RESUMEN
Biomass was assessed as a new approach for evaluating backwashed slow sand filters (BSF). Slow sand filtration (SSF) is a simple technology for water treatment, where biological mechanisms play a key role in filtration efficiency. Backwashed slow sand filters were previously recommended for small-scale filters (~1 m² of filtration area) as an alternative to conventional filters that are usually cleaned by scraping (ScSF). Biomass was never evaluated in BSF, which is a gap in the knowledge of this technology, considering the importance of its biological mechanisms. Therefore, for the first time, two filters operating under the same conditions were used to compare the influence of backwashing on biomass; one filter was cleaned by backwashing and the other by scraping. Biomass along the filter media depth (40 cm) was assessed by different techniques and compared in terms of cellular biomass (by chloroform fumigation), volatile solids, bacterial community (by 16S rRNA gene sequencing), and observations by scanning electron and fluorescence microscopy. Filters were also monitored and compared regarding filtered water quality and headloss; their differences were related to the different cleaning processes. Overall, filtered water quality was acceptable for slow sand filter standards (turbidity < 1 NTU and total coliform removal > 1 log). However, headloss developed faster on scraped filters, and biomass was different between the two filters. Backwashing did not significantly disturb biomass while scraping changed its surface sand layers. Cell biomass was more abundant and spread across the filtration depth, related to lower headloss, turbidity, and cyanobacterial breakthrough. These results agreed with the water quality and microscopy observations. The bacterial community was also less stratified in the backwashed filter media. These results expand the knowledge of backwashing use in slow sand filters, demonstrating that this process preserves more biomass than scraping. In addition, biomass preservation can lead to bacterial selectivity and faster filter ripening. Considering the importance of biomass preservation on slow sand filtration and its biological filtration mechanisms, the results presented in this paper are promising. The novel insight that BSF can preserve biomass after backwashing may contribute to increasing its application in small communities.
Asunto(s)
Arena , Purificación del Agua , Biomasa , Filtración , ARN Ribosómico 16S , Dióxido de SilicioRESUMEN
Chickens, especially if free-range, are frequently exposed to Toxoplasma gondii, and may represent an important reservoir for T. gondii. Poultry products may pose a risk to humans, when consumed undercooked. In addition, chickens are regarded as sensitive indicators for environmental contamination with T. gondii oocysts and have been used as sentinels. The aim of the present study was to determine the suitability of commonly used antibody detection methods, i.e. the modified agglutination test (MAT), IFAT and ELISA to detect T. gondii-infected chickens. Samples of experimentally and naturally infected chickens were used. The infection state of all chickens was determined by Magnetic-Capture (MC-) real-time PCR (RT PCR). Naturally exposed chickens were additionally examined by mouse bioassay and conventional RT PCR on acidic pepsin digests (PD-RT PCR). Blood serum and meat juice of various sources were tested for antibodies to T. gondii. In naturally infected chickens, there was substantial agreement between the mouse bioassay and MC-RT PCR or the mouse bioassay and conventional PD-RT PCR. PD-RT PCR was slightly more sensitive than MC-RT PCR, as all (26/26) bioassay-positive chickens also tested positive in at least one of the tissues tested (heart, drumstick). By MC-RT PCR, 92.3% (24/26) of the naturally infected bioassay-positive chickens were positive. The diagnostic sensitivity of MC-RT PCR was clearly related to the organ examined. Based on a quantitative assessment of the MC-RT PCR results in experimentally infected chickens, brain and heart tissues harbored an at least 100â¯times higher parasite concentration than breast, thigh or drumstick musculature. In naturally infected chickens, only three out of 24 birds, which were MC-RT PCR-positive in heart samples, also tested positive in drumstick musculature. Under experimental conditions, the agreement between MC-RT PCR and the serological techniques revealed 100% diagnostic sensitivity and specificity. Under field conditions, examinations of sera by ELISA, IFAT and MAT showed good performance in identifying chickens that were positive in either a mouse bioassay, MC-RT PCR, or PD-RT PCR as illustrated by diagnostic sensitivities of 87.5%, 87.5% and 65.2%, respectively, and diagnostic specificities of 86.2%, 82.8% and 100%, respectively. The examination of meat juice samples from breast, drumstick or heart musculature revealed similar or even better results in the ELISA. The results in the MAT with meat juice from breast musculature were less consistent than those of ELISA and IFAT because a number of negative chickens tested false-positive in the MAT. The MAT performed similar to ELISA and IFAT when applied to test meat juice samples collected from heart, thigh or drumstick musculature.
