RESUMEN
Precision frequency detection has enabled the suspended microchannel resonator (SMR) to weigh single living cells, single nanoparticles, and adsorbed protein layers in fluid. To date, the SMR resonance frequency has been determined optically, which requires the use of an external laser and photodiode and cannot be easily arrayed for multiplexed measurements. Here we demonstrate the first electronic detection of SMR resonance frequency by fabricating piezoresistive sensors using ion implantation into single crystal silicon resonators. To validate the piezoresistive SMR, buoyant mass histograms of budding yeast cells and a mixture of 1.6, 2.0, 2.5, and 3.0 µm diameter polystyrene beads are measured. For SMRs designed to weigh micron-sized particles and cells, the mass resolution achieved with piezoresistive detection (â¼3.4 fg in a 1 kHz bandwidth) is comparable to what can be achieved by the conventional optical-lever detector. Eliminating the need for expensive and delicate optical components will enable new uses for the SMR in both multiplexed and field deployable applications.
Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentación , Impedancia Eléctrica , Microesferas , Peso Molecular , Reproducibilidad de los Resultados , Saccharomycetales/química , TemperaturaRESUMEN
Type 2 diabetes occurs in special groups of children. One of the highest rates of type 2 diabetes in the world occurs in First Nation people. Screening of First Nation children has been recommended. To diagnose diabetes earlier and prevent complications in adulthood, a program of screening and primary prevention of First Nation people must begin with children. One hundred fifteen school-aged First Nation children were approached to participate in the present study, and 93% of the children completed the study. Eight (7%) of the study participants had abnormal capillary blood glucose levels, but no cases of type 2 diabetes were confirmed. Risk factors related to exercise, diet, blood pressure and body mass index were identified. The present paper describes a collaborative program with a small Paediatric Diabetic Education Center and the Beausoleil First Nation people.
RESUMEN
Studies of the multistage nature of hepatocarcinogenesis in the rat have led to the development of models having significant potential application to carcinogenesis in other tissues as well as other species. Whereas the initial and final stages of carcinogenesis-initiation and progression-involve genetic changes and are operationally irreversible, the intermediate stage of promotion is operationally reversible and can be modulated by a variety of environmental factors. Numerous investigations have demonstrated that chronic caloric restriction modifies neoplastic development, primarily during the stage of promotion, so that fewer lesions develop. Short-term fasting of rats, initiated with a nonnecrogenic dose of diethylnitrosamine (DEN) and promoted with 0.05% phenobarbital (PB) for 4 weeks, results in loss of virtually all of the measurable altered hepatic foci (AHF) after two 5-day periods of fasting with an intermediate 2-day period of feeding. This change was accompanied by a marked decrease in bromodeoxyuridine (BrdU) labeling of hepatocytes within AHF together with a significant increase in apoptosis of such cells measured by nick end-labeling. Similar but lesser effects were noted in surrounding, nonfocal hepatocytes. On refeeding, both the numbers and volume percentage of AHF returned within 2 weeks to values seen in nonfasted controls. Administration of PB during the fasting period did not alter these results, although AHF reappeared more rapidly in such animals on refeeding. Nuclear DNA fragmentation was evident in samples of whole liver from fasted animals. During this same period the expression of c-myc mRNA increased 3- to 9-fold, while levels of albumin and insulin-like growth factor I mRNAs decreased significantly. This study demonstrates a model system in which the reversibility of the effects of promoting agents may be rapidly determined and the effects of chemopreventive inhibitors of promotion may be rapidly evaluated.
