RESUMO
BACKGROUND AND OBJECTIVE: T-helper type 17 (Th17) cells produce interleukin-17 (IL-17) and help to protect against inflammation and infection in periodontal disease. Furthermore, while follicular dendritic cell-secreted protein (FDC-SP) may be involved in the inflammation of periodontal tissue, the biological role of FDP-SP in periodontal disease is still unknown. The purpose of the present study was to clarify the expression of IL-17 and FDC-SP in experimental periodontitis in rats. MATERIAL AND METHODS: Seven-week-old male Wistar rats were divided into baseline control, sham and test groups. Experimental periodontitis was induced by placing a ligature in the mesiopalatal area, and untreated rats served as a baseline control group. Morphological changes in alveolar bone were investigated 7, 14 and 28 d after treatment. Expression of the Rankl, osteoprotegerin (Opg) and Il17 genes was analyzed 5 and 7 d after the induction of experimental periodontitis. RESULTS: Alveolar bone resorption progressed in the test group for 7 d, but not thereafter. At 5 d after the induction of periodontitis, the Rankl/Opg mRNA ratio and the expression of IL-17 in the test group were significantly increased compared with the respective values in the baseline control group; however, there were no significant differences between the test and control groups at 7 d. The expression of FDC-SP was significantly decreased in the test group compared with the baseline control group at 5 and 7 d after the induction of periodontitis, and this value had returned to normal levels at 14 and 28 d. CONCLUSION: These results suggest that both IL-17 and FDC-SP could be involved in the inflammatory response, and FDC-SP in the junctional epithelium might play an important role in the Th17 cell-related immune response.
Assuntos
Células Dendríticas Foliculares/imunologia , Interleucina-17/análise , Osteoprotegerina/análise , Periodontite/imunologia , Proteínas/análise , Ligante RANK/análise , Perda do Osso Alveolar/imunologia , Perda do Osso Alveolar/patologia , Processo Alveolar/imunologia , Processo Alveolar/patologia , Animais , Progressão da Doença , Masculino , Periodontite/patologia , Reação em Cadeia da Polimerase/métodos , Ratos , Ratos Wistar , Células Th17/imunologia , Fatores de Tempo , Microtomografia por Raio-X/métodosRESUMO
UNLABELLED: Oshiro A, Iseki S, Miyauchi M, Terashima T, Kawaguchi Y, Ikeda Y, Shinomura T. Lipopolysaccharide induces rapid loss of follicular dendritic cell-secreted protein in the junctional epithelium. J Periodont Res 2012; 47: 689-694. © 2012 John Wiley & Sons A/S Background and Objective: We have previously reported that mRNA encoding follicular dendritic cell-secreted protein (FDC-SP) is expressed specifically in the junctional epithelium at the gingival crevice. Other tissues, such as tonsil, prostate gland and trachea, also express high levels of FDC-SP. These tissues participate in a range of functions closely related to innate immunity. Therefore, it is hypothesized that FDC-SP plays a crucial role in close association with the host defense system within the gingival crevice. Accordingly, the main aim of this study was to investigate the expression and localization of FDC-SP in and around the junctional epithelium and to observe the dynamic changes of FDC-SP in experimental inflammation. MATERIAL AND METHODS: We examined, immunohistochemically, the expression of FDC-SP in the junctional epithelium using a specific antibody raised in rabbit after immunization with a synthetic peptide derived from the hydrophilic region of FDC-SP. Experimental inflammation was induced in the upper molars of Wistar rats by applying bacterial lipopolysaccharide (LPS; 5 mg/mL in sterile saline) for 1 h. RESULTS: We confirmed that FDC-SP is present in the junctional epithelium in a pattern that is consistent with the expression of FDC-SP mRNA. Of special interest is that no FDC-SP was detectable in the junctional epithelium 3 h after transient topical treatment with LPS. CONCLUSION: The presence of FDC-SP in the junctional epithelium and its loss after LPS treatment strongly support our hypothesis of FDC-SP playing a crucial role in close association with the host defense system within the gingival crevice.