Asunto(s)
Bioensayo/métodos , ADN Protozoario/aislamiento & purificación , Carne/parasitología , Enfermedades de las Aves de Corral/parasitología , Toxoplasma , Toxoplasmosis Animal/sangre , Animales , Pollos , ADN Protozoario/genética , Parasitología de Alimentos , Ratones , Enfermedades de las Aves de Corral/sangre , Enfermedades de las Aves de Corral/diagnósticoRESUMEN
Serological assays are commonly used to determine the prevalence of Toxoplasma gondii infection in livestock, but the predictive value of seropositivity with respect to the presence of infective tissue cysts is less clear. The present study aimed at the identification of seropositive and seronegative free-range laying hens from organic and backyard farms, and the relationship with the presence of viable tissue cysts. In addition, potential risk and protective factors on the selected farms were investigated. An in-house T. gondii surface antigen (TgSAG1, p30, SRS29B) ELISA was validated with sera from experimentally infected chickens and used to examine 470 serum samples collected from laying hens from large organic and small backyard farms at the end of their laying period. A total of 11.7% (55/470) of all chickens tested positive, and another 18.9% (89/470) of test results were inconclusive. The highest seroprevalences were observed on small backyard farms with 47.7% (41/86) of chickens being seropositive while another 20.9% (18/86) of test results were inconclusive. Twenty-nine seropositive, 20 seronegative and 12 laying hens which yielded inconclusive ELISA results, were selected for further examination. Hearts and limb muscles of these hens were examined for T. gondii tissue cysts in a bioassay with IFNÉ£-knockout or IFNÉ£-receptor-knockout mice. Viable T. gondii was isolated from 75.9% (22/29) of the seropositive, 25.0% (3/12) of the inconclusive, and 5.0% (1/20) of the seronegative chickens. All 26 chickens tested positive in heart samples, while drumstick muscles (i.e. limb muscles) tested positive only in three. Data on putative risk and protective factors were collected on the farms using a standard questionnaire. Generalised multilevel modelling revealed farm size, cat related factors ('cats on the premise', 'cats used for rodent control'), hen house/hall related factors ('size category of hen house/hall', 'frequency category of cleaning hen house/hall', 'service period') as significantly associated with seropositivity to T. gondii in hens. The final model, which included the age of the birds as an effect modifier and farm as a random effect variable, revealed that the use of cats for rodent control and an area available per hen in the chicken run of ≥10sqm were statistically significant risk factors for T. gondii seropositivity. Overall this study showed that exposure to T. gondii is common in small backyard farms but is rare on large organic farms with a high density of free-range hens, even when cats were present on the premises.
Asunto(s)
Pollos/parasitología , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de las Aves de Corral/parasitología , Toxoplasma/inmunología , Toxoplasmosis Animal/epidemiología , Crianza de Animales Domésticos/métodos , Animales , Anticuerpos Antiprotozoarios/sangre , Bioensayo/veterinaria , Gatos , Línea Celular , Chlorocebus aethiops , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Genotipo , Alemania/epidemiología , Interferón gamma/genética , Ratones , Ratones Noqueados , Agricultura Orgánica/métodos , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Factores de Riesgo , Estudios Seroepidemiológicos , Encuestas y Cuestionarios , Toxoplasma/clasificación , Toxoplasma/genética , Toxoplasmosis Animal/parasitología , Células VeroRESUMEN
The expression of metallothionein (MT) mRNA and protein was determined in human proximal tubule cells (HPT) following acute exposure to the classic stimulators of the stress response, heat and sodium arsenite (As3+). Treatment of the cells with 100 microM As3+ for 4 h resulted in a significant increase in the MT-1 and MT-2 proteins immediately preceding and following removal of the stress. The level of the MT-3 isoform protein was unchanged as a result of As3+ treatment. An analysis of the MT isoform-specific mRNA demonstrated that control cells express the MT-1E, MT-1F, MT-1X, MT-2A, and MT-3 genes, but not the MT-1A, MT-1B, MT-1C, MT-1H, and MT-4 genes. Treatment with As3+ resulted in a significant increase in the expression of the MT-1X gene and appearance of mRNA for the MT-1A gene. Expression of the other MT genes was unaffected by As3+ exposure, except one isolate expressed a low level of MT-1G mRNA at several time points. It is likely that the increase in MT protein seen in As3+-treated cells is due to the increased expression of the MT-1X gene because its expression is much greater than the MT-1A isoform. Treatment of the HPT cells with heat shock had no marked effect on the levels of MT protein or mRNA. This study demonstrates that acute exposure to As3+ increases the levels of MT protein and that this elevation most likely arises from increased expression of the MT-1X isoform.
