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1.
J Cell Biol ; 129(6): 1691-705, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7790364

RESUMO

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family of growth factors, which interact with EGF receptor to exert mitogenic activity. The membrane-anchored form of HB-EGF, proHB-EGF, is biologically active, providing mitogenic stimulation to neighboring cells in a juxtacrine mode. ProHB-EGF forms a complex with diphtheria toxin receptor-associated protein (DRAP27)/CD9, a tetra membrane-spanning protein that upregulates the juxtacrine mitogenic activity of proHB-EGF. We explored whether other proteins associate with DRAP27/CD9 and proHB-EGF. Immunoprecipitation with anti-DRAP27/CD9 resulted in preferential coprecipitation of integrin alpha 3 beta 1 from Vero cell, A431 cell and MG63 cell lysates. Anti-integrin alpha 3 or anti-integrin beta 1 coprecipitated DRAP27/CD9 from the same cell lysates. Chemical cross-linking confirmed the physical association of DRAP27/CD9 and integrin alpha 3 beta 1. Using Vero-H cells, which overexpress HB-EGF, we also demonstrated the association of proHB-EGF with DRAP27/CD9 and integrin alpha 3 beta 1. Moreover, colocalization of proHB-EGF, DRAP27/CD9, and integrin alpha 3 beta 1 at cell-cell contact sites was observed by double-immunofluorescence staining. At cell-cell contact sites, DRAP27/CD9 was highly coincident with alpha-catenin and vinculin, suggesting that DRAP27/CD9, proHB-EGF, and integrin alpha 3 beta 1 are colocalized with adherence junction-locating proteins. These results indicate that direct interaction of growth factors and cell adhesion molecules may control cell proliferation during the cell-cell adhesion process.


Assuntos
Antígenos CD/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Integrinas/metabolismo , Junções Intercelulares/fisiologia , Animais , Antígenos CD/química , Antígenos CD/isolamento & purificação , Membrana Celular/metabolismo , Chlorocebus aethiops , Reagentes de Ligações Cruzadas , Eletroforese em Gel de Poliacrilamida , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/isolamento & purificação , Imunofluorescência , Heparina/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Integrina alfa3beta1 , Integrinas/química , Integrinas/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intercelular , Glicoproteínas de Membrana/metabolismo , Microscopia Confocal , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tetraspanina 29 , Transfecção , Células Vero
2.
J Cell Biol ; 118(6): 1389-99, 1992 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1522113

RESUMO

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.


Assuntos
Antígenos CD/genética , Glicoproteínas de Membrana/genética , Receptores de Superfície Celular , Receptores Colinérgicos/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/metabolismo , Sequência de Bases , Western Blotting , Clonagem Molecular , Toxina Diftérica/metabolismo , Toxina Diftérica/farmacologia , Imunofluorescência , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ensaio Radioligante , Tetraspanina 29 , Células Vero
3.
J Cell Biol ; 128(5): 929-38, 1995 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7876316

RESUMO

The membrane-anchored heparin-binding EGF-like growth factor precursor (proHB-EGF)/diphtheria toxin receptor (DTR) belongs to a class of transmembrane growth factors and physically associates with CD9/DRAP27 which is also a transmembrane protein. To evaluate the biological activities of proHB-EGF/DTR as a juxtacrine growth factor and the biological significance of its association with CD9/DRAP27, the mitogenic activity of proHB-EGF/DTR was analyzed using stable transfectants of mouse L cells expressing both human proHB-EGF/DTR and monkey CD9/DRAP27, or either one alone. Juxtacrine activity was assayed by measuring the ability of cells in co-culture to stimulate DNA synthesis in an EGF receptor ligand dependent cell line, EP170.7. LH-2 cells expressing human proHB-EGF/DTR stimulated EP170.7 cell growth moderately. However, LCH-1 cells, a stable co-transfectant expressing both human proHB-EGF/DTR and monkey CD9/DRAP27 cDNAs, dramatically unregulated the juxtacrine growth factor activity of proHB-EGF/DTR approximately 25 times over that of LH-2 cells even though both cell types expressed similar levels of proHB-EGF/DTR on the cell surface. Anti-CD9/DRAP27 antibodies which were not able to neutralize the mitogenic activity of soluble HB-EGF suppressed LCH-1 cell juxtacrine growth activity to the same extent as did anti-HB-EGF neutralizing antibodies and CRM 197, specific inhibitors of human HG-EGF. These findings suggest that optimal expression of the juxtacrine growth activity of proHB-EGF/DTR requires co-expression of CD9/DRAP27. These studies also indicate that growth factor potentiation effects which have been observed previously for soluble growth factors also occurs at the level of cell surface associated growth factors.