Assuntos
Células Dendríticas Foliculares/efeitos dos fármacos , Células Dendríticas Foliculares/metabolismo , Inserção Epitelial/imunologia , Gengiva/imunologia , Gengivite/metabolismo , Lipopolissacarídeos/imunologia , Proteínas/metabolismo , Animais , Especificidade de Anticorpos , Células Dendríticas Foliculares/imunologia , Inserção Epitelial/citologia , Inserção Epitelial/metabolismo , Gengiva/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Proteínas/imunologia , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/metabolismoRESUMO
OBJECTIVE: To characterize the in vivo role epiphycan (Epn) has in cartilage development and/or maintenance. METHODS: Epn-deficient mice were generated by disrupting the Epn gene in mouse embryonic stem cells. Epn/biglycan (Bgn) double-deficient mice were produced by crossing Epn-deficient mice with Bgn-deficient mice. Whole knee joint histological sections were stained using van Gieson or Fast green/Safranin-O to analyze collagen or proteoglycan content, respectively. Microarray analysis was performed to detect gene expression changes within knee joints. RESULTS: Epn-deficient and Epn/Bgn double-deficient mice appeared normal at birth. No significant difference in body weight or femur length was detected in any animal at 1 month of age. However, 9-month Epn/Bgn double-deficient mice were significantly lighter and had shorter femurs than wild type mice, regardless of gender. Male Epn-deficient mice also had significantly shorter femurs than wild type mice at 9 months. Most of the deficient animals developed osteoarthritis (OA) with age; the onset of OA was observed earliest in Epn/Bgn double-deficient mice. Message RNA isolated from Epn/Bgn double-deficient knee joints displayed increased matrix protein expression compared with wild type mice, including other small leucine-rich proteoglycan (SLRP) members such as asporin, fibromodulin and lumican. CONCLUSION: Similar to other previously studied SLRPs, EPN plays an important role in maintaining joint integrity. However, the severity of the OA phenotype in the Epn/Bgn double-deficient mouse suggests a synergy between these two proteins. These data are the first to show a genetic interaction involving class I and class III SLRPs in vivo.
Assuntos
Articulação do Joelho/química , Osteoartrite do Joelho/fisiopatologia , Proteoglicanas/análise , Proteoglicanas/deficiência , Animais , Southern Blotting , Peso Corporal , Colágeno/análise , Fêmur/anatomia & histologia , Imuno-Histoquímica , Articulação do Joelho/patologia , Camundongos , Camundongos Knockout , Análise em Microsséries , Osteoartrite do Joelho/genética , Fenótipo , Reação em Cadeia da Polimerase , Proteoglicanas/genética , RNA Mensageiro/análiseRESUMO
We evaluated the effectiveness of intentional hypercapnia against hypotension after induction of anaesthesia with thiopental and isoflurane (TI) or propofol (P). For each group, 24 patients were anaesthetized with thiopental 4 mg kg(-1) (TI) or propofol 2 mg kg(-1) (P) for tracheal intubation and then lightly anaesthetized with isoflurane at 0.6% end-expiratory concentration (TI) or by 6 mg kg(-1) h(-1) infusion of propofol (P). In both anaesthesia groups, patients were randomly assigned to either normocapnia (end-tidal CO(2) = 35 mmHg) or hypercapnia (end-tidal CO(2) = 45 mmHg), which were achieved through adjusting the tidal volume. Systolic arterial pressure (SAP) 15 min after intubation was compared with the preanaesthetic baseline value. Under normocapnia, both TI and P induced a comparable, statistically significant suppression of SAP by approximately 20 mmHg from baseline. Hypercapnia prevented the decrease in SAP in TI but not in P. No patient in the TI-hypercapnia group experienced SAP below 100 mmHg, unlike those in the other groups. In conclusion, mild hypercapnia was effective in the prevention of hypotension in patients receiving thiopental followed by 0.6% end-expiratory isoflurane, but not in patients receiving 6 mg kg(-1) h(-1) propofol.
Assuntos
Anestesia Geral , Hipercapnia/fisiopatologia , Hipotensão/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anestésicos Inalatórios , Anestésicos Intravenosos , Pressão Sanguínea/fisiologia , Dióxido de Carbono/sangue , Feminino , Frequência Cardíaca/fisiologia , Humanos , Hipotensão/etiologia , Isoflurano , Masculino , Pessoa de Meia-Idade , Monitorização Intraoperatória , Propofol , Mecânica Respiratória/fisiologia , TiopentalRESUMO
Twenty-seven patients received boron neutron capture therapy during craniotomy at our research reactor from 1991 to 1999. This is a form of intra-operative radiation therapy, which uses neutrons from a nuclear reactor. There are three additional major problems to anaesthetists: boron neutron capture therapy must be given beside the nuclear reactor, with no hospital facilities; neutrons cannot be shielded effectively by ordinary protectors; and neutrons are detrimental to metal devices and especially to electrical appliances. Boron neutron capture therapy has been adopted as an effective therapy for glioblastoma/astrocytoma, but special considerations are required for anaesthesia.