Asunto(s)
Apoptosis , Ayuno , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , Modelos Biológicos , Animales , Carcinógenos/farmacología , División Celular , Transformación Celular Neoplásica , Femenino , Alimentos , Hígado/efectos de los fármacos , Nafenopina/farmacología , Fenotipo , Ratas , Ratas Sprague-DawleyRESUMEN
Previous work from this laboratory has reported on the effects of two sequential 5 day periods of fasting and subsequent refeeding on tumor promotion in multistage hepatocarcinogenesis in the rat (Carcinogenesis, 18, 159-166, 1997). In the present extension of the earlier study, the sequential fasting-refeeding regimen was begun at later time points (28 and 54 days post-initiation) than the first study. This was done to determine whether larger-sized altered hepatic foci (AHF) exhibited a depletion similar to that of the relatively small AHF in the published experiment and to study concomitant molecular changes during the fasting periods. Groups of animals were fasted in the presence and absence of 0.05% phenobarbital (PB) in the drinking water. During the fasting periods, both body and liver weights decreased dramatically, less in the fast begun at 54 days. This change was accompanied by a significant decrease in the bromodeoxyuridine (BrdU) labeling indices of hepatocytes within AHF. Apoptotic bodies increased dramatically in the non-focal (surrounding the AHF) hepatocytes during the fasting periods. These parameters were slightly lower in hepatocytes of rats administered PB during the fasting periods, most notably during the 54-66 day period. With the nick end-labeling method, the proportion of hepatocytes undergoing apoptosis was significantly higher in cells within AHF at the end of each of the fasting periods in all but one group. Concomitantly, the number of AHF and percentage of liver volume occupied by AHF decreased dramatically during the fasting periods. Refeeding caused a marked increase in BrdU labeling in hepatocytes within and surrounding AHF during the first week or two, most notably in animals not receiving PB during the fasting period. Both the number and volume percentage of liver AHF returned to control values within approximately 2 weeks of the refeeding regimen. Assays of nuclear DNA fragmentation with samples of whole liver indicated that a 'laddering' effect was most noticeable in livers of animals subjected to the fasting-refeeding regimen when phenobarbital was not present during the fasting period. Studies of the levels of mRNA of several genes in the total liver revealed that the expression of c-myc increased 3- to 9-fold during the fasting periods but rapidly returned to normal levels after refeeding. Levels of albumin and insulin-like growth factor I mRNAs decreased significantly during the fasting period, but rapidly reappeared on refeeding. These results indicate that the extensive loss of AHF during the short-term fasting periods occurs even when the number and volume of AHF are 10- to 50-fold greater at the beginning of the fast than the values published previously. Both the decrease in insulin growth factor I and the elevation of c-myc expression during the fasting period may indicate the role of these genes in the transcriptional regulation of hepatocyte apoptosis in both normal and preneoplastic hepatocytes in the rat.
Asunto(s)
Apoptosis , Ingestión de Alimentos , Ayuno , Neoplasias Hepáticas Experimentales/etiología , Fenobarbital/farmacología , Actinas/metabolismo , Animales , Recuento de Células , División Celular , Cocarcinogénesis , Femenino , Expresión Génica , Glutatión Transferasa/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Tamaño de los Órganos , Fragmentos de Péptidos/metabolismo , ARN/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Antibodies raised against a 43 kDa component of a complex of synaptic membrane proteins with ligand binding sites characteristic of glutamate/N-methyl-D-aspartate (NMDA) receptors, were used previously to clone a cDNA for a glycine-, glutamate-, and thienylcyclohexylpirperidine (TCP)-binding protein, pGlyBP (Kumar et al., Biochem. Biophys. Res. Commun. 216, 390-398, 1995). In the present studies, the antibodies were shown to label a 60 kDa protein, in synaptic membranes, that was relatively hydrophilic as demonstrated by its predominant separation in the detergent-depleted phase of proteins solubilized with Triton X-114. A 55-60 kDa protein was purified from rat brain synaptic membranes by chromatographic separation through matrices derivatized with 5,7-di-chlorokynurenic acid (5,7-DCK) followed by chromatography on a matrix derivatized with 8-hydroxyquinoline (8-OHQ). The isolated fractions were highly enriched in strychnine-insensitive [3H]glycine, NMDA- and glutamate-sensitive L-[3H]glutamate, and MK-801-sensitive [3H]TCP binding sites. The purified protein bound [3H]glycine with a stoichiometry of 1.1-1.2 mol glycine per mol protein and exhibited both high (KD = 280 nM) and low affinity (KD = 30 microM) glycine binding sites. Glycine binding was inhibited by D-serine and R-(+)-3-amino-1-hydroxypyrrolidin-2-one(R-(+)-HA-966). The KD values for high and low affinity sites of glycine binding as well as those for the inhibition by R-(+)-HA-966 were very similar to the KDs for glycine binding to the expressed pGlyBP. Both L-glutamate and glycine activated [3H]TCP binding to the isolated proteins, but with relatively low affinity. The anti-43 kDa antibodies reacted strongly with the 55-60 kDa protein. Based on these results, it appears that the 60 kDa glycoprotein in brain synaptic membranes described in the present study is the same protein as the cloned pGlyBP.