Assuntos
Antígenos CD/biossíntese , Glicoproteínas de Membrana , Proteínas de Membrana/biossíntese , Precursores de Proteínas/biossíntese , Receptores de Superfície Celular/biossíntese , Regulação para Cima , Sequência de Aminoácidos , Northern Blotting , Western Blotting , Comunicação Celular , Células Cultivadas , Receptores ErbB/biossíntese , Receptores ErbB/genética , Citometria de Fluxo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Membrana/genética , Mitose/efeitos dos fármacos , Dados de Sequência Molecular , Testes de Neutralização , Precursores de Proteínas/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Tetraspanina 29 , Fator de Crescimento Transformador alfa/biossíntese
4.
Science ; 242(4876): 275-8, 1988 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-3051382

RESUMO

The signal sequence of simian virus 40 (SV40) large T-antigen for translocation into the nucleus is composed of positively charged amino acids Lys-Lys-Lys-Arg-Lys. Rabbit antibodies to a synthetic peptide containing the negatively charged amino acid sequence Asp-Asp-Asp-Glu-Asp were obtained. Indirect immunofluorescence of the antigens recognized by the antibody was punctate at the nuclear rim or the nuclear surface, depending on the plane of focus. The antibody blocked transport of nuclear proteins into the nucleus. The antigens recognized by the antibody were predominantly localized to the nuclear pores.


Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Oligopeptídeos/fisiologia , Fosfoproteínas , Sinais Direcionadores de Proteínas/fisiologia , Sequência de Aminoácidos , Animais , Antígenos/imunologia , Antígenos Transformantes de Poliomavirus , Transporte Biológico , Linhagem Celular , Imunofluorescência , Humanos , Dados de Sequência Molecular , Nucleoplasminas , Oligopeptídeos/imunologia , Ratos
5.
J Ethnopharmacol ; 231: 50-56, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30415057

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Mikania glomerata Spreng. (MG) and Mikania laevigata Sch. Bip. ex Baker (ML), popularly known as guaco, are medicinal plants similar in morphology, chemical composition and medicinal uses. Both species are often used and sold without distinction; however, it is believed that their chemical composition is different. AIM: Thus, the aim of this study is to investigate if the aqueous extract of MG and ML present similar anti-inflammatory activity to the point of being used interchangeably. MATERIAL AND METHODS: Different doses of both extracts and coumarin were given to rats in different experimental models to assess the anti-inflammatory activity between these two species. For this, the animals were submitted to paw edema, pleurisy and degranulation of peritoneal mast cell and the extracts were also characterized by Ultra High Efficiency Liquid Chromatography coupled to Mass Spectrometry (UHPLC-MS). RESULTS: The chromatographic method showed that ML presents ten times more coumarin than MG. Oral administration of MG, ML and coumarin inhibited paw edema induced by carrageenan (400 mg/kg, 55% inhibition; 400 mg/kg, 57% inhibition; 75 mg/kg, 38% inhibition; p < 0.05, respectively). MG, ML and coumarin treatment also inhibited the edema induced by compound 48/80 (400 mg/kg, 56% inhibition; 400 mg/kg, 69% inhibition; 75 mg/kg, 40% inhibition; p < 0.05, respectively). MG, ML and coumarin did not prevent mast cell degranulation and the consequent histamine release in Wistar rat peritoneal mast cells induced by compound 48/80. MG did not inhibit cell infiltration in pleurisy nor the highest dose tested, while ML decreased the leukocyte migration (200 and 400 mg/kg, 23% and 30% inhibition; p < 0.001, respectively) and, to a lesser extent, coumarin also reduced cell infiltration (10, 50 and 75 mg/kg; 15%, 16% and 17% inhibition; p < 0.001, respectively). CONCLUSION: The variation of the results of the anti-inflammatory activity found in M. glomerata and M. laevigata demonstrates that these two species should not be used interchangeably. Coumarin, as already proven, has anti-inflammatory action however, we have suggested that it probably is not the only component responsible for this therapeutic effect in the extracts.


