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Cell differentiation and cell fate determination in sensory systems are essential for stimulus discrimination and coding of environmental stimuli. Color vision is based on the differential color sensitivity of retinal photoreceptors, however the developmental programs that control photoreceptor cell differentiation and specify color sensitivity are poorly understood. In Drosophila melanogaster, there is evidence that the color sensitivity of different photoreceptors in the compound eye is regulated by inductive signals between cells, but the exact nature of these signals and how they are propagated remains unknown. We conducted a genetic screen to identify additional regulators of this process and identified a novel mutation in the hibris gene, which encodes an irre cell recognition module protein (IRM). These immunoglobulin super family cell adhesion molecules include human KIRREL and nephrin (NPHS1). hibris is expressed dynamically in the developing Drosophila melanogaster eye and loss-of-function mutations give rise to a diverse range of mutant phenotypes including disruption of the specification of R8 photoreceptor cell diversity. We demonstrate that hibris is required within the retina, and that hibris over-expression is sufficient to disrupt normal photoreceptor cell patterning. These findings suggest an additional layer of complexity in the signaling process that produces paired expression of opsin genes in adjacent R7 and R8 photoreceptor cells.

We describe a new member of the aldo-keto reductase (AKR) superfamily in the silkworm Bombyx mori. On the basis of its amino acid sequence and phylogenetic tree, this AKR belongs to the AKR1B family and has been designated as bmALD1. In the current study, recombinant bmALD1 was overexpressed, purified to homogeneity and kinetically characterized. We discovered that bmALD1 uses NADPH as a coenzyme to reduce carbonyl compounds such as DL-glyceraldehyde, glucose and 2-nonenal. No NADH-dependent activity was detected. To the best of our knowledge, bmALD1 is only the third AKR characterized in silkworm which, given its substrate specificity, could play a major role in glucose metabolism and antioxidant reactions. Our data provide an increased understanding of insect AKR function.

Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels carry Ih, which contributes to neuronal excitability and signal transmission in the nervous system. Controlling the trafficking of HCN1 is an important aspect of its regulation, yet the details of this process are poorly understood. Here, we investigated how the C-terminus of HCN1 regulates trafficking by testing for its ability to redirect the localization of a non-targeted reporter in transgenic Xenopus laevis photoreceptors. We found that HCN1 contains an ER localization signal and through a series of deletion constructs, identified the responsible di-arginine ER retention signal. This signal is located in the intrinsically disordered region of the C-terminus of HCN1. To test the function of the ER retention signal in intact channels, we expressed wild type and mutant HCN1 in HEK293 cells and found this signal negatively regulates surface expression of HCN1. In summary, we report a new mode of regulating HCN1 trafficking: through the use of a di-arginine ER retention signal that monitors processing of the channel in the early secretory pathway. Electronic supplementary material The online version of this article (doi:10.1007/s00018-014-1705-1) contains supplementary material, which is available to authorized users.

Marine animals, and sea urchin species in particular, have several advantages for use in environmental research. However, the spawned eggs of the sea urchin quickly lose fertility, although the fertile period can be lengthened by the addition of antibiotics to the sea water (Epel et al., 2004). We evaluated five species of Japanese sea urchin and the gametes of Hemicentrotus pulcherrimus could be maintained for 2 weeks or more at low temperature with the addition of antibiotics to sea water. We also demonstrated the practicality of shipping these preserved gametes as experimental material for universities and schools to use immediately for bioassays of physical and chemical impacts on the marine environment.

We aimed to clarify the effects of cold stimulation at various temperatures on mitochondrial activity and vascular endothelial growth factor (VEGF) expression in vitro. Human fibroblast, human mesenchymal stem cell, and rat skeletal muscle myoblast cell lines were used. For each cell type, cells were divided into 4 groups and stimulated in various cold temperatures (0, 4, 17 and 25°C) 3 times for 15 min each by placement on crushed ice or floating on cold water set at each temperature. Control cells were subjected to warm water at 37°C. Factors related to mitochondrial activity, mitochondrial DNA copy numbers, and VEGF expression were analyzed 24 h after the last cold stimulation. In all cell types, significant increases of factors related to mitochondrial activity and mitochondrial DNA copy numbers were seen in the 4°C and 17°C-stimulated cells compared with control cells. In rat skeletal muscle cells stimulated at 4°C, VEGF expression significantly increased compared to the control cells. Our data suggest that cold stimulation at certain temperatures promotes mitochondrial activity, biogenesis and VEGF expression.

Background and Objective: T cells and their cytokines are believed to be key factors in periodontal disease and bone resorption. We previously showed that T cells transferred to nude mice were related to inflammatory bone resorption in vivo. However, it has not been clarified whether T cells can induce bone resorption in the absence of B cells. In this study, we therefore investigated the ability of T cells to induce bone resorption without B cells, using both T cell- and B cell-deficient mice with severe combined immune deficiency (SCID). Material and Methods: Escherichia coli lipopolysaccharide (LPS) was injected into the gingivae of SCID mice reconstituted by T cells (SCID + T mice). Wild-type C.B-17 mice and SCID mice were used as control animals. Alveolar bone resorption and production of cytokines in the gingivae were then compared histopathologically and immunohistologically. Results: The degree of bone resorption in SCID + T mice was significantly greater than that in SCID mice but less than that in wild-type mice. The same tendency was found for expression of receptor activator of nuclear factor kappaB ligand. The number of interferon-gamma-positive cells in SCID + T mice was the highest of the three groups. In contrast, interleukin-4-positive cells were detected in wild-type mice but not in SCID + T and SCID mice. Conclusion: The results suggest that T cells are able to promote LPS-induced bone resorption in the absence of B cells. The expressions of cytokines in the presence of B cells are quite different., Journal of Periodontal Research, 43(5), pp.549-555; 2008