Asunto(s)
Química Encefálica/fisiología , Glicoproteínas de Membrana/aislamiento & purificación , Proteínas del Tejido Nervioso/aislamiento & purificación , Membranas Sinápticas/química , Animales , Clonación Molecular , Detergentes , Inmunoquímica , Masculino , Octoxinol , Polietilenglicoles , Ensayo de Unión Radioligante , RatasRESUMEN
The functional reconstitution of glutamate receptor proteins purified from mammalian brain has been difficult to accomplish. However, channels activated by L-glutamate (L-Glu) and N-methyl-D-aspartate (NMDA) were detected in planar lipid bilayer membranes (PLMs) following the reconstitution of a complex of proteins with binding sites for NMDA receptor (NMDAR) ligands. The presence of glycine was necessary for optimal activation. A linear current-voltage relationship was observed with the reversal potential being zero. Channels activated by L-Glu had conductances of 23, 47 and 65 pS, and were suppressed partially by competitive and fully by noncompetitive inhibitors of NMDARs. Magnesium had little effect on the reconstituted channels.
Asunto(s)
Canales Iónicos/metabolismo , Receptores de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Membranas Sinápticas/química , 2-Amino-5-fosfonovalerato/farmacología , Animales , Ácido Aspártico/farmacología , Sitios de Unión , Encéfalo/metabolismo , Calcio/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/farmacología , Glicina/farmacología , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/química , Canales Iónicos/aislamiento & purificación , Membrana Dobles de Lípidos/química , Liposomas/química , Técnicas de Placa-Clamp , RatasRESUMEN
Cytogenetic changes that occur during the progression of rat hepatocarcinogenesis were assessed with three rat liver epithelial cell lines derived from WB cells. Previously characterized WBneo, WBras, and WBrasIIa cells were grown in culture and analyzed for structural and numerical chromosomal integrity by banded karyotype analysis. The WBneo cells had a low level of aneuploidy with a consistent loss of the Y chromosome by passage 7. The ras-transfected cell line selected for growth in soft agar, WBras, had acquired a loss of chromosome 3 (12%) or 3p (34%), a trisomy of chromosome 1, as well as the chromosome Y loss. The cell line produced from tumors generated by injection of the WBras cells into a syngeneic F344 rat, WBrasIIa, contained additional chromosomal changes. The WBrasIIa line comprised cells retaining a trisomy of chromosome 1 (55%) and cells with two copies of chromosome 1, with a minimal duplication of 1q3.7 to 1q4.3 (45%). This tumor-derived cell line contained, in addition, a higher percentage of cells with a loss of all or part of chromosomes 3 and 6, indicating the possible presence of tumor suppressor genes in this region. The smallest region of duplication of chromosome 1 was bands 1q3.7-4.3. The insulin-like growth factor II (IGF-II) gene is located within the region of duplication on chromosome 1. Because IGF-II is both a rat liver mitogen and an inhibitor of apoptosis, its expression was examined in these three rat liver epithelial cell lines. Northern blot analysis demonstrated an increase in IGF-II mRNA expression in the WBras and WBrasIIa cell lines relative to the WBneo control cell line. Several IGF-II transcripts analogous to those detected in fetal rat liver were observed. An additional IGF-II transcript that migrates above the 28S ribosomal marker was also observed. These results were confirmed at the protein level by immunohistochemical and Western blot analysis. This increased expression of IGF-II may confer a selective growth advantage to rat liver epithelial cells with a duplication of 1q3.7-4.3. This growth advantage may be enhanced by the further sequential loss of putative tumor suppressor genes on chromosomes 3 and 6.