Assuntos
Anti-Inflamatórios/uso terapêutico , Edema/tratamento farmacológico , Mikania , Extratos Vegetais/uso terapêutico , Pleurisia/tratamento farmacológico , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Carragenina , Degranulação Celular/efeitos dos fármacos , Edema/induzido quimicamente , Edema/imunologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/fisiologia , Mikania/química , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/uso terapêutico , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Pleurisia/induzido quimicamente , Pleurisia/imunologia , Ratos Wistar , p-Metoxi-N-metilfenetilamina
6.
Cytokine Growth Factor Rev ; 11(4): 335-44, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10959080

RESUMO

Heparin-binding EGF-like growth factor (HB-EGF), which belongs to the EGF-family growth factors, is synthesized as a membrane-anchored form (proHB-EGF). Proteolytic cleavage of proHB-EGF at the extracellular domain yields the soluble form of HB-EGF (sHB-EGF). ProHB-EGF is not only the precursor molecule for sHB-EGF but also a biologically active molecule itself. Recent studies indicate that proHB-EGF has unique properties distinct from the soluble form. ProHB-EGF forms a complex with membrane proteins including a tetramembrane spanning protein: CD9, an adhesion molecule integrin: alpha3beta1, and heparan sulfate proteoglycans. The complex is localized at the cell-cell contact site, suggesting that proHB-EGF may function in cell-to-cell signaling by a juxtacrine mechanism. In an in vitro model system, proHB-EGF showed growth inhibitory activity, while sHB-EGF was growth stimulatory. Ectodomain shedding, conversion of the membrane-anchored form into the soluble form, is regulated by multiple signaling pathways. All these characteristics imply that proHB-EGF and sHB-EGF are used in different ways. In vivo functions of sHB-EGF and proHB-EGF have been largely undefined, but recent studies implicate them in a variety of physiological processes including blastocyst implantation and wound healing.


Assuntos
Fator de Crescimento Epidérmico/fisiologia , Heparina/fisiologia , Animais , Implantação do Embrião/fisiologia , Fator de Crescimento Epidérmico/química , Feminino , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Membrana/fisiologia , Modelos Biológicos , Gravidez , Precursores de Proteínas/química , Precursores de Proteínas/fisiologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais , Cicatrização/fisiologia
7.
Cancer Res ; 60(24): 6886-9, 2000 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-11156386

RESUMO

To identify the genes located downstream of the activated Ki-Ras signaling pathways in human colon cancer cells, a PCR-based cDNA subtraction library was constructed between HCT116 cells and HCT116-derived activated Ki-ras-disrupted cells (HKe3). One of the genes in HCT116 that was evidently up-regulated was epiregulin, a member of the epidermal growth factor family that is expressed in many kinds of human cancer cells. HKe3-stable transfectants expressing activated Ki-Ras regained over-expression of epiregulin. To further elucidate the biochemical structure and significance of epiregulin expression in tumorigenesis, HKe3-stable transfectants expressing epiregulin (e3-pSE cells) were established. Epiregulin existed as highly glycosylated membrane-bound forms, and TPA rapidly induced ectodomain shedding of epiregulin. Furthermore, the conditioned medium of e3-pSE cells showed more DNA synthesis for 32D cells expressing epidermal growth factor receptor (DER) cells than that of HKe3. Although anchorage-independent growth in soft agar was not observed for e3-pSE cells, tumorigenicity in nude mice was observed evidently, and their growth rate was correlated with each amount of exogenous epiregulin expression. These results suggested that activated Ki-Ras will be one of the factors contributing to the overexpression of epiregulin in human colon cancer cells, and that epiregulin will play a critical role in human tumorigenesis in vivo.


Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Genes ras/genética , Proteína Oncogênica p21(ras)/metabolismo , Transdução de Sinais , Animais , Biotinilação , Northern Blotting , Meios de Cultivo Condicionados/metabolismo , DNA/metabolismo , DNA Complementar/metabolismo , Epirregulina , Biblioteca Gênica , Humanos , Ligantes , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase , Testes de Precipitina , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células Tumorais Cultivadas , Regulação para Cima
8.
FEBS Lett ; 156(1): 33-6, 1983 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-6852253

RESUMO

The proton NMR analysis of D-glucosaminate dehydratase reaction in D2O revealed the incorporation of a deuterium atom at C-3 carbon of the product, 2-keto-3-deoxy-D-gluconate. Based on the chemical shift of C-3 proton of the product and the coupling constant characteristic for the C-3 and C-4 axial-axial coupling in the 2C5 pyranose conformation, the deuterium is in the pro-S position. Thus, the dehydration of D-glucosaminate by the enzyme proceeds in a retention mode at C-3 carbon. Kinetic parameters show that the rate-determining step is the abstraction of alpha-proton from the substrate.