Male Sprague-Dawley rats were underwent superior mesenteric artery occlusion for 45 min followed by reperfusion under urethane anesthesia. Each milk proteins (300 mg/ kg) were injected into the duodenum 1 h before the induction of ischemia. IL-6 release in the blood was measured in course of time. Whey protein isolate (WPI) had the suppressive effect against ischemia/reperfusion-induced IL-6 release, while casein and bovine serum albumin had no effect. Both alpha-lactalbumin (aLA) and beta-lactoglobulin (bLG), major components contained in WPI, significantly inhibited the increase of IL-6. However, aLA had more potent activity compared with bLG. Positive correlation between serum aLA level and efficacy for Ischmia/reperfusion was observed. N(G)-nitro-L-arginine methyl ester, nitric oxide (NO) synthase inhibitor, significantly reversed suppressive effect on IL-6 release by aLA. These findings show that aLA has a marked suppressive effect on pro-inflammatory cytokine release, at least in part, through NO-mediated mechanism.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1, HCN2, HCN3, and HCN4) are differentially expressed in the retina and brain. They are important for multiple neuronal functions including shaping resting membrane potential, modulating synaptic output, and dendritic integration.1 HCN1, which is most abundant in the inner segment (IS) of rods and cones, carries a feedback current that shapes vision at mesopic and photopic conditions.2–4 Trafficking is one of the key regulatory mechanisms for HCN1 function, exemplified by the altered HCN1 trafficking observed in CA1 pyramidal neurons after temporal lobe epilepsy.5 HCN1 trafficking involves a series of signals that are beginning to be unraveled. The large intracellular C-terminal domain of HCN1 is a hot-spot for trafficking signals. This region has two binding sites for the tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an accessory subunit of HCN channels.1 TRIP8b maintains the concentration of HCN1 at the distal dendrites in hippocampal CA1 and neocortical layer V neurons.6–10 However, neither HCN1 compartmentalization nor surface expression in the retina is TRIP8b-dependent.11 The C-terminus of HCN1 also contains the cyclic nucleotide binding domain (CNBD) common to all HCN channels. The CNBD was shown to regulate the surface expression of HCN2 and may participate in the trafficking of other HCN channels.12 Following the CNBD, there is a binding site to filamin-A, which is required for clustering HCN1 at the cell surface and promotes HCN1 internalization in hippocampal neurons.13,14 We have previously identified a di-arginine endoplasmic reticulum (ER) retention signal at the C-terminus of HCN1 using X. laevis rod photoreceptors.15 This ER retention signal negatively regulates the surface expression of HCN1. However, the process of overcoming the retention signal under physiological conditions remains unclear. An ER retention signal is usually counteracted by a forward trafficking signal that promotes the movement of the protein from the endomembrane system to the surface (i.e., plasma membrane). However, the overall effect of fusing the HCN1 C-terminus on a reporter membrane protein is to retain the reporter in the ER. This suggests that the forward trafficking signal is present in other regions of HCN1. One candidate for mediating the forward trafficking signal is protocadherin 15, which interacts with the N-terminus (NT) of HCN1 in inner ear cells.16 However, HCN1 and protocadherin 15 do not colocalize in photoreceptors17,18; raising the question as to which other HCN1 trafficking signals or regulators may promote its trafficking in this cell. The goal of this study was to identify novel forward trafficking signals using the established transgenic X. laevis approach for probing membrane protein trafficking pathways.19 We found that in rods, the intracellular NT of HCN1 is necessary for the protein to target the IS plasma membrane (ISPM). Through investigating a series of truncation mutants, we identified a leucine-based ER export signal that can override the di-arginine ER retention signal. This finding of combinatorial trafficking signals controlling HCN1 localization provides insight into how the amount of HCN1 functioning at the cell surface is regulated under normal and disease conditions.

Total parenteral nutrition (TPN) is associated with cholestasis and hepatic steatosis, which can be lethal in infants who cannot be fed orally. The present animal study focused on the metabolic complications in the liver that may occur due to the excessive administration of fat-free TPN. Thirty infant (3-week-old) male SD rats weighing 60-70 g were randomly allocated to five groups (n = 6): the OD group received an oral diet, the FT group received an oral diet and was fasted overnight on the last day of experiment before sacrifice, the 0% fat group received TPN without fat, the 20% fat group received TPN with 20% of calories from fat emulsion, and the 40% fat group received TPN with 40% of calories from fat emulsion. All TPN regimens were isocaloric, isonitrogenic, and administered for 4 days. In the 0% fat group, plasma levels of liver enzymes were significantly higher than in the other groups. Pathological examination showed hepatomegaly and severe fatty changes without cholestasis in the 0% fat group. The results of this study in infant rats indicate the importance of including fat in the TPN regimen in order to prevent the abnormal hepatic changes associated with the excessive administration of fat-free TPN.

Na(+) /K(+) -ATPase (NKA) participates in setting electrochemical gradients, cardiotonic steroid signaling and cellular adhesion. Distinct isoforms of NKA are found in different tissues and subcellular localization patterns. For example, NKA α1 is widely expressed, NKA α3 is enriched in neurons and NKA α4 is a testes-specific isoform found in sperm flagella. In some tissues, ankyrin, a key component of the membrane cytoskeleton, can regulate the trafficking of NKA. In the retina, NKA and ankyrin-B are expressed in multiple cell types and immunostaining for each is striking in the synaptic layers. Labeling for NKA is also prominent along the inner segment plasma membrane (ISPM) of photoreceptors. NKA co-immunoprecipitates with ankyrin-B, but on a subcellular level colocalization of these two proteins varies dependent on the cell type. We used transgenic Xenopus laevis tadpoles to evaluate the subcellular trafficking of NKA in photoreceptors. GFP-NKA α3 and α1 are localized to the ISPM, but α4 is localized to outer segments (OSs). We identified a VxP motif responsible for the OS targeting by using a series of chimeric and mutant NKA constructs. This motif is similar to previously identified ciliary targeting motifs. Given the structural similarities between OSs and flagella, our findings shed light on the subcellular targeting of this testes-specific NKA isoform.

Objective: This study was designed to elucidate whether there were differences in the hormonal responses of the parameters involving triacylglycerol (TG) deposition and mobilization in adipose tissue among the stages of the estrous cycle in female rats. Measurements: Adipose tissue was obtained from the parametrial region in female rats at each stage of the estrous cycle. Lipoprotein lipase (LPL) activity in the extracts of acetone/ether powders of the tissues was measured as a parameter for TG deposition. Norepinephrine-stimulated lipolysis in isolated fat cells was measured as a parameter for TG mobilization. Results: LPL activity changed periodically during the estrous cycle; the activity level was highest at diestrus, began to decrease at proestrus, reached a minimum at estrus, began to increase again at metestrus-1, and increased further at metestrus-2. At diestrus and proestrus, LPL activity was increased with an increase in plasma insulin levels, suggesting that plasma insulin was the predominant up-regulator of LPL. But at estrus, metestrus-1 and metestrus-2, LPL activity remained low even when plasma insulin levels were high, indicating that it was not up-regulated by plasma insulin. Norepinephrine-stimulated lipolysis in fat cells was high at estrus and metestrus-1 and low at diestrus. Conclusion: The hormonal responses of LPL activity and lipolysis in adipose tissue differed depending on the stage of the estrous cycle.

Transition of hemoglobin (Hb) from larval to adult types during the metamorphosis in a salamander Hynobius retardatus has been reported to occur almost independently of thyroid activity, in contrast to the case with many amphibians. In order to obtain further information on the mechanism of the transition in H. retardatus, larval and adult globin cDNAs were cloned, and the globin gene expression was analyzed in normally developing and metamorphosis-arrested animals. Northern hybridization and RT-PCR revealed that larval globin genes were initially expressed 5 days before hatching, and unexpectedly remained expressed even in juveniles 2 years old. The adult globin gene was expressed 19 days after hatching, much earlier than the initiation of morphological metamorphosis. Furthermore, the pattern of globin gene expression in metamorphosis-arrested larvae was almost identical to that in normal controls, suggesting that the transition occurs independently of thyroid hormones. In larvae recovering from anemia, precocious Hb transition, which occurs in Xenopus laevis and Rana catesbeiana, did not occur in H. retardatus. In situ hybridization convincingly demonstrated that the erythropoietic sites are the ventral blood island and the dorsolateral plate at the prehatching stage. During the ontogeny they changed to the liver, kidney, and spleen and were finally restricted to the spleen. Single erythroid cells expressed concurrently larval and adult globin genes, as demonstrated by double in situ hybridization. Thus the transition occurred within a single erythroid cell population, a unique characteristic of H. retardatus.

An experimental comparative histomorphological study was made on intestinal healing processes following an Albert-Lembert suture with approximation of the serosal surface and Gambee's layer to layer anastomosis of a dog. There was no obvious complications such as postoperative hemorrhage, anastomotic stenosis or anastomotic leakage. Although both anastomoses resulted in a good healing process, Gambee's layer to layer suture, which caused the submucosal layers to face each other, showed better wound healing at the anastomosis in terms of layer to layer attachment. As a conclusion, Gambee's layer to layer anastomosis seemed to be a better anastomotic technique in terms of wound healing for the intestinal anastomosis.