Asunto(s)
Arsenitos/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Metalotioneína/biosíntesis , Teratógenos/toxicidad , Técnicas de Cultivo de Célula , Inducción Enzimática , Humanos , Isoenzimas , Túbulos Renales Proximales/citología , ARN Mensajero/biosíntesisRESUMEN
The UROtsa cell line was isolated from a primary culture of normal human urothelium through immortalization with a construct containing the SV40 large T antigen. It proliferates in serum-containing growth medium as a cell monolayer with little evidence of uroepithelial differentiation. The working hypothesis in the present study was that this cell line could be induced to differentiate and express known features of in situ urothelium if the original serum-containing growth medium was changed to a serum-free formulation. We demonstrated that the UROtsa cells could be successfully placed into a serum-free growth medium consisting of a 1:1 mixture of Dulbeco's modified Eagle's medium and Ham's F-12 supplemented with selenium (5 ng/mL), insulin (5 microg/mL), transferrin (5 microg/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL). Under serum-free growth conditions, confluent UROtsa cells were shown by light microscopy to produce raised, three-dimensional structures. Routine ultrastructural examination disclosed these three-dimensional areas to consist of a stratified layer of cells that strongly resembled in situ urothelium. The cells displayed numerous desmosomal connections, complex interactions of the lateral membranes, and abundant intermediate filaments within the cytoplasm. Freeze fracture analysis demonstrated that the cells possessed tight-junction sealing strands and gap junctions. The overall morphology was most consistent with that found in the intermediate layers of in situ urothelium. The basal expression patterns of the metallothionein (MT) and heat shock proteins 27, 60, and 70 were determined in these cells, and expression was in agreement with that known to occur for in situ urothelium. The cells were also successfully tested for their ability to be stably transfected using expression vectors containing the MT-3 or MT-2A genes. The findings suggest that the UROtsa cells grown with a serum-free medium could be a valuable adjunct for studying environmental insult to the human urothelium in general and for the stress response in particular.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Línea Celular , Modelos Biológicos , Uréter/citología , Urotelio/citología , División Celular , Transformación Celular Viral , Medios de Cultivo , Expresión Génica , Proteínas de Choque Térmico/genética , Humanos , Túbulos Renales Proximales , Metalotioneína/biosíntesis , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , ARN sin Sentido/genética , ARN Mensajero/biosíntesis , Transfección/métodosRESUMEN
The expression of hsp 27, hsp 60, hsc 70, and hsp 70 mRNA and protein was determined in immortalized human proximal tubule cells (HK-2) exposed to heat shock, sodium arsenite, or cadmium chloride (CdCl2) under both acute and extended conditions of exposure. It was demonstrated that the HK-2 cells did not exhibit the classic heat-shock response when subjected to an acute physical (heat) or chemical stress (sodium arsenite or CdCl2). Heat stress, elevated temperature at 42.5 degrees C for 1 h, caused a marked increase only in hsp 70 mRNA and protein, but not hsp 27 or hsp 60 mRNA and protein. Similar results were obtained when the cells were subjected to a classic chemical stress of exposure to 100 microM sodium arsenite for 4 h or CdCl2 for 4 h. These findings were in contrast to those found previously with mortal human proximal tubule (HPT) cells, where acute stress by all three stimuli elicited marked increases in hsp 27, hsp 60, and hsp 70 mRNA and protein. It was shown that the basal levels of expression of hsp 27 and hsp 60 in the HK-2 cells were elevated when compared to those found in unstressed HPT cells and that the basal levels were similar to those found in HPT cells under stress conditions. These results suggest that the failure of the HK-2 cells to increase hsp 27 and hsp 60 levels in response to physical and chemical stress is because they already possess elevated basal levels of these proteins. This would indicate that one or more of the genetic events that resulted in the immortalization of the HK-2 cells also elicited a stress response for hsp 27 and hsp 60, but not for hsp 70, stress response family members. Overall, the results suggest that although there are differences in the regulation of the stress response between the immortal HK-2 and mortal HPT cell lines, as long as these differences are recognized, the HK-2 cell line should be a valuable adjunct to study the stress response of the proximal tubule in general and when exposed to environmental pollutants such as cadmium.