Assuntos
Hidroliases/metabolismo , Deutério , Gluconatos/metabolismo , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Conformação Molecular , Rhizobium/enzimologia
9.
Int J Radiat Oncol Biol Phys ; 31(3): 661-9, 1995 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-7852133

RESUMO

PURPOSE: Pilocarpine hydrochloride administered in either a fixed-dose or in a dose-titration protocol three times a day for 12 weeks was evaluated for its ability to relieve symptoms of postradiation xerostomia and to improve saliva production. The studies were randomized, double-blind, placebo-controlled, multicenter clinical trials. A total of 369 patients who had received at least 40 Gy of radiation to the head and neck with clinically significant xerostomia were enrolled in the two studies. In the dose-titration study, 162 patients were enrolled and they received a thrice daily regimen of 2.5 mg tablets for first 4 weeks, 5.0 mg tablets for the second 4 weeks, and 10.0 mg tablets for last 4 weeks of a 12-week study. Patients in the titration study were allowed to down titrate following at least one dose escalation to alleviate bothersome side effects, if any. In the fixed dose study, 207 patients received either placebo, 5.0 mg, or 10.0 mg tablets t.i.d. for 12 weeks. METHODS AND MATERIALS: Patients were evaluated for symptomatic relief by responding to questionnaires using visual analog scales and categorical questions; and, for saliva production by sialometry. Questionnaires measured relief of intraoral dryness, improvement in overall condition (global response), oral discomfort, difficulty in speaking, chewing and swallowing, denture wearing, and usage of artificial saliva. Evaluations were conducted at baseline, and weeks 4, 8, and 12. RESULTS: There were statistically significant improvements in salivary flow in pilocarpine treatment groups vs. placebo. There was a significant improvement in the overall "global" condition of xerostomia associated with the use of pilocarpine in both studies. In the fixed-dose study, there were significant improvements in oral dryness, mouth comfort, ability to speak, and reduction in the use of oral comfort agents. The dose-titration study showed improvements in dryness that approached significance (p = 0.057) and a decreased use of oral comfort agents (p = 0.045). All pilocarpine dosages (2.5, 5.0, and 10.0 mg three times a day) were judged to be safe. Adverse experiences were those expected for a cholinergic agonist, with the most common being mild to moderate sweating. The incidence of these events increased by dose. CONCLUSION: It is concluded that in these studies pilocarpine produced clinically significant benefits with acceptable side effects and risks for the treatment of symptomatic postradiation xerostomia. The incidence of most adverse events increased with dose. Best results may require continuous treatment for more than 8 weeks with doses greater than 2.5 mg three times a day. A 5.0 mg thrice daily regimen produced the best clinical results when both efficacy and side effects were taken into consideration. There may be some patients who would experience some additional benefit by increasing the dose to 10 mg thrice daily.


Assuntos
Neoplasias de Cabeça e Pescoço/radioterapia , Pilocarpina/uso terapêutico , Xerostomia/tratamento farmacológico , Administração Oral , Adulto , Idoso , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pilocarpina/administração & dosagem , Pilocarpina/efeitos adversos , Radioterapia/efeitos adversos , Xerostomia/etiologia
10.
J Histochem Cytochem ; 49(4): 439-44, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11259446

RESUMO

The tetra-membrane-spanning protein CD9 forms a complex with a membrane-anchored heparin binding epidermal growth factor-like growth factor (HB-EGF) and integrin alpha3beta1 in some human and monkey cell lines. We show here the immunohistochemical distribution of CD9, HB-EGF, and integrin alpha3beta1 in normal human tissues. Distribution of CD9, HB-EGF, and integrin alpha3beta1 was similar in various tissues, including transitional epithelium, squamous epithelium, thyroid follicular epithelium, adrenal cortex, testis, smooth muscle, and stromal fibrous tissue. However, distribution of the three proteins did not coincide in some tissues, such as lung, liver, kidney, gastric and intestinal epithelium, pancreas, salivary gland, and ovary. In striated muscle, including cardiac muscle, CD9 was present not in the muscle cells themselves but in the endomysium and perimysium, whereas HB-EGF was distributed in the muscle cells themselves. CD9 was distributed in the myelin, but HB-EGF was found in the axon of the peripheral and central nervous systems. Coincident distribution of integrin alpha3beta1 with others was not observed in muscles and neural tissues. In conclusion, there is a possibility of complex formation and functional cooperation of CD9 with HB-EGF and/or integrin alpha3beta1 in several tissues.