Assuntos
Anestesia Geral/métodos , Terapia por Captura de Nêutron de Boro , Neoplasias Encefálicas/radioterapia , Glioblastoma/radioterapia , Neoplasias Encefálicas/cirurgia , Terapia Combinada , Craniotomia , Glioblastoma/cirurgia , HumanosRESUMO
To elucidate phytochrome A (phyA) function in rice, we screened a large population of retrotransposon (Tos17) insertional mutants by polymerase chain reaction and isolated three independent phyA mutant lines. Sequencing of the Tos17 insertion sites confirmed that the Tos17s interrupted exons of PHYA genes in these mutant lines. Moreover, the phyA polypeptides were not immunochemically detectable in these phyA mutants. The seedlings of phyA mutants grown in continuous far-red light showed essentially the same phenotype as dark-grown seedlings, indicating the insensitivity of phyA mutants to far-red light. The etiolated seedlings of phyA mutants also were insensitive to a pulse of far-red light or very low fluence red light. In contrast, phyA mutants were morphologically indistinguishable from wild type under continuous red light. Therefore, rice phyA controls photomorphogenesis in two distinct modes of photoperception--far-red light-dependent high irradiance response and very low fluence response--and such function seems to be unique and restricted to the deetiolation process. Interestingly, continuous far-red light induced the expression of CAB and RBCS genes in rice phyA seedlings, suggesting the existence of a photoreceptor(s) other than phyA that can perceive continuous far-red light in the etiolated seedlings.
Assuntos
Genes de Plantas , Oryza/crescimento & desenvolvimento , Oryza/genética , Fitocromo/fisiologia , Southern Blotting , Proteínas de Transporte , Escuridão , Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica de Plantas , Genes Reporter , Gravitropismo/genética , Gravitropismo/fisiologia , Luz , Mutação , Oryza/efeitos da radiação , Fatores de Terminação de Peptídeos , Fenótipo , Complexo de Proteínas do Centro de Reação Fotossintética/biossíntese , Fitocromo/genética , Fitocromo/isolamento & purificação , Fitocromo/efeitos da radiação , Fitocromo A , Raízes de Plantas/crescimento & desenvolvimento , Retroelementos , Transdução de SinaisRESUMO
The pharmacological profile of metabotropic glutamate receptor (mGluR) activation of phospholipase D (PLD), and the associated signalling pathways, were examined in rat cerebrocortical synaptosomes. The assay was conducted using a transphosphatidylation reaction in synaptosomes which were pre-labelled with either [(3)H]-arachidonic acid or [(32)P]-orthophosphate. The mGluR agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S, 3R-ACPD) and (RS)-3,5-dihydroxyphenylglycine (DHPG), both activated PLD, while phorbol 12,13-dibutyrate (PDBu) treatment caused receptor-independent activation of PLD and had an additive effect on 1S,3R-ACPD induced PLD activity. A protein kinase C (PKC) inhibitor, GF109203X, failed to antagonize mGluR receptor-coupled PLD activity. We could not detect any increase in the products of PI (phosphoinositide)-specific phospholipase C (PI-PLC), inositol(1,4, 5)trisphosphate or diacylglycerol, by 1S, 3R-ACPD at 15 s. However, diacylglycerol increased monophasically in response to mGluR agonists and remained elevated for at least 15 min. Phosphatidic acid phosphohydrolase (PAP) activity, which converts PA to DAG, was present in the synaptosomes. These data suggest that, in rat cerebrocortical synaptosomes, the 1S,3R-ACPD-sensitive mGluR is coupled to PLD through a mechanism that is independent of both PKC and PI-PLC.