Asunto(s)
Arsenitos/farmacología , Cloruro de Cadmio/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas de Choque Térmico/efectos de los fármacos , Proteínas de Choque Térmico/genética , Túbulos Renales Proximales/efectos de los fármacos , ARN/aislamiento & purificación , Compuestos de Sodio/farmacología , Análisis de Varianza , Western Blotting , Células Cultivadas , Chaperonina 60/efectos de los fármacos , Chaperonina 60/genética , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Calor , Humanos , Túbulos Renales Proximales/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The third isoform (MT-3) of the metallothionein gene family is unique in that it has a limited tissue distribution, is not induced by metals, has a neuronal growth inhibitory activity, and sequesters zinc more effectively under zinc-depleted conditions. The goal of the present study was to determine whether MT-3 was absent in normal breast tissue, was overexpressed in breast cancers, and if MT-3 overexpression would be associated with disease outcome. A combination of immunohistochemistry and reverse-transcription polymerase chain reaction was used to demonstrate that the normal breast had no detectable expression of MT-3 mRNA or protein. Using immunohistochemistry, it was shown that MT-3 was overexpressed in 25 of 34 cases of breast cancer. In all cases of positive staining, MT-3 was diffusely localized to the cytoplasm. The tumors from these 34 cases were divided as to outcome based on known 5-year survival, with 20 patients being disease free at 5 years (good outcome) and the other 14 having recurring disease within 5 years (bad outcome). When analyzed for MT-3 staining, it was shown that there was a trend for increased MT-3 immunoreactivity in the group having bad outcomes. However, when the tumor subgrouping was further defined on the basis of carcinoma in situ (CIS), there was a marked significant difference in MT-3 staining between patients with good and bad outcomes. Limited to DCIS, MT-3 staining was significantly increased in patients with bad outcomes compared to those with good outcomes. Thus, these studies demonstrate that MT-3 is overexpressed in selected breast cancers and that overexpression is associated with tumors having a poor prognosis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama Masculina/metabolismo , Carcinoma in Situ/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Metalotioneína 3 , Persona de Mediana Edad , Invasividad Neoplásica , Recurrencia Local de Neoplasia , Proteínas del Tejido Nervioso/genética , Pronóstico , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Supervivencia , Distribución TisularRESUMEN
The goals of this study were to determine the expression of metallothionein isoform 1 and 2 proteins (MT-1 and MT-2) in bladder cancer and then to determine which MT isoform-specific genes promoted the expression of these proteins. Immunohistochemical analysis disclosed no immunoreactivity for MT-1 and MT-2 (designated as MT-1/2 to reflect the nonspecificity of the antibody for the two isoforms) in cells comprising the normal bladder or in nonmalignant bladder disorders, such as cystitis and interstitial cystitis. Immunohistochemical analysis demonstrated that MT-1/2 was overexpressed in all samples of carcinoma in situ and in high-grade bladder cancer, with variable overexpression in low-grade bladder cancer and dysplastic lesions. The intensity and frequency of MT-1/2 staining correlated with the grade of the tumor. The MT-1 and MT-2 proteins are encoded by a family of eight genes (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-IH, MT-1X, and MT-2A), and reverse transcriptase-polymerase chain reaction was used to determine which genes were expressed in the normal bladder and in bladder cancer. This analysis demonstrated that both normal and cancerous bladder tissue expressed mRNA for the MT-2A and MT-1X genes. The expression of MT-1E mRNA was variable in both normal bladder and bladder cancer specimens. Comparison of expression relative to that of beta-actin demonstrated that the level of MT-1X mRNA was overexpressed greatly in bladder cancer as compared to the level in normal bladder tissue. In contrast, the level of MT-2A mRNA was similar in both the normal and the bladder cancer specimens. The level of MT-1X expression did not vary with tumor grade. These studies suggest that the overexpression of MT-1/2 protein in bladder cancer is a result of the overexpression of the MT-1X gene.
Asunto(s)
Carcinoma de Células Transicionales/genética , Metalotioneína/genética , ARN Mensajero/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Adulto , Expresión Génica , Humanos , Inmunohistoquímica , Metalotioneína/metabolismo , Isoformas de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
The expression of hsp 60 mRNA and protein were determined in human proximal tubule cells (HPT) exposed to lethal and sub-lethal concentrations of Cd(2+) under both acute and extended conditions of exposure. It was demonstrated that HPT cells exhibited the classic heat shock response when subjected to a physical (heat) or chemical stress (sodium arsenite). Heat stress, elevated temperature at 42.5 degrees C for 1 h, caused an increase in both hsp 60 mRNA and protein following removal of the stress. Similar results were obtained when the cells were subjected to a classic chemical stress of exposure to 100 microM sodium arsenite for 4 h. Acute exposure of HPT cells to 53.4 microM CdCl(2) for 4 h also resulted in an increase in hsp 60 mRNA and protein following removal of the metal. An extended exposure to Cd(2+) was modeled by treating the cells continuously with Cd(2+) at both lethal and sub-lethal levels over a 16-day time course. It was demonstrated that chronic exposure to Cd(2+) failed to increase either hsp 60 mRNA or protein expression in HPT cells, even at concentrations of Cd(2+) that were lethal to the cells during the time course. In fact, hsp 60 protein levels were decreased compared to controls at lethal levels of Cd(2+) exposure. These findings suggest that hsp 60 expression may have two distinct roles when the human proximal tubule cell is exposed to Cd(2+). A protective role through hsp 60 induction when the proximal tubule cell is acutely exposed to Cd(2+) and a deleterious role when hsp 60 protein is down-regulated during extended exposure to Cd(2+).
