identical to those of the adult animals by PO 54d, the number of regenerated PGP 9.5-IR nerve fibers did not recover the adult levels even by PO 56d. A small number of Schwann cells migrated into the shear zone, the border between the alveolus-related part (ARP) and the tooth-related part (TRP), but did not enter into the TRP. Following transection of the IAN at PN 14d or PN 28d, when clusters of apparent terminal Schwann cells could be recognized, axon regeneration started around PO 5d. Individual axon terminals of the regenerating Ruffini endings ramified and became identical to those of the adult animals around PO 28d, but the number of regenerated Ruffini endings was smaller than that of the adult animals. Similar to the adult animals, the migration of Schwann cells into the shear zone and TRP occurred, and disappeared prior to the completion of the axonal regeneration. The present results indicate that the migration of the Schwann cells into TRP during the regeneration of the periodontal nerve fibers following nerve injury to the IAN depends on the maturation of the terminal Schwann cells of the periodontal Ruffini endings, not on post-operative time.

Decrease in analgesic effect of nifedipine following chronic morphine administration.

The analgesic effect of nifedipine, a calcium antagonist, was compared in the acetic acid-induced writhing test with control and morphine-tolerant mice. Nifedipine inhibited the writhing syndrome less effectively in mice made tolerant to morphine. The results support the notion that inhibition of the calcium influx is one of the causes of the analgesic action of morphine and that its chronic administration causes an increase of calcium entry.

Morphine prevents peroxynitrite-induced death of human neuroblastoma SH-SY5Y cells through a direct scavenging action.

N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC12), a nitric oxide donor, 3-morpholinosydnonimine (SIN-1), a generator of peroxynitrite (ONOO-), and peroxynitrite induced cell death accompanied by DNA fragmentation in human neuroblastoma SH-SY5Y cell cultures. Morphine prevented the cell death induced by SIN-1 or peroxynitrite, but not that induced by NOC12. The protective effect of morphine was concentration-dependent (10-100 microM), but was not antagonized by naloxone. The selective ligands for opioid receptor subtypes, [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO, micro-opioid receptor agonist), [D-Pen2,5]enkephalin (DPDPE, delta-opioid receptor agonist) and trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)benze neacetamide (U-50488, kappa-opioid receptor agonist) even at the concentration of 100 microM did not prevent the cell death induced by SIN-1. From measurement of the absorbance spectrum of peroxynitrite, the decomposition of peroxynitrite in 0.25 M potassium phosphate buffer (pH 7.4) was very rapid and complete within seconds. However, the absorbance was very stable in the presence of morphine. In addition, morphine inhibited peroxynitrite-induced nitration of tyrosine in a concentration-dependent manner. These results indicate that morphine rapidly reacts with peroxynitrite. The present study showed that morphine prevented peroxynitrite-induced cell death through its direct scavenging action, suggesting that morphine can protect cells against damage caused by peroxynitrite.

Effect of prolonged administration of clonidine on [3H]PN 200-110 and [125I]omega-conotoxin binding in mouse brain.

The effect of chronic exposure to clonidine or morphine on clonidine- and morphine-induced analgesia in mice was examined. Binding of L- or N-type calcium channel antagonist to cortical membrane fractions was also compared between these groups of mice. A decrease in the analgesic effect of clonidine and morphine was observed following prolonged administration of clonidine or morphine. Binding of [3H]PN 200-110, an L-type calcium channel antagonist, decreased following prolonged administration of clonidine whereas it increased after morphine treatment. On the other hand, a significant increase of [125I]omega-conotoxin, an N-type calcium channel antagonist, binding was observed after chronic clonidine or morphine treatment. These results will be discussed in relation with the possible development of cross-tolerance between clonidine and morphine through the change in calcium channels, more specifically in N-type channels.

The elastic modulus of the temporomandibular joint disc from adult dogs.

The present study was designed to measure the elastic properties of temporomandibular joint (TMJ) discs from six adult dogs. Each disc was divided mediolaterally into medial, middle, and lateral parts. Under tension, the articular disc exhibited a non-linear stress-strain relationship, which could be represented as two lines (two moduli of elasticity) connected at a point of stress around 1.5 MPa. These two elastic moduli of the disc were approximately 44 MPa and 92 MPa in the lower- and higher-stress regions, respectively. Elastic moduli of the articular disc in the middle area were significantly different from that in the lateral area of the disc. The reaction to external force appeared to be different in the medial, middle, and lateral regions of the disc.

Effect of a pulsing electromagnetic field on demineralized bone-matrix-induced bone formation in a bony defect in the premaxilla of rats.

A 2-mm non-healing bony defect was prepared in the premaxilla of male Wistar rats weighing about 180 g as a simulation of an alveolar cleft, for determination of whether a pulsing electromagnetic field (PEMF) could promote regeneration of bone induced by demineralized bone matrix (DBM). The defect was either treated with 7 mg DBM or was left as a non-grafted control. The rats were exposed to a PEMF with a frequency of 100 Hz, a 10-ms-wide burst with 100 microseconds-wide quasi-rectangular pulses, repeating at 15 Hz, and magnetic field strength of 1.5-1.8 G. Alkaline phosphatase activity increased significantly from day 7 in the DBM-graft-plus-PEMF group and from day 10 in the DBM-graft group, reaching a maximum on day 14. A greater-than-two-fold rise in alkaline phosphatase activity and a three-fold rise in the amount of 45Ca incorporation in the DBM-graft-plus-PEMF group were attained compared with those of the DBM-graft group. The DBM-graft-plus-PEMF group produced more bone with almost complete osseous bridging in the defect sites than did the group treated with DBM only on day 35. The findings indicate that PEMF had an enhancing effect on the bone-inductive properties of the DBM through the stimulation of osteoblast differentiation induced by DBM.

EMG approach to assessing tongue activity using miniature surface electrodes.

Miniature surface electrodes were developed to examine the EMG activity of the tongue during various functions under normal physiological conditions with minimal fear and discomfort to human subjects. An outline of the electrodes, which consist of electrically high-conductive silver paste, small rubber caps, and fine wires, is introduced, and the practicability of their clinical application is discussed.

Effect of centrifugal force on growth of mouse osteoblastic MC3T3-E1 cells in vitro.

The effect of biomechanical force on growth of skeletal tissue was studied in monolayer cultures of mouse osteoblastic MC3T3-E1 cells which were centrifuged at 320 g for 15 min to 72 h in a CO2 incubator. Centrifugation of the cells for 30 min in low concentrations (0.3 or 1%) of fetal bovine serum (FBS) caused a two-fold increase of [3H]thymidine incorporation at 20 h from the start of centrifugation. However, centrifugation under 10% FBS caused no increase in [3H]thymidine incorporation into DNA. Under 0.3% FBS, [3H]thymidine incorporation increased in a manner dependent on the period of centrifugation and reached a maximum when the cells were centrifuged for 3 h. Stimulation of DNA synthesis by centrifugation was abolished in the presence of H-7, an inhibitor of protein kinase C. Moreover, conditioned medium collected from the centrifuged cultures increased [3H]thymidine incorporation by two-fold over the basal when added to a quiescent culture of MC3T3-E1 cells. These results suggest that centrifugal force stimulates growth of osteoblastic cells through autocrine secretion of some diffusible growth-promoting activity. On the other hand, centrifugation of the cells inhibited induction by FBS of alkaline phosphatase activity and calcium-uptake, two indices of the differentiated phenotype of osteoblasts.