Assuntos
Córtex Cerebral/enzimologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Fosfolipase D/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Sinaptossomos/enzimologia , Animais , Diglicerídeos/biossíntese , Ativação Enzimática/efeitos dos fármacos , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/farmacologia , Dibutirato de 12,13-Forbol/farmacologia , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Proteína Quinase C/fisiologia , Ratos , Ratos Wistar , Fosfolipases Tipo C/metabolismoRESUMO
It has been proposed that hyaluronan-binding proteoglycans play an important role as guiding cues during neural crest (NC) cell migration, but their precise function has not been elucidated. In this study, we examine the distribution, structure and putative role of the two major hyaluronan-binding proteoglycans, PG-M/versicans and aggrecan, during the course of avian NC development. PG-M/versicans V0 and V1 are shown to be the prevalent isoforms at initial and advanced phases of NC cell movement, whereas the V2 and V3 transcripts are first detected following gangliogenesis. During NC cell dispersion, mRNAs for PG-M/versicans V0/V1 are transcribed by tissues lining the NC migratory pathways, as well as by tissues delimiting nonpermissive areas. Immunohistochemistry confirm the deposition of the macromolecules in these regions and highlight regional differences in the density of these proteoglycans. PG-M/versicans assembled within the sclerotome rearrange from an initially uniform distribution to a preferentially caudal localization, both at the mRNA and protein level. This reorganization is a direct consequence of the metameric NC cell migration through the rostral portion of the somites. As suggested by previous in situ hybridizations, aggrecan shows a virtually opposite distribution to PG-M/versicans being confined to the perinotochordal ECM and extending dorsolaterally in a segmentally organized manner eventually to the entire spinal cord at axial levels interspacing the ganglia. PG-M/versicans purified from the NC migratory routes are highly polydispersed, have an apparent M(r) of 1,200-2,000 kDa, are primarily substituted with chondroitin-6-sulfates and, upon chondroitinase ABC digestion, are found to be composed of core proteins with apparent M(r )of 360-530, 000. TEM/rotary shadowing analysis of the isolated PG-M/versicans confirmed that they exhibit the characteristic bi-globular shape, have core proteins with sizes predicted for the V0/V1 isoforms and carry relatively few extended glycosaminoglycan chains. Orthotopical implantation of PG-M/versicans immobilized onto transplantable micromembranes tend to 'attract' moving cells toward them, whereas similar implantations of a notochordal type-aggrecan retain both single and cohorts of moving NC cells in close proximity of the implant and thereby perturb their spatiotemporal migratory pattern. NC cells fail to migrate through three-dimensional collagen type I-aggrecan substrata in vitro, but locomote in a haptotactic manner through collagen type I-PG-M/versican V0 substrata via engagement of HNK-1 antigen-bearing cell surface components. The present data suggest that PG-M/versicans and notochordal aggrecan exert divergent guiding functions during NC cell dispersion, which are mediated by both their core proteins and glycosaminoglycan side chains and may involve 'haptotactic-like' motility phenomena. Whereas aggrecan defines strictly impenetrable embryonic areas, PG-M/versicans are central components of the NC migratory pathways favoring the directed movement of the cells.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/fisiologia , Proteínas da Matriz Extracelular , Ácido Hialurônico/metabolismo , Crista Neural/citologia , Proteoglicanas/fisiologia , Agrecanas , Animais , Anticorpos/química , Western Blotting , Bovinos , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Embrião de Galinha , Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/metabolismo , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Epitopos , Fibronectinas/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Membranas Intracelulares , Lectinas Tipo C , Microscopia Eletrônica , Crista Neural/embriologia , Isoformas de Proteínas , Proteoglicanas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Distribuição Tecidual , Células Tumorais Cultivadas , VersicanasRESUMO
Midkine is a heparin-binding growth factor with survival-promoting and migration-enhancing activities. In order to understand the regulation of midkine signaling, we isolated midkine-binding proteoglycans from day 13 mouse embryos, when midkine is intensely expressed. Deglycosylation followed by SDS/PAGE revealed various protein bands; one of these was identified as PG-M/versican by in gel trypsin digestion and sequencing the resulting peptides. PG-M/versican isolated from day 13 mouse embryos bound midkine with a Kd of 1.0 nM. Pleiotrophin/heparin-binding growth-associated molecule, which has a structure related to midkine, was also bound similarly. Digestion with chondroitinase ABC, AC-I or B abolished the binding to midkine. Heparin as well as chondroitin sulfate D and E inhibited the binding. After chondroitinase ABC digestion, the midkine-binding PG-M/versican released 4-sulfated, 6-sulfated, 2, 6-disulfated and 4,6-disulfated unsaturated disaccharides. These results suggest that midkine binds to a polysulfated domain in the chondroitin sulfate chain with a region of dermatan sulfate structure. This proteoglycan may modulate the midkine activity, as binding to midkine can enhance midkine action by concentrating it to the cell periphery or inhibit the action by competing with the binding to a signaling receptor.