Asunto(s)
Aberraciones Cromosómicas , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Hígado/metabolismo , Hígado/fisiología , Animales , Células Cultivadas , Progresión de la Enfermedad , Epitelio/química , Epitelio/metabolismo , Epitelio/fisiología , Expresión Génica , Inmunohistoquímica , Factor II del Crecimiento Similar a la Insulina/análisis , Factor II del Crecimiento Similar a la Insulina/genética , Cariotipificación , Hígado/química , Ratas , Ratas Endogámicas F344 , Transfección , Proteínas ras/análisis , Proteínas ras/biosíntesis , Proteínas ras/genéticaRESUMEN
Long-term treatment of breast cancer patients with tamoxifen has prompted concern over potential toxicity of this drug with chronic administration. Since tamoxifen has estrogenic action in the rat liver and estrogenic agents can increase hepatoma incidence in rats, tamoxifen and two non-isomerizable, fixed-ring analogs (FRT1 and FRT2) were evaluated as promoting agents in a two-stage model of hepatocarcinogenesis in female Fischer F344 rats. The rats were subjected to 70% partial hepatectomy and half of the animals were administered the initiating agent, diethylnitrosamine (DEN; 10 mg/kg body wt), while the other half were not initiated. Groups of initiated and uninitiated animals were allowed to recover for 2 weeks and were then administered tamoxifen or one of the fixed-ring analogs admixed into AIN-76A diet at 25, 100 or 250 mg/kg diet. After 6 months of anti-estrogen administration the rats were sacrificed and uterine weights, blood levels of anti-estrogen, and liver histopathology were assessed. Uterine weights were decreased 2- to 3-fold by each of the agents, consistent with an anti-estrogenic action in the rat. The serum levels in rats administered 250 mg anti-estrogen/kg diet for 6 months were 320+/-20 ng/ml for tamoxifen, 320+/-10 for FRT1 and 350+/-20 for FRT2. The liver levels after a 6 month administration of 250 mg anti-estrogen/kg diet were 13 870+/-860 ng/g for tamoxifen, 13 300 +/-860 for FRT1 and 26 900+/-1900 for FRT2. A dose-dependent increase in serum and liver level of each compound was noted when measured at the 6 month time period. The number and percentage of the liver occupied by altered hepatic foci (AHF) were determined by quantitative stereology. A dose-dependent increase above initiated controls was observed in the initiated, tamoxifen-treated rats. Both fixed-ring analogs also increased the number and size of AHF compared with initiated controls, but were less potent than tamoxifen, suggesting that tamoxifen has an intrinsic promoting action in the liver that is independent of its ability to isomerize to more potent estrogenic compounds. In addition, the fixed-ring analogs have a weaker promoting activity in the rat liver than does tamoxifen. This may be due to pharmacokinetic differences at the lower two doses, but it is independent of achieved serum level at the highest dose and hence may reflect differences in intrinsic activity of these compounds. Thus tamoxifen and the two fixed-ring analogs promote the development of rat hepatocarcinogenesis.
Asunto(s)
Anticarcinógenos/toxicidad , Antagonistas de Estrógenos/toxicidad , Neoplasias Hepáticas Experimentales/inducido químicamente , Hígado/efectos de los fármacos , Tamoxifeno/toxicidad , Animales , Anticarcinógenos/sangre , Anticarcinógenos/farmacocinética , Peso Corporal/efectos de los fármacos , Dietilnitrosamina , Relación Dosis-Respuesta a Droga , Antagonistas de Estrógenos/sangre , Antagonistas de Estrógenos/farmacocinética , Femenino , Hígado/metabolismo , Hígado/ultraestructura , Microcuerpos/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Endogámicas F344 , Tamoxifeno/análogos & derivados , Tamoxifeno/sangre , Tamoxifeno/farmacocinética , Útero/efectos de los fármacosRESUMEN
Polyclonal antibodies (Ab's) were raised against a 43-kDa component of a protein complex that has ligand recognition sites similar to those of brain N-methyl-D-aspartate (NMDA) receptors. The Ab's were used to immunopurify from brain synaptic membranes a 60-kDa glycine (Gly), glutamate (Glu) and thienylcyclohexylpiperidine (TCP)-binding protein and to screen a rat hippocampal cDNA expression library. A 1.85-kb clone, pGlyBP, coding for a protein of 470 amino acids (52.7 kDa) was identified. Northern blot analyses performed on poly(A+) RNA from brain revealed hybridization of the labeled cDNA probes to transcripts of 1.9 kb. E. coli transformed with the pGyBP expressed a protein that was recognized by the anti-43 kDa Ab's and had recognition sites for Gly, Glu and TCP. The cloned protein has 2 glycosylation sites, 3 hydrophobic domains, 4 cysteine-rich motifs (C-X2-C-X16-20-C-X5-11), and 2 regions homologous to the NR1 receptor protein.