Asunto(s)
Cloruro de Cadmio/toxicidad , Chaperonina 60/biosíntesis , Túbulos Renales Proximales/efectos de los fármacos , Arsenitos/farmacología , Cloruro de Cadmio/farmacología , Línea Celular , Chaperonina 60/genética , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Calor , Humanos , Túbulos Renales Proximales/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Compuestos de Sodio/farmacologíaRESUMEN
The goal of the present study was to determine if the expression of metallothionein isoform 3 (MT-3) might serve as a biomarker for human bladder cancer. To accomplish this goal, we defined the localization and expression of MT-3 protein and mRNA using fresh and archival biopsy specimens obtained from patients undergoing differential diagnosis for a variety of bladder disorders. We used immunohistochemistry, immunoblot, and RT-PCR analysis to define the localization and expression of MT-3 protein and mRNA. Immunohistochemical analysis disclosed no immunoreactivity for MT-3 in normal bladder cells. The absence of MT-3 expression in the normal bladder was further confirmed by demonstrating that MT-3 mRNA could not be detected using reverse transcriptase-polymerase chain reaction (RT-PCR) or MT-3 protein using immunoblot. Immunohistochemistry also disclosed no immunoreactivity for MT-3 in archival biopsy specimens from patients with interstitial cystitis and related disorders. Immunohistochemical analysis demonstrated that MT-3 was expressed in carcinoma in situ (CIS), high-grade bladder cancer, low-grade bladder cancer, and dysplastic lesions. MT-3 immunostaining was intense in both CIS and high-grade bladder cancer, and low to moderate in low-grade bladder cancer and dysplastic lesions. We determined MT-3 mRNA expression in a subset of these bladder cancer specimens; expression was elevated as compared to that of the housekeeping gene, ss-actin. The cDNA from the RT-PCR reaction primed for MT-3 contained a FokI restriction site, a site unique for MT-3 as compared to other MT family members. In conclusion, this study demonstrates that MT-3 is up-regulated in human bladder cancer and that this up-regulation increases with increasing tumor grade. The finding that MT-3 expression is minimal in normal bladder suggests that MT-3 might be developed into an effective biomarker for bladder cancer.
Asunto(s)
Biomarcadores de Tumor/genética , Metalotioneína/genética , Neoplasias de la Vejiga Urinaria/genética , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/metabolismo , Carcinoma de Células Transicionales/patología , Cartilla de ADN/genética , Expresión Génica , Humanos , Inmunohistoquímica , Metalotioneína/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
BACKGROUND: Studies have shown an association of metallothionein (MT) overexpression with tumor type and grade. However, a family of genes underlies the expression of these proteins. The goals of this study were to define the expression of MT genes and protein in normal human prostate and to provide evidence that the expression of the MT isoforms is altered in prostate cancer. METHODS: Immunohistochemistry was used to localize MT protein, reverse transcription-polymerase chain reaction (RT-PCR) to determine the MT isoform-specific mRNAs, and immunoblot analysis to determine MT protein levels. RESULTS: The localization of MT in the prostate was further defined using the E9 antibody. Using normal prostate tissue dissected from glands removed for prostate cancer, it was demonstrated that MT protein expression in the normal prostate is supported by mRNA from the MT-1A, MT-1E, MT-1X, and MT-2A genes. No expression of the MT-1X gene was demonstrated in cases of advanced prostate cancer. The expression of MT-1 and MT-2 isoform-specific mRNA varied among three commonly utilized prostate cancer cell lines. CONCLUSIONS: MT protein in the normal human prostate is supported by transcription of mRNA from the MT-1A, MT-1E, MT-1X, and MT-2A genes. Expression of MT-1X mRNA is downregulated in advanced prostate cancer. Variable expression of MT mRNA in prostate cell lines provides evidence that MT gene expression may be altered among individual prostate cancers.
Asunto(s)
Expresión Génica , Metalotioneína/genética , Próstata/fisiología , Línea Celular , Regulación hacia Abajo , Humanos , Inmunohistoquímica , Masculino , Metalotioneína/metabolismo , Próstata/metabolismo , Neoplasias de la Próstata/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We determined the expression of the constitutive (hsc 70) and inducible (hsp 70) forms of heat shock protein 70 mRNA and protein in human proximal tubule (HPT) cells exposed to lethal and sublethal concentrations of Cd(+2) under both acute and extended conditions of exposure. The HPT cells exhibited the classic heat shock response when subjected to a physical (heat) or chemical stress (sodium arsenite); hsc 70 mRNA and protein levels were constant or slightly increased, whereas hsp 70 mRNA and protein were greatly elevated. Acute exposure to 53.4 microM CdCl(2) for 4 hr failed to increase either hsc 70 mRNA or protein, a finding similar to that observed under classic conditions of stress. However, under identical conditions of acute exposure to Cd(2+), the expected increase in hsp 70 protein level was suppressed as compared to that found under classic conditions of physical or chemical stress. The decrease in hsp 70 protein level correlated to the reduced expression of mRNA from the hsp 70B gene. The expression of mRNA from the hsp 70A and hsp 70C genes was similar to that found when the cells were treated with heat shock or sodium arsenite. We modeled an extended exposure to Cd(2+) by treating the cells continuously with Cd(2+) at both lethal and sublethal levels over a 16-day time course. Chronic exposure to Cd(2+) failed to increase either hsc 70 mRNA or protein levels in the HPT cells at a nonlethal dosage level and decreased hsc 70 mRNA and protein levels late in the time course of lethal exposure. Under identical conditions, the expression of hsp 70 protein remained at basal levels that were only marginally detectable throughout the time course. Hsp 70A and hsp 70C mRNA levels were unaltered by extended exposure to Cd(2+), and hsp 70B mRNA was not detected during the 16-day time course. Cd(2+) is a poor inducer of hsc 70 and hsp 70 in the proximal tubule under both acute and long-term exposure. These results reinforce the fact that the expression of hsp 70 protein does not result from the transcription of a single gene, but is derived from what may be a complex interplay of several underlying genes.