Assuntos
Antígenos CD/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Integrinas/metabolismo , Glicoproteínas de Membrana , Western Blotting , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Imuno-Histoquímica , Integrina alfa3beta1 , Peptídeos e Proteínas de Sinalização Intercelular , Especificidade de Órgãos , Tetraspanina 29
11.
J Biochem ; 109(1): 66-9, 1991 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1849886

RESUMO

Pseudomonas fluorescens (Migula) (IFO 14808) has both a membrane-bound PQQ-dependent D-glucose (D-Glc) dehydrogenase [EC 1.1.99.17] [which also acts on D-glucosamine (D-GlcN)] and a PLP-dependent D-glucosaminate (D-GlcNA) dehydratase [EC 4.2.1.26]. Further, these two enzymes were induced when D-GlcN was added to the culture medium. However, D-glucosamine-6-phosphate (D-GlcN-6-P) isomerase [EC 5.3.1.10], another enzyme involved in the metabolism of D-GlcN, was only present at a low level in this bacterium. The bacterium was able to grow in a minimal medium containing D-GlcN or D-GlcNA as the sole source of carbon and nitrogen. Intact cells of P. fluorescens (Migula) converted D-GlcN to D-GlcNA and then to 2-keto-3-deoxy-D-gluconate (KDGA). These results demonstrate that D-GlcN is metabolized via D-GlcNA to KDGA in P. fluorescens (Migula) (Entner-Doudoroff pathway). In contrast, Enterobacter cloacae(IFO 13535) and Agrobacterium radiobacter (IAM 1526) have significant amounts of D-GlcN-6-P isomerase with low levels of the D-Glc dehydrogenase and D-GlcNA dehydratase. Further, only the isomerase activity was induced on the addition of D-GlcN to the culture medium. These results demonstrate that there is a new route (Entner-Doudoroff pathway), i.e., in addition to the known one (Embden-Meyerhof pathway), for the metabolism of D-GlcN in bacteria and one of the two routes is predominant in the each of bacteria examined.


Assuntos
Aldose-Cetose Isomerases , Bactérias/metabolismo , Glucosamina/metabolismo , Cálcio/farmacologia , Carboidratos Epimerases/metabolismo , Enterobacter/metabolismo , Glucose 1-Desidrogenase , Glucose Desidrogenase/metabolismo , Hidroliases/metabolismo , Cofator PQQ , Pseudomonas fluorescens/metabolismo , Quinolonas/farmacologia , Rhizobium/metabolismo
12.
J Biochem ; 95(1): 13-8, 1984 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-6706902

RESUMO

The holoenzyme of D-glucosaminate dehydratase [EC 4.2.1.26] from Agrobacterium radiobacter showed absorption peaks at 280 and 415 nm with a shoulder in the region of 320 to 330 nm. The treatment of the enzyme with hydroxylamine followed by dialysis led to disappearance of both the absorption peak at 415 nm and the shoulder, giving the apoenzyme. The fluorescence excitation maximum of the holoenzyme was at 320 nm with a shoulder at 420 nm (emission at 510 nm), and the emission maxima were at 420 nm with a shoulder at 370 nm (excitation at 320 nm) and at 510 nm (excitation at 420 nm). The holoenzyme showed a negative circular dichroic band at 418 nm and a positive shoulder at around 320 nm. Reduction of the holoenzyme with sodium borohydride caused a loss of the absorption peak at 415 nm with a concomitant increase of 325 nm absorbance and an irreversible loss of the activity. The occurrence of epsilon-N-pyridoxyllysine in the acid hydrolysate of the reduced enzyme showed that D-glucosaminate dehydratase contains a catalytically essential lysine residue whose epsilon-amino group binds the 4-formyl group of pyridoxal 5'-phosphate to form a Schiff base. The plots of absorption of the apoenzyme against the amount of pyridoxal 5'-phosphate added showed that four and two molar equivalents of the cofactor bind to the apoenzyme and subunit, respectively. The biphasic nature of the spectrometric titration curve of the apoenzyme with pyridoxal 5'-phosphate and the two Km values obtained for the cofactor suggest the occurrence of two distinct types of binding sites for pyridoxal 5'-phosphate in the enzyme.