Assuntos
Proteínas de Transporte/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Citocinas , Proteoglicanas/metabolismo , Sequência de Aminoácidos , Animais , Proteoglicanas de Sulfatos de Condroitina/isolamento & purificação , Sulfatos de Condroitina/metabolismo , Humanos , Lectinas Tipo C , Camundongos , Midkina , Dados de Sequência Molecular , VersicanasRESUMO
Elementary processes of photoperception by phytochrome A (PhyA) for the high-irradiance response (HIR) of hypocotyl elongation in Arabidopsis were examined using a newly designed irradiator with LED. The effect of continuous irradiation with far-red (FR) light could be replaced by intermittent irradiation with FR light pulses if given at intervals of 3 min or less for 24 h. In this response, the Bunsen-Roscoe law of reciprocity held in each FR light pulse. Therefore, we determined the action spectrum for the response by intermittent irradiation using phyB and phyAphyB double mutants. The resultant action spectrum correlated well with the absorption spectrum of PhyA in far-red-absorbing phytochrome (Pfr). Intermittent irradiation with 550 to 667 nm of light alone had no significant effect on the response. In contrast, intermittent irradiation with red light immediately after each FR light pulse completely reversed the effect of FR light in each cycle. The results indicate that neither red-absorbing phytochrome synthesized in darkness nor photoconverted Pfr are physiologically active, and that a short-lived signal is induced during photoconversion from Pfr to red-absorbing phytochrome. The mode of photoperception by PhyA for HIR is essentially different from that by PhyA for very-low-fluence responses and phytochrome B for low-fluence responses.
Assuntos
Arabidopsis/fisiologia , Hipocótilo/crescimento & desenvolvimento , Fitocromo/metabolismo , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis , Hipocótilo/efeitos da radiação , Luz , Fitocromo ARESUMO
Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K(m) values for the two substrates UDP-GlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 x 10(5) to 1 x 10(6) Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 x 10(6) Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions.
Assuntos
Glucuronosiltransferase/química , Glicosiltransferases , Proteínas de Membrana , Transferases , Proteínas de Xenopus , Animais , Linhagem Celular , Estabilidade Enzimática , Expressão Gênica , Glucuronosiltransferase/genética , Hialuronan Sintases , Ácido Hialurônico/biossíntese , Ácido Hialurônico/química , Isoenzimas/química , Cinética , Microscopia de Contraste de Fase , Proteínas Recombinantes/química , Especificidade por Substrato , Transfecção , Uridina Difosfato Ácido Glucurônico/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
UNLABELLED: Glutamate is a major neural transmitter of noxious stimulation in the spinal cord. We measured glutamate release from rat spinal synaptoneurosomes by using an enzyme-linked fluorimetric assay. Glutamate was released from spinal cord synaptoneurosomes in response to the addition of 30 mM potassium chloride, 1 mM 4-aminopyridine, or 1 microM ionomycin in the presence of external calcium. There was less release of glutamate in the absence, versus the presence, of external calcium. Clonidine significantly reduced the level of glutamate released from the spinal cord synaptoneurosomes. Tetradecanoyl phorbol acetate, an activator of protein kinase C, enhanced glutamate release. Forskolin, a protein kinase A activator, had no effect on the glutamate efflux. Our data indicate that glutamate released in the spinal cord is dependent on protein kinase C but is independent of the protein kinase A pathway. They also suggest that the inhibition of glutamate release may be the underlying mechanism of antinociception by clonidine at the spinal cord level. IMPLICATIONS: We demonstrated that synaptoneurosomes from rat spinal cord could release glutamate in response to depolarization. We showed that an activator of protein kinase C increased glutamate released from spinal cord synaptoneurosomes but that clonidine decreased it. Glutamate release may be one of the mechanisms of antinociception at the spinal cord level.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Clonidina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ácido Glutâmico/metabolismo , Proteína Quinase C/metabolismo , Medula Espinal/metabolismo , Sinaptossomos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , 4-Aminopiridina/farmacologia , Animais , Cálcio/fisiologia , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Glutamato Desidrogenase/metabolismo , Técnicas In Vitro , Masculino , Cloreto de Potássio/farmacologia , Ratos , Ratos Wistar , Espectrometria de Fluorescência , Medula Espinal/efeitos dos fármacos , Medula Espinal/ultraestrutura , Sinaptossomos/efeitos dos fármacosRESUMO
We have examined the expression pattern of the PG-Lb/epiphycan gene that encodes a small leucine-rich repeat proteoglycan during mouse embryonic development. PG-Lb/epiphycan mRNA transcripts were first detected at E12.5 days postcoitus (dpc) at high levels in structures that were developing cartilage elements. The gene is expressed in a very specific temporal and spatial fashion in cartilaginous structures. To examine PG-Lb/epiphycan gene expression during cartilage development in more detail, we performed in situ hybridization on hindlimb sections at specific stages of mouse embryonic development. The expression of PG-Lb/epiphycan was compared to that of collagen type II and collagen type X, which are early and late markers for cartilage development, respectively. The expression of PG-Lb/epiphycan occurs later than collagen type II in cartilage development, but its expression appears in the growth plate before and is excluded from the zone of hypertrophic chondrocytic cells expressing collagen type X. An antibody against PG-Lb/epiphycan localized the protein within the entire growth plate of the E17.5 dpc embryonic hindlimb cartilage including the hypertrophic zone where PG-Lb/epiphycan gene expression is turned off. Our results show that PG-Lb/epiphycan gene expression is an intermediate marker for chondrogenesis, and that the protein can be localized to the extracellular matrix surrounding resting, proliferating, and hypertrophic chondrocytes by immunofluorescence histochemistry.