Asunto(s)
Arsenitos/efectos adversos , Cloruro de Cadmio/efectos adversos , Inhibidores Enzimáticos/efectos adversos , Proteínas HSP70 de Choque Térmico/biosíntesis , Túbulos Renales Proximales/efectos de los fármacos , Compuestos de Sodio/efectos adversos , Arsenitos/farmacología , Cloruro de Cadmio/farmacología , Técnicas de Cultivo de Célula , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Proteínas HSP70 de Choque Térmico/genética , Calor , Humanos , Túbulos Renales Proximales/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Compuestos de Sodio/farmacología , Transcripción GenéticaRESUMEN
BACKGROUND: Development of the culture of renal epithelial cells in a serum-free growth medium was driven by the need to examine the effects of hormones and other effector molecules on differentiated cell function without interference from the complex mixture of substances in serum. The present report details this laboratory's cumulative experience in the use of a defined growth medium for the propagation of epithelial cells from adult, fetal, and malignant human renal tissue. METHODS: Routine cell culture technology was used to determine the capability of a defined growth medium to support the growth of renal epithelial cells isolated by collagenase dissociation of tissue from adult and fetal kidneys, renal cell carcinoma, and Wilms' tumors. RESULTS: The defined growth medium formulation consistently allows the isolation and growth of transporting renal epithelial cells from both normal adult and fetal kidneys. This growth medium only rarely supports the growth of epithelial cells from renal cell carcinomas and Wilms' tumors. CONCLUSIONS: The method developed for the culture of human proximal tubule cells requires minimal cell culture expertise and equipment, and results in the repeatable isolation of transporting epithelial cell cultures that retain features of differentiated proximal tubule cells.
Asunto(s)
Técnicas de Cultivo de Célula , Medio de Cultivo Libre de Suero , Riñón/citología , 1-Metil-3-Isobutilxantina/farmacología , Arginina/farmacología , Carcinoma de Células Renales/patología , Diferenciación Celular , División Celular , AMP Cíclico/metabolismo , Embrión de Mamíferos , Células Epiteliales/citología , Técnica de Fractura por Congelación , Humanos , Corteza Renal/citología , Neoplasias Renales/patología , Túbulos Renales Proximales/citología , Microscopía Electrónica , Microscopía de Contraste de Fase , Hormona Paratiroidea/farmacología , Tumor de Wilms/patologíaRESUMEN
BACKGROUND: Expression of metallothionein isoform 3 (MT-3) was initially reported to be confined to neural tissues. However, it was recently demonstrated that MT-3 is expressed in epithelial cells of the human kidney. This motivated the current examination of the expression of MT-3 in the human prostate. METHODS: Immunohistochemistry (IHC) was used to localize the expression of MT-3, RT-PCR to determine the expression of MT-3 mRNA, and Western blot analysis to determine the level of MT-3 protein. RESULTS: Selected epithelial and stromal cells of the normal human prostate were shown to have low levels of MT-3 expression. MT-3 was increased in prostatic intraepithelial neoplasia (PIN) lesions and further increased in a highly variable fashion in prostatic adenocarcinoma. In some adenocarcinomas, MT-3 expression exceeded that of nerve. Three cell culture models of prostate cancer were also shown to variably express MT-3. Restriction enzyme analysis confirmed the expression of MT-3 in the cells and tissues. CONCLUSIONS: MT-3 is expressed in the normal human prostate, and expression is enhanced and highly variable in PIN lesions and primary prostate cancer cells. The variable nature of MT-3 expression was also noted in commonly utilized prostate cancer cell lines.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Inhibidores de Crecimiento/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Neoplasias de la Próstata/genética , Secuencia de Aminoácidos , Células Epiteliales/fisiología , Inhibidores de Crecimiento/genética , Humanos , Inmunohistoquímica , Masculino , Metalotioneína 3 , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
The objective of the present study was to determine the expression of MT-3 in the human kidney. To accomplish this, an antibody was generated against a unique 8 amino acid sequence present in MT-3 that is not shared by any other MT family member. Western analysis demonstrated that the resulting antibody reacted with a protein band of approximately 6 kDa, corresponding to the known molecular weight of MT-3. Immunohistochemical staining using this antibody demonstrated reactivity with several epithelial components of the nephron. In the glomerulus, moderate intensity was demonstrated in parietal epithelial cells of Bowman's capsule and in visceral epithelial cells of the glomerular tuft. Proximal convoluted tubule cells exhibited moderate cytoplasmic MT-3 reactivity. Distal tubules showed strong cytoplasmic staining for MT-3, particularly in the medullary rays. In the medulla, MT-3 staining was the most variable, with weak to moderate staining in the medullary collecting ducts and a general absence of staining in the thin loops of Henle and in the transitional epithelium of the renal pelvis. The finding that MT-3 is constitutively expressed in several glomerular and tubular epithelial elements of the human kidney warrants consideration of an expanded role for this protein family in maintaining renal homeostasis.