The term for re-entrainment to a new light-dark cycle in streptozotocin (STZ)-induced diabetic rats was significantly longer than that in control rats. In STZ-induced diabetic rats, the same level of phase delay in the suprachiasmatic nucleus neuronal firing rhythm was observed in control rats after glutamate application. Therefore, 5-HT function in the hypothalamus is thought to be decreased in STZ-induced diabetic rats. These results suggest that postsynaptic neuronal function is still maintained in the suprachiasmatic nucleus of STZ-induced diabetic rats. Therefore, 5-HT mechanisms may play an important role in the maintenance of this function.

The effect of essential trace metals on bone metabolism was investigated in the femoral-metaphyseal tissues obtained from skeletal-unloaded rats. Skeletal unloading was designed by using the model of hindlimb suspension in rats; the animals were fed for 4 days with the unloading. Femoral-metaphyseal tissues were cultured for 24 hours in a medium containing either vehicle (control), nickel, manganese, cobalt, copper, zinc, or zinc-chelating dipeptide (beta-alanyl-L-histidinato zinc; AHZ) in the concentration range of 10(-6) to 10(-4) M. Bone biochemical components (alkaline phosphatase activity, glucose consumption, and DNA content) were significantly decreased by skeletal unloading. The presence of zinc sulfate or AHZ (10(-5) and 10(-4) M) caused a significant increase of alkaline phosphatase activity in the bone tissues from unloaded rats. This effect was not seen by nickel, manganese, cobalt and copper (10(-6) to 10(-4) M). The culture medium glucose was clearly consumed by the bone tissues. This consumption was inhibited by nickel, manganese, or copper (10(-5) and 10(-4) M), while cobalt, zinc, and AHZ had no effect. DNA content in the bone tissues from unloaded rats was significantly increased by all metal compounds (10(-5) M). The effect of AHZ on bone components was greater than zinc sulfate. The AHZ (10(-5) M)-increased alkaline phosphatase activity in the bone tissues from unloaded rats was clearly blocked by the presence of cycloheximide (10(-6) M), staurosporine (10(-7) M), dibucaine (10(-4) M), or okadaic acid (10(-7) M). The present study demonstrates that, of various essential trace metals, zinc compounds have an unique anabolic effect on bone metabolism in the femoral-metaphyseal tissues of rats with skeletal unloading. Zinc-chelating dipeptide may stimulate bone protein synthesis through the mechanism that is involved in protein kinases.

Regucalcin (RGN/SMP30) was discovered in 1978 and is a unique calcium-binding protein contains no EF-hand motif calcium-binding domain. Its name, regucalcin, was proposed as it suppresses activation of enzymes related to calcium signalling. The regucalcin gene (rgn) is localized on the X chromosome. Regucalcin plays its role of suppressor protein in intracellular signalling pathways, including of protein kinases and protein phosphatase activities, protein synthesis, and DNA and RNA synthesis in liver cells. Overexpression of endogenous regucalcin has a suppressive effect on cell proliferation in modelled rat hepatoma H4-II-E cells, which are induced by various signalling stimulations in vitro. This suppressive effect is independent of apoptosis. Endogenous regucalcin plays a suppressive role on overproduction of proliferating cells in regenerating rat liver in vivo. Regucalcin mRNA expression is uniquely down-regulated in development of carcinogenesis in liver of rats in vivo. Regucalcin mRNA and protein expressions are also depressed in human hepatoma HepG2 cells, MCF-7 breast cancer cells, and prostate cancer LNCaP cells. Depression of regucalcin expression may be associated with activity progression of carcinogens. Regucalcin may be a key molecule suppressor protein in cell proliferation and carcinogenesis.

The purpose of this study was to determined whether fibers in the anterior latissimus dorsi (ALD) muscle from aged Japanese quail have decreased hypertrophic or proliferative responses to 30 days of stretch overload compared with fibers from adult birds. Two groups of quail were studied, 12-wk-old quail (adult; n = 16) and 90-wk-old quail (aged; n = 16). The left wing of each bird was overloaded with a weight corresponding to 10% of the bird's body weight, and the right wing served as the intra-animal control. Quails were killed after 30 days of stretch overload. Total fiber number was quantified by counting all the fibers in a transverse section from the midbelly of the ALD muscle. ALD muscles in aged quails retained the capacity to increase their muscle mass (145%), total fiber number (49%), and fiber cross-sectional area (54%) in response to stretch overload. The ALD muscle in aged quail had a significantly lower increase in muscle mass (33%) and mass corrected for nonmuscle tissue (36%) compared with the ALD from young adult birds. Age had no effect on fiber type distribution shifts with stretch. These results suggest that although muscles in old birds have a substantial ability to adapt to enlarge, stretch-induced hypertrophy is attenuated in muscles from old quail.

The aim of this study was to investigate how T-helper 17 cells (Th17 cells), interleukin (IL)-17, and interleukin-6 contribute to root resorption during orthodontic tooth movement.Fifteen male 6-week-old Wistar rats were subjected to orthodontic force of 10 or 50 g to induce a mesially tipping movement of the upper first molars for 7 days. The expression levels of TRAP, IL-17, the IL-17 receptor (IL-17R), and IL-6 proteins were determined in periodontal ligament (PDL) by immunohistochemical analysis. Moreover, the fluorescent localization immunoassay was performed to detect Th17 cells. Furthermore, the effects of IL-17 on IL-6 release were investigated using human PDL cells in vitro. The effect of IL-17 on osteoclastogenesis was evaluated by TRAP staining, actin ring staining, and the pit formation assay.The immunoreactivity for Th17, IL-17, IL-17R, and IL-6 was detected in PDL tissue subjected to the orthodontic force on day 7. IL-17 increased the release of IL-6 from human periodontal ligament cells in a time-dependent manner. Moreover, IL-17 stimulated osteoclastogenesis from human osteoclast precursor cells, and these effects were partially suppressed by an anti-IL-6 antibody.These results suggest that Th17 cells may aggravate the process of orthodontically induced inflammatory root resorption.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:04:21Z No. of bitstreams: 1 S0036-46651994000300005.pdf: 595431 bytes, checksum: f73d8a209f65720a2cf30b914c19be5e (MD5) Made available in DSpace on 2013-08-22T19:04:21Z (GMT). No. of bitstreams: 1 S0036-46651994000300005.pdf: 595431 bytes, checksum: f73d8a209f65720a2cf30b914c19be5e (MD5) Previous issue date: 1994-06-01 Made available in DSpace on 2013-09-30T20:07:10Z (GMT). No. of bitstreams: 2 S0036-46651994000300005.pdf: 595431 bytes, checksum: f73d8a209f65720a2cf30b914c19be5e (MD5) S0036-46651994000300005.pdf.txt: 7 bytes, checksum: 212b0306580d4f0044d18f9a3edcc832 (MD5) Previous issue date: 1994-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:17:10Z No. of bitstreams: 2 S0036-46651994000300005.pdf: 595431 bytes, checksum: f73d8a209f65720a2cf30b914c19be5e (MD5) S0036-46651994000300005.pdf.txt: 7 bytes, checksum: 212b0306580d4f0044d18f9a3edcc832 (MD5) Made available in DSpace on 2014-05-20T15:17:10Z (GMT). No. of bitstreams: 2 S0036-46651994000300005.pdf: 595431 bytes, checksum: f73d8a209f65720a2cf30b914c19be5e (MD5) S0036-46651994000300005.pdf.txt: 7 bytes, checksum: 212b0306580d4f0044d18f9a3edcc832 (MD5) Previous issue date: 1994-06-01 Estudou-se sequencialmente, à microscopia eletrônica de transmissão, a interação entre Paracoccidioides brasiliensis (Pb) e células inflamatórias em hamsters inoculados por via intratesticular. Seis horas após inoculações havia predominância de neutrófilos, estando presentes algumas células mononucleares e eosinófilos. Os neutrófilos foram progressivamente substituídos por células mononucleares. Fungos viáveis apresentavam-se fagocitados ou circunscritos por células inflamatórias, geralmente com ampla interface hospedeiro-parasita. Fungos mortos ou degenerados eram acompanhados de interfase estreita. A camada externa da parede do Pb era às vezes quebrada quando em contacto com neutrófilos, em vários pontos, sendo os fragmentos dessa parede descamados e fagocitados. Células fúngicas pequenas com um único núcleo se relacionavam com ampla interfase enquanto as células maiores e multinucleadas apresentavam paredes irregulares, por vezes, contendo lomasoma e/ou estrutura semelhante à mielina. Diferentes padrões de interação do Pb com células do hospedeiro podem ser decorrentes do a fluxo de células inflamatórias funcionalmente diferentes ao local de inoculação ou à idade dos fungos ou ambos os fatores. Interaction between Paracoccidioides brasiliensis (Pb) and inflammatory cells in hamster testis was studied sequentially by transmission electron microscopy. In early lesions (six hours after inoculation), polymorphonuclear neutrophils (PMNs) were the major and mononuclear cells and eosinophils were the minor constituents of the inflammatory cells. PMNs were later replaced by mononuclear cells. Viable Pb cells were phagocytosed or surrounded by inflammatory cells. Preserved Pb cells usually had broad host-parasite interphases, whereas dying ones had narrow interphases. The outer layer of the fungus wall was sometimes broken by PMN in some focal points, broken pieces being peeled off and phagocytosed. Small Pb cells were uninuclear, and were often related to broad interphase. Large Pb cells were multinucleated with irregularly shaped wall, and sometimes had lomasome and/or myelin like structures. Different interaction patterns of Pb with inflammatory cells may be due to functionally different host cell flow to the inoculation site or due to the age of Pb cells or both. UNESP Faculty of Medicine Department of Pathology Jikei Medical University Institute of Basic Medicine UNESP Faculty of Medicine Department of Pathology