Asunto(s)
Encéfalo/metabolismo , Glicoproteínas de Membrana/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Membranas Sinápticas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Clonación Molecular/métodos , Cisteína , ADN Complementario/metabolismo , Biblioteca de Genes , Ácido Glutámico/metabolismo , Glicina/metabolismo , Hipocampo/metabolismo , Drogas Ilícitas , Cinética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Fenciclidina/análogos & derivados , Fenciclidina/metabolismo , ARN Mensajero/biosíntesis , Ratas , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
After a 70% partial hepatectomy (PH), the steady-state levels of Connexin (Cx)32, Cx26, and Cx43 messenger RNA (mRNA) transcripts each displayed unique patterns of temporal expression. Within 1 hour after surgical resection, increased expression of all three Cx mRNAs was observed. Subsequently, the level of Cx32 mRNA transcripts transiently decreased to a nadir at 12 hours. Comparisons of the spatial changes with previously reported hepatocyte proliferation kinetics induced by PH demonstrated that hepatocytes before S-phase "remodel" their GJs. Within 1 to 5 hours post-PH, midzonal hepatocytes exhibited diffuse membrane staining different from the normal punctate distribution. Subsequently, midzonal hepatocytes expressed colocalized punctate Cx32 and Cx26 immunostaining. Because the changes occurred in midzonal hepatocytes before 24 hours post-PH, near the peak of hepatocyte DNA synthesis, these findings indicate that Cx26 is enhanced in hepatocytes before the onset of S-phase. In contrast to the restricted expression of Cx43 in Glisson's capsule in adult liver, Cx43 protein and mRNA were enhanced specifically in proliferating bile duct and perisinusoidal cells post-PH. PH performed during continuous administration of 2-acetylaminofluorene (AAF) prevented changes in Cx32 and Cx26 staining observed in the absence of AAF. Proliferating oval cells were found to express diffuse Cx43 immunoreactivity. On day 11 post-PH and AAF, basophilic hepatocytes displayed both punctate Cx32 and Cx26 staining, whereas bile ducts and perisinusoidal cells expressed Cx43. These findings indicate that alterations in Cx32 and Cx26 expression occur rapidly in hepatocytes stimulated to proliferate and that several nonparenchymal liver cell types upregulate Cx43 expression when induced to proliferate. Differentiation of oval cells into basophilic hepatocytes resulted in their expression of Cx32 and Cx26.
Asunto(s)
Uniones Comunicantes/fisiología , Expresión Génica , Hígado/citología , Hígado/fisiología , Animales , Northern Blotting , División Celular , Conexinas/genética , Conexinas/metabolismo , Femenino , Uniones Comunicantes/genética , Hepatectomía/métodos , Inmunohistoquímica , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
A method was developed for the solubilization of approximately 50% of proteins in synaptic membranes that have ligand-binding characteristics of N-methyl-D-aspartate (NMDA) receptors. Affinity chromatographic separation of the solubilized proteins through L-glutamate-derivatized matrices and subsequent elution by NMDA-containing buffers led to the purification of four predominant proteins with estimated sizes of 67-70, 53-62, 41-43, and 28-36 kDa. The co-purification of NMDA-sensitive L-glutamate binding, dizocilpine-sensitive thienylcyclohexyl piperidine (TCP)-binding, and strychnine-insensitive glycine-binding proteins was achieved by this affinity chromatographic procedure. Glutamate, glycine, and the polyamine spermidine increased both the "on" rate and the equilibrium level of [3H]TCP binding to the isolated proteins. The group of proteins eluted by NMDA from the glutamate-derivatized matrices could be further purified through size exclusion chromatography of the NMDA-sensitive glutamate-binding from the dizocilpine-sensitive TCP-binding proteins. Polyclonal and monoclonal antibodies to the cloned NMDA receptor protein NMDAR1 did not react with any proteins in the solubilized membrane proteins or the purified fractions. However, immunoreaction of antibodies raised against a glutamate-binding protein and a phosphonoaminocarboxylic acid-binding protein indicated that these are two of the major proteins in the purified fractions. These studies indicate that these two proteins might be components of a complex that has some of the characteristics of NMDA receptors and that neuronal membranes may contain varieties of NMDA-like receptors composed of protein subunits that differ from the NMDAR1 and NMDAR2 receptor proteins.