Assuntos
Cartilagem/embriologia , Colágeno/genética , Desenvolvimento Embrionário e Fetal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteoglicanas/genética , Animais , Membro Posterior/embriologia , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Proteoglicanos Pequenos Ricos em LeucinaRESUMO
The physiological role of nitric oxide (NO) on the mechanism of insulin secretion is unknown, but some studies suggest that NO affects glucose metabolism in pancreatic beta-cells. We have aimed at clarifying the physiological role of endogenous NO and its target in the glucose metabolism of beta-cells. The expression of brain-type NO synthase (bNOS) was detected in pancreatic islets by Western blotting. Under the condition of elevated intracellular Ca2+ concentration induced in the beta-cells by high glucose and forced depolarization by 40 mM K+, the generation of NO from the islets was enhanced. This increase was suppressed by the NOS blockers, N-iminoethyl-l-ornithine (L-NIO), and exposure to Ca2+-free extracellular solution. In addition, the NOS blockers L-NIO and 7-nitro indazole (7-NI) enhanced glucose-induced but not glyceraldehyde- or KIC-induced insulin secretion. In an in vitro enzyme study, the NO donor sodium nitroprusside (SNP) suppressed phosphofructokinase activity and activated glucokinase and glucose-6-phosphate isomerase activity, but SNP significantly inhibited the combined activity of the enzymes. This suggests that endogenous NO has an inhibitory role on insulin release induced by glucose and that its underlying mechanism is the suppression of phosphofructokinase activity in glycolysis.
Assuntos
Glucose/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Óxido Nítrico/fisiologia , Fosfofrutoquinase-1/metabolismo , Animais , Cálcio/farmacologia , Cerebelo/enzimologia , Inibidores Enzimáticos/farmacologia , Glucose/farmacologia , Técnicas In Vitro , Indazóis/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Isoenzimas/biossíntese , Isoenzimas/genética , Masculino , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/genética , Nitroprussiato/farmacologia , Ornitina/análogos & derivados , Ornitina/farmacologia , Fosfofrutoquinase-1/antagonistas & inibidores , Ratos , Ratos WistarRESUMO
Arabidopsis thaliana seeds imbibed for a short duration show phytochrome B (PhyB)-specific photo-induction of germination. Using this system, the relationship was determined between the amount of PhyB in seeds and photon energy required for PhyB-specific germination in two transgenic Arabidopsis lines transformed with either the Arabidopsis PhyB cDNA (ABO) or the rice PhyB cDNA (RBO). Immunochemical detection of PhyB apoprotein (PHYB) showed that the expression level of PHYB in ABO seeds was at least two times higher than that in the wild-type seeds, but in RBO seeds the PHYB level was indistinguishable from that in wild-type seeds. The photon fluence required for induction and photoreversible inhibition of germination was examined using the Okazaki large spectrograph. At the wavelengths of 400-710 nm, the ABO seeds required significantly less photon fluence than wild-type seeds for induction of germination, whereas the RBO seeds required similar fluence to wild-type seeds. A critical threshold wavelength for either induction or inhibition of germination of ABO seeds shifted towards the longer wavelengths relative to wild-type seeds. By assuming that PhyA and PhyB are similar in their photochemical parameters, amounts of Pfr at each wavelength were calculated. The photon fluence required for 50% germination was equivalent to the fluence generating a Pfr/Ptot ratio of 0.21-0.43 in wild-type seeds, and of 0.035-0.056 in ABO seeds. These results indicate that PhyB-specific seed germination is not strictly a function of the Pfr/Ptot ratio, but is probably a function of the absolute Pfr concentration.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Células Fotorreceptoras , Fitocromo/metabolismo , Fatores de Transcrição , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis , Imuno-Histoquímica , Luz , Fitocromo/genética , Fitocromo B , Plantas Geneticamente Modificadas , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Sementes/efeitos da radiaçãoRESUMO