Ferritin synthesis is known to be regulated translationally by specific mRNA-protein interactions between an iron-responsive element (IRE) localized in the 5′ untranslated region of ferritin mRNA and IRE-binding protein (IRE-BP). Binding of IRE-BP to IRE depresses its translation. In the present study, we demonstrated that ferritin synthesis in macrophages is strongly induced under hypoxic conditions by diethylmaleate, a sulfhydryl-reactive agent. The induction by diethylmaleate decreased as the oxygen tension rose. O − 2 is involved in this oxygen effect, because the induction was prevented when O − 2 -generating agents were present. Actinomycin D did not inhibit the ferritin synthesis induced by diethylmaleate under hypoxi. These results suggest that O − 2 is ivolved in post-transcriptional regulation of ferritin sythesis.

The aim of this study was to investigate how interleukin (IL)-8 (cytokine-induced neutrophil chemoattractant; CINC-1) and monocyte chemotactic protein (MCP)-1/CCL2 contribute to root resorption during orthodontic tooth movement.Forty 6-week-old male Wistar rats were subjected to orthodontic force of 10 or 50 g to induce a mesially tipping movement of the upper first molars for 7 days. We determined the expressions of CINC-1, CXCR2, and MCP-1 proteins in root resorption area using immunohistochemistry. Furthermore, we investigated the effects of compression forces (CF) on IL-8 and MCP-1 production by human periodontal ligament (hPDL) cells. We observed an effect of chemokine treatment on rat odonto/osteoclasts in dentin slices that recapitulated root resorption.The immunoreactivity for CINC-1/CXCR2 and MCP-1 was detected in odontoclasts and PDL fibroblasts by the orthodontic force of 50 g on day 7. CF increased the secretion and the expression of mRNA of IL-8 and MCP-1 from PDL cells in a magnitude-dependent manner. Moreover, CINC-1 and MCP-1 stimulated osteoclastogenesis from rat osteoclast precursor cells.IL-8 (CINC-1) and MCP-1 may therefore facilitate the process of root resorption because of excessive orthodontic force.

Single fibers from toe or anterior tibialis muscle contracted transiently and then tonically in the presence of 1.8 mM Ca2+ on addition of 10 microM Ag+. Exposure of fibers to Cd2+ completely inhibited tonic contraction and modified phasic contraction to some extent. Nifedipine at 10 microM initially potentiated and then completely inhibited twitch tension; subsequently, fibers no longer contracted phasically in response to 20 microM Ag+, whereas slight tonic contraction still occurred. Fibers with membrane potential clamped at -90 mV produced maintained inward current on application of Ag+. Simultaneous administration of 1 mM Cd2+ and 10 microM Ag+ to fibers voltage clamped with the double mannitol gap technique almost completely blocked the inward current. Removal of Cd2+ elicited a rapid and large inward current. Ag(+)-induced inward current was inhibited when 1 mM Cd2+ was applied to fibers during development of the inward current. In fibers paralyzed with 10 microM nifedipine, the inward current induced by 10 microM Ag+ was partially inhibited. These results suggest that phasic contraction induced by Ag+ is controlled by L-type Ca2+ channels (probably voltage sensors) located in the T-tubular membrane, whereas tonic contraction involves Ca2+ channels sensitive and/or insensitive to dihydropyridine in the surface and T-tubular membranes.

This study was undertaken to determine whether epidermal growth factor (EGF) regulates the secretion of mouse placental lactogen (mPL)-I and mPL-II. Primary cell cultures were prepared from placentas from days 7, 9, and 11 of pregnancy and cultured for up to 5 days. Addition of EGF (20 ng/ml) to the medium resulted in significant stimulation of mPL-I secretion by the second day of culture in cells from days 7 and 9 of pregnancy and significant inhibition of mPL-II secretion by the third or fourth day of culture in cells from days 7, 9, and 11. Dose-response studies carried out with cells from day 7 of pregnancy demonstrated that the minimum concentration of EGF that stimulated mPL-I secretion and inhibited mPL-II secretion was 1.0 ng/ml. EGF did not affect the DNA content of the cells or cell viability, assessed by trypan blue exclusion, nor did it have a general effect on protein synthesis. There are three types of PL-containing giant cells in mouse placental cell cultures: cells that contain either mPL-I or mPL-II and cells that contain both hormones. Immunocytochemical analysis and the reverse hemolytic plaque assay indicated that EGF treatment was accompanied by a significant increase in the number of cells that produce mPL-I, but among the PL cells that contained mPL-I, there was no change in the fraction of cells that contained only mPL-I or the fraction that contained both mPL-I and mPL-II. In contrast, EGF treatment did affect the distribution of mPL-II among PL cells. In control cultures, about 75% of the cells that contained mPL-II also contained mPL-I, but in EGF-treated cultures, all of the cells that contained mPL-II also contained mPL-I. These data suggest that EGF regulates mPL-I and mPL-II secretion at least partly by regulating PL cell differentiation.