Assuntos
Hidroliases/isolamento & purificação , Fosfato de Piridoxal/isolamento & purificação , Rhizobium/enzimologia , Dicroísmo Circular , Ligação Proteica , Espectrometria de Fluorescência , Espectrofotometria
13.
J Biochem ; 91(1): 283-9, 1982 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7068563

RESUMO

The bacterial distribution of D-glucosaminate dehydratase [EC 4.2.1.26] was investigated and Agrobacterium radiobacter (IAM 1526) was found to have the highest enzyme activity. The enzyme was formed inducibly in a glycerol-urea medium by D-glucosamine, D-galactosamine, and D-glucosamine, but not by D-mannosamine. The enzyme purified from the cells grown in the glucosamine-glycerol-urea medium was shown to be homogeneous by ultracentrifugation. The molecular weight was determined to be about 66,000 by the sedimentation equilibrium method, and 72,800 by the gel permeation chromatography low-angle light scattering method. The pH optimum is 8.3-9.0. The enzyme catalyzed the dehydration of D-glucosaminate (relative activity: 100, Km: 2.8 mM), D-galactosaminate (31.5, 5.0 mM), D-mannosaminate (17.5, 29 mM), D-threonine (5.1, 4.8 mM), D-serine (3.2, 0.026 mM), and L-serine (1.1, ND), but not L-threonine. The reverse reaction does not occur. The enzyme is inhibited by typical inhibitors of pyridoxal 5'-phosphate enzymes, such as L'penicillamine, and also by carbonyl reagents, thiol reagents, divalent metals, and several D-amino acids and D-amino sugars.


Assuntos
Hidroliases/metabolismo , Rhizobium/enzimologia , Estabilidade de Medicamentos , Indução Enzimática/efeitos dos fármacos , Gluconatos/metabolismo , Glucosamina/farmacologia , Hidroliases/antagonistas & inibidores , Hidroliases/isolamento & purificação , Concentração de Íons de Hidrogênio , Fosfato de Piridoxal/farmacologia , Especificidade por Substrato , Temperatura
14.
Oncology (Williston Park) ; 10(3 Suppl): 12-5, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8723428

RESUMO

Xerostomia during and following a course of head and neck irradiation profoundly impacts the quality of life of many patients. Xerostomia not only affects mucous membranes and teeth but also interferes with patient comfort, nutrition, and activities of daily living. A thorough evaluation of xerostomia is essential and should include providing anticipatory guidance to the patient and family. In addition, education on prophylactic oral care is necessary during the initial phases of treatment. As symptoms occur, various palliative interventions are tailored to the patient's and family's needs, promoting adherence to mouth care regimens and enhancing patient comfort. Long-term follow-up with education and counseling is critical for optimal patient management.


Assuntos
Lesões por Radiação/enfermagem , Radioterapia/efeitos adversos , Xerostomia/enfermagem , Atividades Cotidianas , Aconselhamento , Seguimentos , Neoplasias de Cabeça e Pescoço/radioterapia , Promoção da Saúde , Humanos , Estudos Longitudinais , Fenômenos Fisiológicos da Nutrição , Cuidados Paliativos , Educação de Pacientes como Assunto , Qualidade de Vida , Xerostomia/etiologia , Xerostomia/prevenção & controle
15.
Acta Med Okayama ; 51(1): 19-23, 1997 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9057931

RESUMO

This study was conducted to evaluate the frequency of DNA transfection into human cells following X-ray irradiation. We transfected plasmid DNA (pSV2neo) into human cells, HeLa and PA-1, by either calcium phosphate precipitation or the lipofection method immediately after irradiating the cells with various doses of X-rays. The transfection frequency was evaluated by counting the number of G418-resistant colonies. When circular plasmid DNA was used, irradiation up to a dose of 2 Gy dose-dependently increased the transfection frequency, which reached a maximum of 5 to 10-fold that of the control unirradiated cells. When linear plasmid DNA was used, the transfection frequency was 2 times higher than that of circular DNA. All five of the clones that were randomly chosen expressed the transfected neo gene. In addition, the pSV2neo gene was randomly integrated into the genomic DNA of each clone. These findings indicate that X-ray treatment can facilitate foreign DNA transfer into human cells and that radiation-induced DNA breaks may promote the insertion of foreign DNA into host DNA. The enhancement of DNA transfection with X-rays may be instrumental in practicing gene therapy.