Asunto(s)
Riñón/metabolismo , Metalotioneína/análisis , Western Blotting , Encéfalo/metabolismo , Humanos , Inmunohistoquímica , Metalotioneína/biosíntesisRESUMEN
The expression of hsp 27 mRNA and protein was determined in cultured human proximal tubule (HPT) cells exposed to lethal and sublethal concentrations of Cd2+ under both acute and extended conditions. Initial procedures demonstrated that HPT cells display the classic stress response following physical and chemical stress. Heat stress (42.5 degrees C for 1 hr) caused an increase in both hsp 27 mRNA and protein as well as a shift in the protein to a more phosphorylated state. Results were similar when the cells were subjected to chemical stress (exposure to 100 microM sodium arsenite for 4 hr). Acute exposure to 53 microM CdCl2 for 4 hr also resulted in an increase in hsp 27 mRNA and protein and a shift to the more phosphorylated protein isoform. Extended Cd2+ exposure involved continuous treatment with Cd2+ at both lethal and sublethal levels over a 16-day time course. The results of this treatment showed that chronic exposure to Cd2+ failed to increase either hsp 27 mRNA or protein expression in HPT cells, even at lethal Cd2+ concentrations. In fact, hsp 27 protein levels decreased as compared to controls at both lethal and sub-lethal exposure to Cd2+. These findings imply that hsp 27 expression in human proximal tubule cells may have two distinct modes depending on the nature (acute vs. chronic) of the stress.
Asunto(s)
Cloruro de Cadmio/toxicidad , Proteínas de Choque Térmico/biosíntesis , Túbulos Renales Proximales/efectos de los fármacos , Apoptosis/efectos de los fármacos , Arsenitos/toxicidad , Células Cultivadas , Humanos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , FosforilaciónRESUMEN
In contrast to the single metallothionein (MT)-1 gene of the mouse, the human MT-1 gene family is composed of seven active genes and six pseudogenes. In this study, the expression of mRNA representing the seven active human MT-1 genes was determined in cultured human proximal tubule (HPT) cells under basal conditions and after exposure to the metals Cd2+, Zn2+, Cu2+, Hg2+, Ag2+, and Pb2+. Basal expression of MT-1X and MT-1E mRNA in HPT cells was similar to expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. In contrast, mRNAs representing the basal expression of MT-1A and MT-1F were a minor transcript in HPT cells. Treatment of HPT cells with Cd2+, Zn2+, or Cu2+ increased the levels of MT-1E and MT-1A mRNA, but not the levels of MT-1X or MT-1F mRNA. The increase in MT-1E mRNA appeared to be influenced mainly by exposure to the various metals, whereas the increase in MT-1A mRNA was influenced more by exposure to a metal concentration eliciting a loss of cell viability. Treatment of HPT cells with the metals Hg2+, Ag2+, and Pb2+ was found to have no effect on the level of MT-1 mRNA at either sublethal or lethal concentrations. Using HPT cells as a model, these results suggest that new features of MT gene expression have been acquired in the human due to the duplication of the MT-1 gene.