Surfactant rich lipid (lipid) was extracted from cell free 10 000 × g pellets of amniotic fluid. White blood cells (WBC) were isolated from human donors. 36 × 10 7 WBC and 5 g rabbit lung were incubated with pretreated lipid or dipalmitoyl lecithin (lecithin). Leukotrienes (LTs) were identified by high performance liquid chromatography (HPLC) and bioassay, and quantified by radioimmunoassay. Peaks of LTC 4 and LTD 4 on HPLC and guinea-pig ileum contraction could be identified in lipid and lecithin groups, but not in the control group. LTC 4 production by lipid and lecithin groups was significantly higher than that by the control group. An involvement of amniotic fluid surfactant in leukotriene production is suggested.

The primary objective of this study was to develop a cell culture system for assessing effects of putative secretagogues on mouse PL-I (mPL-I) secretion. Trophoblast from days 7 to 11 of pregnancy was dispersed in collagenase, and the cells were fractionated on a Percoll gradient and plated on collagen gels in serum-free medium. Cells from days 7-9 of pregnancy yielded five bands on Percoll gradients and those from days 10 and 11 yielded six. mPL-I was present in four of the bands of cells from each day of pregnancy. Cells from day 7 of pregnancy that banded at a density of 1.044 g/ml secreted the largest amount of mPL-I during 5 days of culture. The mPL-I concentration of the medium of these cells increased for the first 3 or 4 days of culture and then declined on the fifth day. mPL-II could not be detected in the medium until the third or fourth day of culture, and its concentration increased thereafter. Cell viability was about 90% at the time of plating, remained at about 80% between days 1 and 4, and then declined on day 5. The cell type that produced mPL-I was identified with the reverse hemolytic plaque assay and by staining with anti-mPL-I antiserum. Both methods indicated that mPL-I was produced by giant cells. The ability of the cells to respond to putative secretagogues was examined using mPL-II and progesterone. mPL-II, at concentrations ranging between 10 ng/ml and 10 micrograms/ml, had no effect on the mPL-I concentration of the medium when it was present for up to 3 days of culture, which suggests that mPL-II does not inhibit mPL-I secretion in vitro. Incubation of the cells in the presence of 100-1000 ng/ml progesterone caused a dose- and time-dependent reduction in the mPL-I concentration of the medium and a decrease in the number of cells that stained with anti-mPL-I antiserum. The effect of progesterone on both endpoints was not apparent until the second day of treatment. These data suggest that progesterone inhibits mPL-I secretion at least in part by inhibiting the differentiation of mPL-I-producing giant cells. The fact that the mPL-I-producing cells responded to progesterone indicates that this culture system will be useful in assessing effects of putative secretagogues on mPL-I secretion.

Cerebral lesions accompanying stroke in male stroke-prone spontaneously hypertensive rats (SHRSP, n = 10) were examined by both magnetic resonance imaging (MRI) and histological evaluation. T2-weighted MR images (T2-WI), taken 1-2 days after animals showed behavioral hyperactivity, indicated hyperintense regions in the occipital cortex, caudate putamen and/or thalamus. The areas of hyperintensity on T2-WI corresponded to neurodegenerative regions including edema, gliosis, and softening of the tissue. T1-weighted images (T1-WI) did not show any hyperintense regions. However T1-weighted images enhanced by the contrast media Gd-DTPA (Gd-T 1-WI) showed hyperintense spots within some of the hyperintense areas on T2-WI, which exhibited neurodegenerative regions such as thrombus, angionecrosis and hemorrhage in addition to the edematous formation. The hyperintense areas on Gd-T1-WI were smaller than those on T2-WI. In some animals, hypointense spots on T2-, T1- and Gd-T1-WI were found within the hyperintense areas, which corresponded to clots. Extensive histological examination did not reveal any additional cerebral degeneration which had not been detected on the MR images. These findings indicate that MRI is useful for detecting and differentiating various types of cerebrovascular disease in this model.

Parathyroid hormone-related protein (PTHrP), which causes hypercalcemia associated with malignant tumors, is known to be present in milk. Gene expression of PTHrP in the mammary gland increases markedly during parturition and with the onset of lactation. Even when circulating PTHrP levels are extremely low or below the detection limit, milk PTHrP levels are remarkably high. Parathyroid hormone-related protein derived from the mammary gland is assumed to play a role in maintaining the maternal calcium homeostasis and calcium transport from blood to milk. In previous studies that determined the PTHrP concentrations in milk, the pretreatments and diluent composition were not standardized. Here, we investigated the effect of various pretreatment procedures and diluent constitutions and the consequent PTHrP concentrations in commercial milk and milk products in Japan. Significant differences were found in PTHrP concentrations in raw milk samples subjected to different combinations of pretreatments (mixing, centrifugation, acidification, and heating) and diluents (0 pM standard solution of PTHrP, plasma treated with protease inhibitors, and original diluent). We measured the PTHrP concentrations in normal liquid milk, processed milk, milk drinks, formulated milk powders, and skim milk powder by using the appropriate combination of pretreatment (acidification) and diluent (plasma treated with protease inhibitors). The PTHrP concentration in normal liquid milk, processed milk, and skim milk powder was as high as that in raw milk (>5 nM), whereas that in milk drinks differed considerably. The PTHrP concentration in infant formulas (

Recent studies have demonstrated that apoptosis in cerebral arteries could play an essential role in cerebral vasospasm after subarachnoid hemorrhage (SAH) and that SP600125, an inhibitor of c-Jun N-terminal kinase (JNK) could suppress apoptosis. The present study examined whether SP600125 could reduce cerebral vasospasm through the suppression of apoptosis.Fifteen dogs were assigned to 3 groups: control, SAH, and SAH + SP600125 (30 micromol/l). SAH was induced by the injection of autologous blood into the cisterna magna on day 0 and day 2. Angiograms were evaluated on day 0 and day 7. The activation of the JNK pathway and caspase-3 were also evaluated using Western blot. To determine the distribution, TUNEL staining and immunohistochemistry for phosphorylated c-jun and cleaved caspase-3 were performed.Severe vasospasm was observed in the basilar artery of the SAH dogs. SP600125 reduced angiographic and morphological vasospasm and reduced the expression of cleaved caspase-3, thereby suppressing apoptosis.These results demonstrate that SP600125 attenuates cerebral vasospasm through the suppression of apoptosis, which may provide a novel therapeutic target for cerebral vasospasm.

We used immunohistochemistry to localize canine Apolipoprotein B-100 (CApoB-100) and glutathione peroxidase (GSH-PO) in canine atherosclerotic lesions. CApoB-100 was deposited in the tunica intima and cytoplasms of infiltrating macrophages in early atherosclerotic lesions. In advanced atherosclerotic lesions, the cystic space of the lesions contained a large amount of CApoB-100 immunoreaetive material. Expression of GSH-PO was recognized in the foamy cytoplasm of macrophages and smooth muscle cells in the early and advanced atherosclerotic lesions. These results indicate that expression of GSH-PO is closely associated with the deposition of CApoB-100. In addition, they suggest that, as in human atheromas, low-density lipoprotein (LDL) is peroxidized and changed into modified LDL. Deposition of modified LDL (oxidized or acetylated) may be a critical step in the formation of canine atherosclerotic lesions.

Age-related changes in haematology and serum chemistry values were examined in male and female Weiser–Maples guineapigs ( Cavia porcellus). Haematological changes that significantly ( P < 0.01) correlated with ageing were increased white blood cell and neutrophil counts in both sexes, decreased lymphocyte counts in both sexes, decreased reticulocyte and platelet counts in males, and decreased basophil counts in females. For serum chemistry, increases in total protein, triglycerides, blood urea nitrogen and creatinine were seen in both sexes, along with increases in total cholesterol in males and sodium in females. Decreased alkaline phosphatase in both sexes and decreased chloride in males were significantly ( P < 0.01) associated with age. These age-related changes are compared with the published literature.