Assuntos
DNA/genética , Plasmídeos/genética , Transfecção/genética , Linhagem Celular , DNA/efeitos da radiação , Células HeLa , Humanos , Doses de Radiação , Transfecção/efeitos da radiação
16.
Semin Oncol Nurs ; 17(2): 90-100, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11383249

RESUMO

OBJECTIVES: To review the advances in radiation therapy for prostate cancer and the nursing care of patients with prostate cancer. DATA SOURCES: Peer-reviewed journal articles, including research studies and review articles. CONCLUSIONS: Radiation therapy is used to cure early stage prostate cancer, control locally advanced disease, and effectively palliate symptoms of metastasis. The three forms of treatment used include external beam radiation therapy, brachytherapy; and radiopharmaceutical treatments. IMPLICATIONS FOR NURSING PRACTICE: Nursing care of patients receiving radiation therapy for prostate cancer includes managing the symptoms associated with the disease and treatment, educating patients and families about self-care measures, and providing support throughout the course of the disease.


Assuntos
Braquiterapia/métodos , Neoplasias da Próstata/radioterapia , Radioterapia Conformacional/métodos , Antineoplásicos Hormonais/uso terapêutico , Braquiterapia/efeitos adversos , Braquiterapia/psicologia , Terapia Combinada , Humanos , Masculino , Estadiamento de Neoplasias , Enfermagem Oncológica/educação , Cooperação do Paciente/psicologia , Educação de Pacientes como Assunto , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/psicologia , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/uso terapêutico , Radioterapia Conformacional/efeitos adversos , Radioterapia Conformacional/psicologia , Sexualidade/efeitos da radiação , Resultado do Tratamento
17.
J Clin Anesth ; 13(7): 504-8, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11704448

RESUMO

STUDY OBJECTIVE: To test the hypothesis that core temperature is well preserved when atropine and midazolam are combined. DESIGN: Randomized, blinded study. SETTING: Department of Anesthesia, Yamanashi Medical University. PATIENTS: 40 elderly, ASA physical status I and II patients (aged more than 60 years). INTERVENTIONS: Patients were randomly assigned (n = 10 per group) to premedication with: 1) saline control; 2) midazolam 0.05 mg/kg; 3) atropine 0.01 mg/kg; and 4) midazolam 0.05 mg/kg combined with atropine 0.01 mg/kg. All premedication was given on the ward at approximately 8:30 am, approximately 30 minutes before induction of anesthesia. MEASUREMENTS AND MAIN RESULTS: Core temperatures were measured at the right tympanic membrane. Mean skin temperature was calculated as 0.3 x (T(chest) + T(arm)) + 0.2 x (T(thigh) + T(calf)). Fingertip perfusion was evaluated using forearm minus fingertip and calf minus toe, skin-surface temperature gradients. Temperatures were evaluated at the time of premedication and 30 minutes later, just before induction of anesthesia. Core temperature remained nearly constant in the control patients (0.1 +/- 0.2 degrees C; mean +/- SD), whereas it decreased significantly in the patients given midazolam alone (-0.3 +/- 0.1 degrees C). Atropine alone increased core temperature (0.3 +/- 0.2 degrees C), although the increase was not statistically significant. The combination of midazolam and atropine attenuated the hypothermia induced by midazolam alone (0.0 +/- 0.2 degrees C). Initial skin-temperature gradients exceeded 0 degrees C in all groups, indicating that the patients were vasoconstricted. The gradients were unchanged by premedication with saline or atropine. Midazolam significantly decreased the gradient (-1.8 +/- 1.1 degrees C), as did the combination of midazolam and atropine (-1.4 +/- 0.9 degrees C). CONCLUSIONS: The thermoregulatory effects of benzodiazepine receptor agonist and cholinergic inhibitors oppose each other, and the combination leaves core temperature unchanged.


Assuntos
Atropina/farmacologia , Hipotermia/prevenção & controle , Midazolam/efeitos adversos , Pré-Medicação , Idoso , Regulação da Temperatura Corporal/efeitos dos fármacos , Feminino , Hemodinâmica/efeitos dos fármacos , Humanos , Hipotermia/induzido quimicamente , Masculino
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