The extracellular matrix plays an integral role in the pivotal processes of development, tissue repair, and metastasis by regulating cell proliferation, differentiation, adhesion, and migration. This review is focused on a family of related glycoproteins represented by at least one member in all specialized extracellular matrices. This family currently comprises nine members grouped together on the basis of their presence in the extracellular matrix and by virtue of a leucine-rich repeat motif that dominates the structure of the core protein. It is likely that most, if not all the members of this group exist as proteoglycans in some tissues, and thus have been termed the Small Leucine-Rich Proteoglycan family, or SLRPs. The leucine-rich repeat (LRR) is usually present in tandem array and has been described in an increasing number of proteins, giving rise to a LRR-superfamily. The LRR domain of the SLRP family is unique within the superfamily in that it is flanked by cysteine clusters, and the 24 amino acid consensus for SLRP members is x-x-I/V/L-x-x-x-x-F/P/L-x-x-L/P-x-x-L-x-x-L/I-x-L-x-x-N-x-I/L, where x is any amino acid. Enormous progress has been made in describing the membership, structure and localization of this family, and recently new insight has emerged into the putative function of these molecules not just as modulators of matrix assembly but also on their intriguing role in regulating cell growth, adhesion, and migration. Determination of membership, structure and putative function of this fascinating class of molecules is summarized in this review.
Assuntos
Proteínas da Matriz Extracelular/genética , Glicoproteínas/genética , Leucina/genética , Sequência de Aminoácidos , Animais , Biglicano , Decorina , Humanos , Dados de Sequência Molecular , Proteoglicanas/genéticaRESUMO
Mild depolarisation (20 mM KCl) synergistically enhances the ability of a muscarinic agonist to activate phosphoinositide turnover and to elevate inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in cerebellar granule cells in primary culture. The effects of lithium on this intense stimulation of phosphoinositide turnover was studied. Lithium causes depletion of cytoplasmic inositol and phosphoinositides, which results in the inhibition of phosphoinositide turnover within 15 min and the return of Ins(1,4,5)P3 to basal levels at this time. This inhibition could not be reversed by culturing and preincubating cerebellar granule cells in concentrations of inositol similar to those detected in the CSF. Inositol concentrations substantially in excess of those in the CSF not only reversed the effects of lithium on stimulated Ins(1,4,5)P3 levels, but significantly enhanced this level in comparison with stimulation in the absence of lithium. sn-1,2-Diacylglycerol elevation during stimulated phosphoinositide turnover was also disrupted by lithium, but in contrast to Ins(1,4,5)3, the presence of lithium resulted in a transient enhancement of the elevation evoked by carbachol plus mild KCl depolarisation, which was reversed by 500 microM inositol, but not by 200 microM inositol. The implications of these phenomena in relation to the mechanism of action of lithium in the treatment of manic depression are discussed.
Assuntos
Cerebelo/efeitos dos fármacos , Cerebelo/fisiologia , Lítio/farmacologia , Neurônios/fisiologia , Fosfatidilinositóis/fisiologia , Transdução de Sinais/efeitos dos fármacos , Animais , Carbacol/farmacologia , Células Cultivadas , Cerebelo/citologia , Diglicerídeos/metabolismo , Espaço Extracelular/metabolismo , Inositol/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Agonistas Muscarínicos/farmacologia , Neurônios/efeitos dos fármacos , Cloreto de Potássio/farmacologia , RatosRESUMO
PG-Lb was originally characterized as a small chondroitin/dermatan sulphate proteoglycan expressed preferentially in the zones of flattened chondrocytes in developing chick limb cartilage. The occurrence of this proteoglycan in mammalian cartilage has been shown by the isolation of a cDNA clone from mouse cartilage cDNA library [Kurita, Shinomura,Ujita, Zako, Kida, Iwata and Kimata (1996) Biochem. J. 318, 909-914]. To understand the regulation mechanisms for such a unique expression, we have investigated a genomic DNA structure of the PG-Lb gene. The gene is composed of seven exons and six introns spanning more than 50 kb. The leucine-rich repeats are encoded from exon V to exon VII. The transcription initiation site has been determined by rapid amplification of the cDNA ends ('5'-RACE'). The possible TATA box was detected about 90 bp upstream of the adenosine residue that was numbered as position +1. Further analyses of 1.5 kb of the 5' flanking region and 2.2 kb of the first intron have revealed several potential binding motifs for transcription factors such as Sox 5 and 9. The presence of those sequences in the PG-Lb gene was discussed in relation to the unique expression of this proteoglycan. The chromosomal localization of the murine PG-Lb gene was determined to be on the mouse chromosome 10 by the fluorescence-in-situ-hybridization ('FISH') method.