Zinc, an essential trace element, has been demonstrated to stimulate bone growth in animal and human. The cellular mechanism by which zinc stimulates bone growth has not been fully clarified. The effect of hormone and zinc on protein tyrosine phosphatase activity in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers, and then exchanged to culture medium containing either vehicle, zinc sulfate or various hormones in the absence of 10% FBS. After medium change, cells were cultured for 48 h. Protein tyrosine phosphatase activity in the lysate of cells was significantly increased by culture with zinc (10(-6) - 10(-4) M). The effect of zinc in increasing the enzyme activity was completely blocked by culture with cycloheximide (10(-7 )M), an inhibitor of protein synthesis, or 5, 6-dichloro-l-beta-D- riboifuranosylbenzimidarzole (DRB) (10(-6) M), an inhibitor of translational activity. Addition of calcium chloride (10 microM) into the reaction mixture caused a significant increase in protein tyrosine phosphatase activity; this increase was completely blocked in the presence of trifluoperazine (50 microM), an antagonist of calmodulin. Culture with zinc caused a significant increase in Ca2+/calmodulin-dependent protein tyrosine phosphatase activity in osteoblastic cells. Protein tyrosine phosphatase activity was significantly raised by culture with parathyroid hormone (human PTH [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33]; 10(-7) M), 17beta-estradiol (10(-7) M), insulin-like growth factor-I (IGF-I; 10(-8) M) or insulin (10(-8) M). The enzyme activity was not significantly enhanced by the addition of calcium (10 microM) into the reaction mixture. The effect of PTH or IGF-I in increasing protein tyrosine phosphatase activity was completely blocked by culture with DRB. The IGF-I-induced increase in enzyme activity was significantly enhanced by culture with zinc. Such an effect was not seen in the case of PTH. Moreover, the effect of IGF-I in increasing proliferation of osteoblastic cells was significantly enhanced by culture with zinc. The effect of PTH was not enhanced by zinc. This study demonstrates that protein tyrosine phosphatase activity in osteoblastic cells is enhanced by various bone anabolic factors, and that zinc modulates the effect of IGF-I on protein tyrosine phosphatase activity and cell proliferation.

This report describes a case of smooth muscle hamartoma of the abomasum in a 6-month-old steer humanely killed because of severe pneumonia. At necropsy, marked thickening of the abomasal wall in the area of the pylorus was found. On cut section, the thickness of the submucosal layer, extending from the submucosa to the muscularis propria, was seen to be increased to 3 cm. The upper (i.e., nearest to the gut lumen) half of the sectioned thickening was composed mainly of adipose-like tissue and the lower half mainly of muscle-like tissue. Histologically, the submucosal layer was composed of fibroadipose tissue, within which were embedded, to varying degrees, numerous well-defined, haphazardly oriented, thin to thick bundles of smooth muscle fibres. This appears to be the first report of smooth muscle hamartoma of the stomach or abomasum in animals, including man.

The effect of menaquinone-7 (MK-7; vitamin K2) on osteoclast-like cell formation and osteoclastic bone resorption in rat femoral tissues in vitro was investigated. The bone marrow cells were cultured for 7 days in a a-minimal essential medium (alpha-MEM) containing a well-known bone resorbing agent [parathyroid hormone (1-34) (PTH) or prostaglandin E2 (PGE2)] with an effective concentration. Osteoclast-like cells were estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of PTH (10(-8) M) or PGE2 (10(-6) M) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were significantly inhibited by MK-7 (10(-8) - 10(-5) M). MK-7 (10(-7) and 10(-6) M) significantly inhibited phorbol 12-myristate 13-acetate-induced osteoclast-like cell formation, whereas MK-7 did not inhibit dibutyryl cyclic adenosine monophosphate (DcAMP) (10(-5) M)-induced osteoclast-like cell formation. These results suggest that the inhibitory action of MK-7 is partly involved in protein kinase C signaling. The bone cells isolated from rat femoral tissues were cultured for 48 h in an alpha-MEM containing either vehicle or MK-7 (10(-8) - 10(-5) M). The presence of MK-7 (10(-6) and 10(-5) M) caused a significant decrease in the number of mature osteoclasts. Such a decrease was also seen in the presence of calcitonin (10(-10) - 10(-8) M), DcAMP (10(-6) and 10(-5) M), or calcium chloride (10(-4) and 10(-3) M). The effect of MK-7 (10(-6) M) in decreasing the number of osteoclasts was not further enhanced in the presence of calcitonin (10(-8) M), DcAMP (10(-5) M), or calcium chloride (10(-3) M), and was completely abolished by the presence of dibucaine (10(-6) M) or staurosporine (10(-7) M), which are inhibitors of Ca2+-dependent protein kinases. These results suggested that MK-7 has a suppressive effect on osteoclasts. Moreover, the femoral-metaphyseal tissues obtained from rats were cultured for 48 h in Dulbecco's modified Eagle's medium containing either vehicle, PTH (10(-7) M), orPGE2 (10(-5) M) in the absence or presence of MK-7 (10(-7) - 10(-5) M). The presence of PTH or PGE2 induced a significant decrease in bone calcium content. These decreases were significantly blocked by MK-7 (10(-7) - 10(-5) M). This study demonstrates that MK-7 has an inhibitory effect on osteoclastic bone resorption in vitro.

The effect of zinc, an essential trace element, on insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 production was investigated to determine the role of this metal in bone growth of newborn rats. Femoral-diaphyseal and metaphyseal tissues were obtained between 1 and 28 days after birth of newborn rats, and cultured for 24 h in a serum-free Dulbecco's modified Eagle's medium containing either vehicle or zinc sulfate (10(-6) - 10(-4) M). Protein concentration in the medium was significantly increased by culture with bone tissues of newborn rats with increasing age (14 and 21 days). Medium IGF-I and TGF-beta1 concentration was gradually reduced with increasing age after birth. The presence of zinc (10(-5) and 10(-4) M) caused a significant increase in protein, IGF-I, and TGF-beta1 concentrations in the medium cultured with the diaphyseal or metaphyseal tissues obtained at 7 and 14 days after birth. The expression of IGF-I and TGF-beta1 mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in the diaphyseal and metaphyseal tissues cultured for 24 h using rat IGF-I or TGF-beta1-specific primers. These expressions were significantly raised in the presence of zinc (10(-4) M) in culture medium. The present study demonstrates that zinc has a stimulatory effect on IGF-I and TGF-beta1 production in the femoral tissues with bone growth of newborn rats.

The effect of regucalcin, a regulatory protein of Ca2+ signaling, on guanosine-5'-triphosphatase (GTPase) activity in isolated rat liver plasma membranes was investigated. GTPase activity was significantly increased by the addition of Ca2+ (25-100 microM) in the enzyme reaction mixture. Such an increase was not seen by other metals (Mg, Co, Zn, Cu, Ni and Mn) with 50 microM. The activatory effect of calcium (50 microM) was significantly decreased by calmodulin (2.5 and 5 microg/ml), indicating that it does not depend on calmodulin. The presence of regucalcin (0.1-0.5 microM) in the enzyme reaction mixture caused a significant increase in GTPase activity. This increase was not significantly enhanced by calcium (50 microM). GTPase activity was significantly increased by dithiothreitol (DTT; 5 mM), a protecting reagent of thiol (SH)-groups, while it was decreased by N-ethylmaleimide (NEM; 5 mM), a modifying reagent of SH-groups. The effect of calcium or regucalcin in increasing GTPase activity was not seen in the presence of NEM. Also, the activatory effect of calcium or regucalcin on GTPase was not seen in the presence of vanadate, an inhibitor of protein phosphorylation, which could inhibit GTPase activity. Moreover, the effect of regucalcin was not seen in the presence of digitonin (0.01%), a solubilizing reagent of membranous lipids, while the effect of calcium was not inhibited by digitonin. The present study demonstrates that regucalcin has an activatory effect on GTPase activity independently of Ca2+ in rat liver plasma membranes.