Asunto(s)
Receptores de N-Metil-D-Aspartato/química , Membranas Sinápticas/química , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Maleato de Dizocilpina/metabolismo , Glutamatos/metabolismo , Ligandos , Datos de Secuencia Molecular , Peso Molecular , Ratas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/aislamiento & purificaciónRESUMEN
Direct intercellular signal transduction is achieved by the passage of small molecules through gap junctions (GJ). Previous studies in our laboratory showed that the liver tumor promoter phenobarbital (PB) reversibly decreases the abundance of the GJ protein connexin32 (Cx32) in both preneoplastic-altered hepatic foci and centrolobular hepatocytes (M. J. Neveu et al., Cancer Commun., 2: 21-31, 1990). Because the inhibitory effects of PB on GJ intercellular communication are prevented by the nonspecific cytochrome P-450 inhibitor SKF-525A (J. E. Klauning, et al., Toxicol. Appl. Pharmacol., 102: 533-563, 1990), we investigated whether alterations in Cx32 are coincident with changes in the major PB-inducible cytochrome P-450, termed b/e or IIB1/2. Immunostaining of liver cryosections from rats fed dietary PB demonstrated that centrolobular hepatocytes that exhibit reduced Cx32 express enhanced cytochrome P450IIB1/2 protein. In contrast, no change in the periportal distribution of connexin26 immunoreactivity was found in PB-treated rats. In addition, rats were treated with the structurally related barbiturates pentobarbital, amobarbital, barbital, and barbituric acid. We found that the extent of the hepatic lobule occupied by coincident centrolobular alterations in Cx32 and P-450 staining correlates with the ability of the compounds to promote liver oncogenesis. To determine the molecular mechanisms responsible for the modification in Cx32 staining, we examined the mRNA and protein levels of Cx32 and P450IIB1/2 in total-tissue homogenates from PB-treated rats. Northern blotting demonstrated thatdietary PB dramatically induced P-450IIB1 mRNA, but the same RNA samples failed to show alterations in Cx32 steady-state transcripts. Consistent with these findings, the level of Cx32 protein in total liver homogenates did not change in rats chronically fed PB. Examination of Cx32 solubility in 20 mM NaOH demonstrated that PB treatment results in the generation of a NaOH-soluble form of Cx32 (i.e., 47 kDa). In addition, trypsinized paraffin-embedded liver sections from PB-treated rats exhibited diffuse cytoplasmic Cx32 staining that was restricted to centrolobular cells. Our results show that PB and related barbiturate tumor promoters reversibly down-regulate punctate Cx32 staining in centrolobular hepatocytes posttranslationally, possibly through modification(s) in the transport, assembly, and/or turnover of GJs.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Carcinógenos/farmacología , Conexinas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Fenobarbital/farmacología , Esteroide Hidroxilasas/metabolismo , Animales , Barbitúricos/farmacología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Conexinas/fisiología , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/fisiología , Inducción Enzimática , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/fisiología , Immunoblotting , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/enzimología , Neoplasias Hepáticas Experimentales/metabolismo , Masculino , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Esteroide Hidroxilasas/biosíntesis , Esteroide Hidroxilasas/fisiología , Acetato de Tetradecanoilforbol/farmacología , Proteína beta1 de Unión ComunicanteRESUMEN
Although several abnormalities in gap junction (GJ) structure and/or function have been described in neoplasms, the molecular mechanisms responsible for many of the alterations remain unknown. The identification of a family of GJ proteins, termed connexins, prompted this study of connexin32 (Cx32), connexin26 (Cx26) and connexin43 (Cx43) expression during rat hepatocarcinogenesis. Using antibody, cDNA and cRNA probes, we investigated connexin mRNA and protein expression in preneoplastic and neoplastic rat livers. In normal liver, Cx32 is expressed in hepatocytes throughout the hepatic acinus, Cx26 is restricted to periportal hepatocytes, and Cx43 is expressed by mesothelial cells forming Glisson's capsule. Most preneoplastic altered hepatic foci generated by diethylnitrosamine (DEN) initiation and either phenobarbital (PB) or 2,3,7,8-dichlorodibenzo-p-dioxin (TCDD) promotion