Asunto(s)
Expresión Génica/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Metalotioneína/genética , Metales Pesados/toxicidad , Células Cultivadas , Relación Dosis-Respuesta a Droga , Duplicación de Gen , Humanos , Túbulos Renales Proximales/metabolismo , Metalotioneína/efectos de los fármacos , Metales Pesados/farmacología , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The human metallothionein 3 (MT-3) gene has recently been identified and characterized as a brain-specific MT having growth inhibitory activity for neuronal cells. One objective of the present study was to determine if MT-3 is brain-specific or also present in the renal system, a site for chronic toxicity due to heavy metal exposure. Using RT-PCR methodology, MT-3 mRNA was shown to be expressed in the human renal system at levels below mRNA for the beta-actin gene. MT-3 mRNA was shown to be expressed in all samples obtained from both the developing and adult renal systems, from 20 weeks of fetal age to 72 years. Cultures of human proximal tubule (HPT) cells were used to determine if MT-3 mRNA expression is influenced by metal exposure. Exposure of HPT cells to either Zn2+ or Cd2+ resulted in an early (within 24 h), but unsustained increase in MT-3 mRNA. The demonstration of MT-3 mRNA expression in the kidney indicates that MT-3 may play an important early role in the response of the cell to metal exposure. MT-3 mRNA expression was also examined in tissues and cells from three cases of renal cell carcinoma. MT-3 was found to be expressed in all three cases at levels similar to those found for normal kidney, providing evidence that MT-3 mRNA expression is not altered in this cancer.
Asunto(s)
Carcinoma de Células Renales/enzimología , Neoplasias Renales/enzimología , Túbulos Renales Proximales/enzimología , Metaloendopeptidasas/biosíntesis , ARN Mensajero/biosíntesis , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Secuencia de Bases , Carcinoma de Células Renales/patología , Células Cultivadas , Niño , Preescolar , Cartilla de ADN/química , Expresión Génica , Humanos , Lactante , Recién Nacido , Neoplasias Renales/patología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/embriología , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
The organization of the metallothionein (MT) gene family has been demonstrated to be much more complex in humans than in the mouse, and possibly rodents in general. For humans, the MTs are encoded by a family of genes located at 16q13 representing 10 functional and 7 non-functional MT isoforms. In the present study, the 5' and 3' untranslated region sequences of the highly conserved, functional MT genes were utilized to generate primer pairs for the analysis of isoform-specific MT mRNA using reverse transcriptase-polymerase chain reaction (RT-PCR). Human kidneys from 13 weeks gestation through adulthood were examined for the expression of MT protein and mRNA. Immunohistochemical analysis demonstrated MT immunoreactivity to be confined exclusively to the proximal tubules of the adult and developing kidney. For all MT-positive cells, MT was localized in the cytoplasm and nuclear localization was variable. There was no correlation between nuclear staining and stage of development. Of the 10 MT genes examined (MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X, MT-2A, MT-3, and MT-4), mRNAs representing the MT-1E, MT-1F, MT-1X, and MT-2A genes were consistently expressed in all samples regardless of gestational age. There was no indication of a 'fetal form' of MT analogous to that noted to occur in human liver. Messenger RNA for the MT-1A gene was detected in 2 of 6 renal samples without correlation to gestational age. In no instance was mRNA for the MT-1B, MT-1G, MT-1H, MT-3 or MT-4 genes detected. These studies detail the initial determination of MT gene expression in the human renal system and provide the PCR primers for testing and determination of MT gene expression in other organ systems.
Asunto(s)
Expresión Génica , Túbulos Renales Proximales/metabolismo , Riñón/metabolismo , Metalotioneína/genética , Adolescente , Adulto , Anciano , Secuencia de Bases , Western Blotting , Preescolar , Feto/metabolismo , Humanos , Inmunohistoquímica , Lactante , Riñón/química , Riñón/embriología , Riñón/crecimiento & desarrollo , Túbulos Renales Proximales/química , Túbulos Renales Proximales/embriología , Túbulos Renales Proximales/crecimiento & desarrollo , Metalotioneína/análisis , Metalotioneína/biosíntesis , Metalotioneína 3 , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
The expression of heat shock protein (hsp) 27 was examined in the developing and adult human kidney. Immunolocalization using a monoclonal antibody against human hsp 27 demonstrated immunoreactivity in both the developing and adult kidney. Low to moderate immunoreactivity for hsp 27 was observed in the fetal and adult proximal tubule, distal tubule, and mesangial cells of the glomeruli. Intense immunoreactivity for hsp 27 was localized to the cortical and medullary collecting ducts in both the adult and fetal kidney, with the most intense staining in the medullary regions. The loop of Henle demonstrated no immunoreactivity for hsp 27. The blastemal element of the developing kidney showed no hsp immunostaining and the ureteric bud demonstrated moderate staining. Western, northern, and reverse transcription-polymerase chain reaction (RT-PCR) analyses disclosed no significant differences in hsp 27 mRNA or protein level as a function of gestational age. An analysis of the phosphorylation state of hsp 27 showed the majority of hsp 27 to be present in the unphosphorylated isoform for both adult and fetal samples. These studies are the first to demonstrate the presence of hsp 27 in the human kidney. It is suggested that this pool of hsp 27 is constitutive as it appears in an inactivated state; localized to the cytoplasm and in an unphosphorylated state.