The molecular cloning and sequencing of the cDNA coding for a novel regucalcin gene promoter region-related protein (RGPR) was investigated using rat, mouse and human liver cDNA library with a yeast one-hybrid system and a rapid amplification of cDNA ends (RACE) method. The clone coding an unknown protein was isolated, and a novel protein was identified. This protein was termed as RGPR-p117. RGPR-p117 in rat, mouse and human liver consisted of 1058, 1051 and 1060 amino acid residues with calculated molecular mass of 117, 115 and 117 kDa and estimated pI of 5.69, 5.70 and 5.71, respectively. The homologies of amino acids among rat, mouse and human RGPR-p117 were at least 70%. RGPR-p117 had a leucine zipper motif. The expression of RGPR-p117 mRNA was found in the liver, kidney, heart, spleen, and brain of rats. The database search of the human RGPR-p117 showed that its gene consisted of at least 26 exons spanning approximately 4.1 kbp and localized on human chromosome 1q25.2. Furthermore, we found a cDNA clone which was highly identical to a front half part of the human RGPR-p117 cDNA, using the BLAST search of human RGPR-p117. This cDNA clone was a splicing variant of human RGPR-p117, which derived from human placental choriocarcinoma. Our study demonstrates that a novel gene coding RGPR-p117 is present in rat, mouse and human.

DNA replication-related element binding factor (DREF) has been suggested to be involved in regulation of DNA replication- and proliferation-related genes in Drosophila. While the effects on the mutation in the DNA replication-related element (DRE) in cultured cells have been studied extensively, the consequences of elevating wild-type DREF activity in developing tissues have hitherto remained unclear.We over-expressed DREF in the wing imaginal disc using a GAL4-UAS targeted expression system in Drosophila. Over-expression of DREF induced a notching wing phenotype, which was associated with ectopic apoptosis. A half reduction of the reaper, head involution defective and grim gene dose suppressed this DREF-induced notching wing phenotype. Furthermore, this was also the case with co-expression of baculovirus P35, a caspase inhibitor. In addition, over-expression of the 32 kDa boundary element-associated factor (BEAF-32), thought to compete against DREF for common binding sites in genomic regions, rescued the DREF-induced notching wing phenotype, while a half reduction of the genomic region, including the BEAF-32 gene, exerted enhancing effects. To our knowledge, this is the first evidence for a genetic interaction between DREF and BEAF-32.The DREF-induced notching wing phenotype is caused by induction of apoptosis in the Drosophila wing imaginal disc.

The effect of zinc on in vitro deoxyribonucleic acid (DNA) synthesis activity in the femoral-diaphyseal and metaphyseal tissues of newborn rats was investigated to determine a role of zinc in bone growth. In vitro DNA synthesis was assayed in a reaction mixture containing the 100 g centrifugation supernatant, which includes the nucleus of bone cells, of bone issue homogenate with incorporation of [3H]-deoxythymidine 5'-triphosphate (dTTP). DNA synthesis activity in the femoral-diaphyseal and metaphyseal tissues of newborn rats was significantly raised with increasing age (1-21 days) after birth. The presence of dipicolinate (10(-3) M), a chelator of zinc, in the reaction mixture caused a significant decrease in DNA synthesis activity in the diaphyseal and metaphyseal tissues of newborn rats at 7 and 14 days after birth. The addition of zinc sulfate (10(-6) - 10(-4) M) resulted in a significant increase in DNA synthesis activity in the diaphyseal and metaphyseal tissues. When the diaphyseal and metaphyseal tissues of newborn rats at 7 days after birth were cultured for 24 hours in a serum-free medium containing either vehicle, zinc sulfate (10(-4) M), insulin-like growth factor-I (IGF-I; 10(-8) M) or transforming growth factor-beta (TGF-beta; 10(-10) M), bone DNA synthesis activity was significantly elevated. Culture with both zinc and IGF-I enhanced additively bone DNA synthesis activity. Such an effect was not seen in the case of zinc and TGF-beta. The effect of zinc, IGF-I, or zinc plus IGF-I in increasing bone DNA synthesis activity was completely prevented by culture with PD98059 (10(-5) M), an inhibitor of mitogen-activated protein (MAP) kinase. Also, the effect of zinc, TGF-beta. or zinc plus TGF-beta in elevating bone DNA synthesis activity was significantly inhibited by culture with staurosporine (10(-6) M), an inhibitor of protein kinase C. The present study demonstrates that zinc, like bone growth factors, has a stimulatory effect on bone DNA synthesis in newborn rats.

Menaquinone-7, which is vitamin K2 (menatetrenone) with seven isoprene units, is highly contained in the fermented soybean. The effect of menaquinone-7 (MK-7) on osteoblastic bone formation was investigated. Femoral-diaphyseal and metaphyseal tissues of young male rats (4 weeks old) were cultured for 48 h in a medium containing either vehicle or MK-7 (10(-7)-10(-5) M). Calcium content, alkaline phosphatase activity, and deoxyribonuclic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased in the presence of MK-7 (10(-6) and 10(-5) M). The effect of MK-7 in increasing the diaphyseal and metaphyseal calcium content and alkaline phosphatase activity was completely prevented in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. Moreover, osteoblastic MC3T3-E1 cells after subculture were cultured for 24 h in a serum-free medium containing MK-7 (10(-7)-10(-5) M). Protein content, alkaline phophatase activity, osteocalcin and DNA content in the cells was significantly increased in the presence of MK-7 (10(-6) and 10(-5) M). The effect of MK-7 in increasing protein content, alkaline phosphatase activity, and osteocalcin production in the cells was completely blocked by cycloheximide. This study demonstrates that MK-7 has an anabolic effect on bone tissue and osteoblastic MC3T3-E1 cells in vitro, suggesting that the compound can stimulate osteoblastic bone formation.

The effect of zinc, a stimulator of bone formation, on bone protein components in the femoral-diaphyseal tissues with fracture healing was investigated. Rats were sacrificed between 1 and 7 days after the femoral fracture, and the diaphyseal tissues were cultured in a serum-free Dulbecco's modified Eagle's medium for 24 h. Protein content in the femoral-diaphyseal tissues was markedly elevated by fracture healing. The amount of protein in the medium cultured with the diaphyseal tissues obtained from fracture healing rats was markedly elevated as compared with that of normal rats, indicating that bone protein components were secreted into culture medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel elecrophoresis (SDS-PAGE) showed that many protein molecules were secreted from the diaphyseal tissues with fracture healing. Especially, protein molecule of about 66 kDa was markedly secreted by fracture healing. The presence of zinc acexamate (10(-5) and 10(-4) M) in culture medium induced a significant elevation of medium protein content; the zinc effect was enhanced by culture with the diaphyseal tissues of fracture healing rats. Also, the culture of diaphyseal tissues with fracture healing caused a significant increase in insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta1) in culture medium. The production of IGF-I and TGF-beta1 from bone tissues with fracture healing was significantly enhanced in the presence of zinc acexamate (10(-6)-10(-4) M). Moreover, the addition of IGF-I (10(-8) M) or TGF-beta1 (10(-10) M) in a culture medium caused a significant elevation of protein content in the medium cultured with the femoral-diaphyseal tissues from normal and fracture healing rats. The effect of IGF-I or TGF-beta1 was significantly enhanced in the presence of zinc acexamate (10(-4) M). Also, deoxyribonucleic acid (DNA) content in the diaphyseal tissues from normal and fracture healing rats was significantly raised by the culture with IGF-I or TGF-beta1. The effect of IGF-I was significantly enhanced by zinc. The present study demonstrates that fracture healing increases production of bone IGF-I and TGF-beta1, and that this elevation is enhanced by zinc treatment.