exhibited decreased Cx32 or increased Cx26 staining. Foci from either protocol failed to display Cx43 immunoreactivity. In the majority of PB-promoted foci, Cx32 immunoreactivity decreased independently of changes in mRNA abundance. Continuous thymidine labeling, following cessation of PB promotion, showed that downregulation of Cx32 staining is reversible in foci that are promoter-dependent for growth, but irreversible in lesions that are promoter-independent for growth. Hepatic neoplasms from rats initiated with DEN and promoted with PB or TCDD also displayed modified connexin expression. While all 24 neoplasms studied were deficient in normal punctate Cx32 and Cx26 staining, altered cellular localization of these proteins was apparent in some tumors. Immunoblotting of crude tissue extracts revealed that neoplasms with disordered Cx32 staining showed immunoreactive bands with altered electrophoretic mobility. These observations show that hepatomas may downregulate Cx32 expression through changes in the primary structure of Cx32 or by post-translational modifications. Northern blotting of total tumor mRNAs failed to demonstrate consistent changes in the abundance of Cx32, Cx26 or Cx43 transcripts. Some tumors expressed steady-state transcripts without observable immunoreactivity, indicating that some hepatomas downregulate connexin immunoreactivity independently of mRNA abundance. Increased levels of Cx43 mRNA and protein were found in several neoplasms, but immunostaining was always localized to nonparenchymal cells. Areas of bile duct proliferation and cholangiomas displayed Cx43 staining, whereas, cholangiocarcinomas were deficient in immunoreactivity. These findings show that alterations in the expression of connexins, by either downregulation or differential induction, represent common modifications during hepatocarcinogenesis. Although our results imply that connexins represent useful markers for the boundary between tumor promotion and progression, preneoplastic and neoplastic rat hepatocytes fail to use a common mechanism to modify connexin expression.
Asunto(s)
Transformación Celular Neoplásica , Conexinas/biosíntesis , Expresión Génica , Neoplasias Hepáticas Experimentales/metabolismo , Hígado/metabolismo , Lesiones Precancerosas/metabolismo , Animales , Northern Blotting , Conexina 26 , Conexina 43/biosíntesis , Dietilnitrosamina/toxicidad , Hibridación in Situ , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/patología , Fenobarbital/toxicidad , Dibenzodioxinas Policloradas/toxicidad , Lesiones Precancerosas/patología , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Transcripción Genética , Proteína beta1 de Unión ComunicanteAsunto(s)
Alcoholismo/fisiopatología , Encéfalo/fisiología , Canales Iónicos/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Membranas Sinápticas/fisiología , Alcoholismo/genética , Animales , Encéfalo/fisiopatología , Clonación Molecular , Canales Iónicos/genética , Ratas , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/aislamiento & purificaciónRESUMEN
The water soluble benzodiazepine derivative, midazolam, is used almost exclusively at our institution to produce sedation for numerous surgical procedures. Mild arterial oxygen desaturation has been reported in patients who have received as little as .04 mg/kg. A time series design study was undertaken to determine if there was any correlation between the decline in arterial oxygen percent saturation (SaO2) and the time at which sedation occurred and to establish the presence of any statistical significance in this decline. Thirty-one ASA I and II patients consisting of 8 females and 23 males requiring various minor orthopedic and general surgical procedures were studied. The total mean age of the population was 32.29 +/- 12.43 years (mean +/- SD). Fourteen patients had a smoking history, while 15 patients did not (2 patients were eliminated from the study for failure to demonstrate sedation, as characterized by either Verrill's sign or thickened speech following intravenous administration of midazolam). All patients arrived in the operating room unpremedicated and were administered .04 mg/kg midazolam intravenously. Arterial oxygen saturation was measured over a 10-minute period using pulse oximetry. Results were analyzed using regression analysis, a t-test for independent groups, and a one-way analysis of variance. There was no statistically significant difference in the decline in SaO2 between smokers and nonsmokers. Our study has shown that the mean onset of sedation using a dose of .04 mg/kg occurred between 3 and 4 minutes, with the peak fall in SaO2 occurring at the 3-minute interval irrespective of smoking history. The greatest mean drop in SaO2 was 95.84%. Midazolam, like its parent drug, diazepam, alters ventilatory mechanics.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Midazolam/farmacología , Oximetría , Respiración/efectos de los fármacos , Adulto , Femenino , Humanos , Inyecciones Intravenosas , Masculino , Midazolam/administración & dosificación , Midazolam/farmacocinéticaRESUMEN