Assuntos
Dermatan Sulfato/genética , Proteoglicanas/genética , Animais , Sequência de Bases , Sulfatos de Condroitina/genética , Mapeamento Cromossômico , Clonagem Molecular , Éxons/genética , Hibridização in Situ Fluorescente , Íntrons/genética , Camundongos , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Regiões Promotoras Genéticas/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
PURPOSE: In the presence of carbon dioxide absorbents, sevoflurane is degraded to CF2 = C(CF3)OCH2F, an olefin compound A. There remains some concern of the hepatic and renal toxicity that compound A poses when using low-flow anaesthetic techniques. We investigated a device to decrease the concentration of compound A products by decreasing the temperature of exhaled air and soda lime in semi-closed low-flow anaesthesia technique in surgical patients. METHODS: Ten patients, ASA 1 or 2, were studied. Five received anaesthesia using a cooling circuit, that consisting of an anaesthetic circuit and an intercooler device interposed in the expiratory tube. The intercooler was dipped in an iced water tank. Anaesthesia was given through this circuit from induction to emergence. Another five patients received anaesthesia without cooling. Anaesthesia was maintained with sevoflurane and O2 50%/N2O during four to six hours of operation. A fixed concentration of sevoflurane 2% at a total flow of 1 L.min-1 was administered. Gas samples were taken every hour and compound A was quantitated by gas chromatography. The temperatures of canister, circuit and body were measured every hour. RESULTS: The device effectively lowered the temperatures [24 +/- 3.4 to 5 +/- 1.3 degrees C] and the concentrations of compound A [27.1 +/- 3.8 ppm to 16.3 +/- 2.08 ppm, P < 0.05] in the circuit. The body temperatures were not lowered. CONCLUSION: Compound A concentrations were reduced by cooling the anaesthetic circuit in clinical settings.
Assuntos
Anestesia por Inalação/instrumentação , Anestésicos Inalatórios/análise , Temperatura Baixa , Éteres/análise , Hidrocarbonetos Fluorados/análise , Éteres Metílicos/administração & dosagem , Absorção , Anestésicos Inalatórios/administração & dosagem , Anestésicos Inalatórios/química , Temperatura Corporal , Compostos de Cálcio/química , Dióxido de Carbono/química , Cromatografia Gasosa , Desenho de Equipamento , Éteres/química , Feminino , Seguimentos , Humanos , Hidrocarbonetos Fluorados/química , Gelo , Masculino , Éteres Metílicos/química , Pessoa de Meia-Idade , Óxido Nitroso/administração & dosagem , Óxidos/química , Oxigênio/administração & dosagem , Respiração , Sevoflurano , Hidróxido de Sódio/química , ÁguaRESUMO
We investigated the occurrence of alternatively spliced forms (V0, V1, V2, and V3) of PG-M/versican, a large chondroitin sulfate proteoglycan in developing chicken retinas, using the reverse transcription-polymerase chain reaction. We characterized the PLUS domain, which is apparently unique to the chicken molecule and is regulated by alternative splicing. PG-M in chicken retinas consisted of four forms with (V0, V1, V2, and V3) and two forms without (V1 and V3) the PLUS domain (PG-M+ and PG-M-, respectively). The four forms of PG-M+ were found in all samples examined, but the occurrence of the two PG-M- forms was regulated developmentally. Genomic analysis has revealed that the PLUS and CS-alpha domains are encoded by a single exon, and this exon has an internal alternative 5'-splice donor site, allowing alternative spliced forms that do not include the 3'-end of the exon. Sequences corresponding to the chicken PLUS domain (plus) were not found in mouse and human and may have disappeared during evolution. Sequence similarity suggests that the PLUS domain corresponds to the keratan sulfate attachment domain of aggrecan and that it has a distinct function in the chicken eye.