It is now clear that the mast cell's functional response to IgE-dependent stimulation can be influenced significantly by the level of expression of the high-affinity IgE receptor (Fc epsilon RI) on the cell's surface. Thus, modulation of Fc epsilon RI surface expression represents a potentially important mechanism for regulating mast cell activity in allergic reactions. In this study, we examined whether a glucocorticoid, dexamethasone (DEX), can influence levels of mast cell Fc epsilon RI expression either in the presence or absence of IgE, an up-regulator of the mast cell surface Fc epsilon RI level. In the absence of IgE, DEX decreased the surface Fc epsilon RI levels in mouse peritoneal mast cells, mouse bone marrow-derived cultured mast cells and a mouse mast cell line, Cl.MC/C57.1. Moreover, DEX also partially suppressed the ability of IgE to enhance surface expression of Fc epsilon RI in these cells. Three different glucocorticoids, DEX, methylprednisolone and hydrocortisone, suppressed Fc epsilon RI expression in mast cells, whereas sex steroids, i.e. estradiol, progesterone and testosterone, did not, indicating that the Fc epsilon RI-suppressing effect is glucocorticoid specific. On the other hand, DEX did not affect levels of Fc epsilon RI alpha, beta or gamma mRNA, suggesting that its ability to decrease surface Fc epsilon RI reflects a post-transcriptional mechanism. Finally, DEX-treated mast cells showed a reduced degranulation response to antigenic stimulation through down-regulation of surface Fc epsilon RI expression in addition to DEX-induced changes in downstream signals. These results show that mast cell surface Fc epsilon RI expression is suppressed by glucocorticoids in both the presence and absence of IgE, and suggest that reduction of mast cell surface Fc epsilon RI levels may be one of the favorable anti-allergic actions of glucocorticoids.

The binding of nuclear factor on the promoter region of the regucalcin gene and the expression of regucalcin in the kidney cortex of rats was investigated. Nuclear extracts from kidney cortex were used for oligonucleotide competition gel mobility shift assay. An oligonucleotide between position -523 and -506 in the 5'-flanking region of the rat regucalcin gene, which contains a nuclear factor 1 (NF1) consensus motif TTGGC(N)6CC, competed with the probe for the binding of the nuclear protein from kidney cortex. The mutation of TTGGC in the consensus sequence caused an inhibition of the binding of nuclear factors. The binding of nuclear factor on the 5'-flanking region was clearly reduced in the kidney cortex obtained at 1, 2, and 3 days after a single intraperitoneal administration of cisplatin (1.0 mg/100 g body wt) to rats. Moreover, cisplatin administration caused a remarkable decrease in regucalcin mRNA levels and regucalcin concentration in the kidney cortex. Also, serum regucalcin concentration was significantly decreased by cisplatin administration. Meanwhile, serum urea nitrogen concentration was markedly elevated by cisplatin administration. The present study demonstrates that the specific nuclear factor binds to the NF1-like sequence in the promotor region of regucalcin gene in the kidney cortex of rats, and that the nuclear factor binding and regucalcin expression are suppressed by cisplatin administration.

The alteration in Ca(2+)-ATPase activity in the brain plasma membrane of rats with increasing age was investigated. Calcium content in the brain tissues was significantly raised in aged rats (50 weeks old) as compared with that of young rats (5 weeks old). Increasing age caused a significant decrease in Ca(2+)-ATPase activity in the brain plasma membranes. The presence of N-ethylmaleimide (2.5 or 5 mM), a modifying reagent of thiol (SH)-groups, in the reaction mixture caused a significant decrease in the brain plasma membrane Ca(2+)-ATPase activity of young and aged rats, while dithiothreitol (2.5 or 5 mM), a protecting reagent of SH-groups, produced a significant increase in the enzyme activity, indicating that the SH-group is an active site of Ca(2+)-ATPase. The active site of Ca(2+)-ATPase may not be impaired by ageing. The brain plasma membrane Ca(2+)-ATPase activity of young rats was significantly reduced in the presence of dibutyryl cyclic AMP (10(-7)-10(-5) M) or inositol 1, 4, 5-trisphosphate (10(-7)-10(-5) M) in the reaction mixture. Such an decrease was not seen in aged rats. The responsibility for signaling factors seemed to be weakened by ageing. Calmodulin (2.5 and 5 microg/ml) or regucalcin (10(-8) and 10(-7) M), a Ca(2+)-regulating protein, did not have an effect on Ca(2+)-ATPase activity. This study demonstrates that ageing induces a decrease in Ca(2+)-ATPase activity in the brain plasma membranes. This finding suggests a cellular mechanism by which ageing causes calcium accumulation in brain.

Novel in vivo 31P-nuclear magnetic resonance spectoroscopy(NMR) and NMR-imaging techniques for accurate and noninvasive assessment of teratogenicity induced by environmental endocrine disrupters(EDs) were developed. Mice with pregnancy were administered ED at extremely low dose, and then in vivo 31P-NMR spectra of embryos were acquired noninvasively and quantitatively to evaluate the energy metabolism. A significant decrease in embryo ATP level was seen, but no significant changes were detected by conventional histological and biochemical methods. In conclusion, in vivo NMR techniques are highly sensitive(at least 100-fold more sensitive than conventional methods) and are useful for toxicological assessment of environmental pollutants.

The effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells in vitro was investigated to determine a cellular mechanism by which the isoflavones stimulate bone formation. Cells were cultured for 48 h in alpha-minimal essential medium containing either vehicle, genistein (l0(-7) - 10(-5) M) or daidzein (10(-7) - 10(-5) M). The 5,500 g supernatant of cell homogenate was used for assay of protein synthesis with [3H]leucine incorporation in vitro. The culture with genistein or daidzein caused a significant elevation of protein synthesis in the cell homogenate. The effect of genistein ( 10(-5) M) or daidzein ( 10(-5) M) in elevating protein synthesis was significantly prevented, when cells were cultured for 48 h in a medium containing either actinomycin D (10(-7) M) or cycloheximide (10(-6) M) in the absence or presence of isoflavones. Moreover, when genistein (10(-7) 10(-5) M) or daidzein (10(-6) and 10(-5) M) was added to the reaction mixture containing the cell homogenate obtained from osteoblastic cells cultured without isoflavone, protein synthesis was significantly raised. This increase was markedly blocked by the addition of cycloheximide (10(-7) M). In addition, [3H]leucyl-tRNA synthetase activity in the cytosol of osteoblastic cells was significantly increased by the addition of genistein (10(-6) and 10(-5) M) or daidzein (10(-5) M) into the enzyme reaction mixture. The present study demonstrates that genistein or daidzein can stimulate protein synthesis in osteoblastic MC3T3-E1 cells. The isoflavones may have a stimulatory effect on osteoblastic bone formation due to increasing protein synthesis.