Rat serine dehydratase cDNA clones were isolated from a lambda gt11 cDNA library on the basis of their reactivity with monospecific immunoglobulin to the purified enzyme. Using the cDNA insert from a clone that encoded the serine dehydratase subunit as a probe, additional clones were isolated from the same library by plaque hybridization. Nucleotide sequence analysis of the largest clone obtained showed that it has 1444 base pairs with an open reading frame consisting of 1089 base pairs. The deduced amino acid sequence contained sequences of several portions of the serine dehydratase protein, as determined by Edman degradation. Rat liver serine dehydratase mRNA virtually disappeared from livers of rats fed a protein-free diet for 5 days. Several genomic clones were isolated from two libraries. Determinations of the transcription start site and the structure of the 3' flanking region of the gene indicated that the coded mRNA is 1504 nucleotides long. The 5' promoter region contained a variety of sequences similar to several consensus sequences believed to be important for the regulation of specific gene expression.
Asunto(s)
ADN/análisis , L-Serina Deshidratasa/genética , ARN Mensajero/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/aislamiento & purificación , Dieta , Glucocorticoides/farmacología , Hígado/enzimología , Datos de Secuencia Molecular , RatasRESUMEN
The CSHP Saskatchewan Branch created the position of Small Hospital Representative on their Executive Committee. This appointed person's major function was to improve representation on the Executive Committee for hospitals outside of Saskatoon and Regina. The representative sent a survey to these hospitals to establish the status of pharmacy practice in these hospitals and how to best serve them. There were 90 of 128 (70.3%) surveys completed. Survey results were separated into hospitals which employed pharmacists and those which did not. Elements of practice reported by hospitals with a pharmacist, which showed some degree of deficiency, included drug distribution systems, medication profiles and clinical pharmacy programs. Hospitals which did not employ a pharmacist obtained their pharmacy services from either a larger hospital or a local retail pharmacist. There were 11 hospitals which had no access to a pharmacist, depending on the Director of Nursing for medication-related activities. The Small Hospital Representative of CSHP Saskatchewan Branch, in co-operation with the professional associations used this study as a starting point in attempts to improve pharmacy practice in Saskatchewan in hospitals outside of Saskatoon and Regina.
Asunto(s)
Sistemas de Medicación en Hospital/normas , Servicio de Farmacia en Hospital/normas , Recolección de Datos , Hospitales con menos de 100 Camas , Farmacéuticos , SaskatchewanRESUMEN
Approval for capital equipment funding and appropriate staffing was granted in August, 1982 for conversion to a unit dose drug distribution system at Melfort Union Hospital. All necessary packaging equipment, kardexes, forms and supplies were ordered in September and had arrived by November 1982, the same time a second pharmacist was hired. Prepackaging began in December for the proposed unit dose starting date of January 6, 1983. Inservices were performed to nursing three times prior to the implementation of unit dose on our 30 bed medical floor on January 6, 1983. During implementation one pharmacist spent a great deal of time with the nurses to ensure proper use of the system. Numerous meetings were held to correct the minor flaws inherent in any new system. Normal resistance to change was experienced, taking about 3 weeks to subside. Clinical services such as patient counselling, a monthly drug information bulletin and a formulary have been developed. These changes were not difficult and have provided a superior pharmacy service at a reasonable cost.