RESUMEN
Our previous report showed that supernatants of Lactobacillus acidophilus (LS) cultures possessed chemotactic and angiogenic properties. Specifically, LS stimulated gene expression and the secretion of tumor necrosis factor-alpha (TNF-alpha), the proliferation of immune cells in vitro, and blood vessel formation. Chemotaxis and proliferation of inflammatory cells in vivo were also stimulated by LS. In the current study, we hypothesized that LS stimulates the growth and development of other rapidly dividing cells, including embryonic cells. The stimulatory effects of LS on a neuroblastoma cell line (Neuro-2a), chicken embryos, and bovine embryos were examined. The addition of LS to Neuro-2a cultures caused a proliferation of cells in a concentration-dependent manner. Pretreatment of LS at 56 degrees C for 30 mins did not affect its stimulatory activity. The administration of LS to the chorioallantoic membrane (CAM) of chicken-embryonated eggs for 1-2 days resulted in extensive thickening of the membrane. The thickening was due to the influx and proliferation of fibroblasts and inflammatory cells, the accumulation of loose connective tissue composed primarily of mucopolysaccharides, and/or the formation of blood vessels. Stimulatory effects of LS on bovine embryos were also observed. The treatment with LS significantly promoted the development of zygotes to the four-cell stage and from the four-cell stage to blastocysts. These results have confirmed our hypothesis that LS exerts a stimulatory effect on the cells of embryonic stages including neuroblastoma cells, the CAM of chicken embryos, and bovine embryos from zygotes to blastocysts.
Asunto(s)
Inductores de la Angiogénesis/farmacología , Embrión de Mamíferos/efectos de los fármacos , Lactobacillus acidophilus/química , Probióticos/farmacología , Animales , Bioensayo , Vasos Sanguíneos/citología , Vasos Sanguíneos/efectos de los fármacos , Bovinos , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Membrana Corioalantoides/efectos de los fármacos , Embrión de Mamíferos/citología , RatonesRESUMEN
Antillatoxin is a potent ichthyotoxin and cytotoxin previously discovered from the marine cyanobacterium Lyngbya majuscula. Ensuing studies of its mechanism of action showed it to activate the mammalian voltage-gated sodium channel at a pharmacological site that is distinct from any previously described. The structure of antillatoxin, initially formulated from spectroscopic information, was subsequently corrected at one stereocenter (C-4) as a result of synthesis of four different antillatoxin stereoisomers (all possible C-4 and C-5 diastereomers). In the current study these four stereoisomers, (4R,5R)-, (4S,5R)-, (4S,5S)-, and (4R,5S)-antillatoxin, were characterized in five different biological assay systems: ichthyotoxicity to goldfish, microphysiometry using cerebellar granule cells (CGCs), lactose dehydrogenase efflux from CGCs, monitoring of intracellular Ca(2+) concentrations in CGCs, and cytotoxicity to Neuro 2a cells. Across these various biological measures there was great consistency in that the natural antillatoxin (the 4R,5R-isomer) was greater than 25-fold more potent than any of the other stereoisomers. Detailed NMR studies provided a number of torsion and distance constraints that were modeled using the MM2 force field to yield predicted solution structures of the four antillatoxin stereoisomers. The macrocycle and side chain of natural (4R,5R)-antillatoxin present an overall "L-shaped" topology with an accumulation of polar substituents on the external surface of the macrocycle and a hydrogen bond between N(H)-7' and the C(O)-1 carbonyl. The decreased potency of the three non-naturally occurring antillatoxin stereoisomers is certainly a result of their dramatically altered overall molecular topologies.
Asunto(s)
Cianobacterias/química , Toxinas de Lyngbya/farmacología , Toxinas Marinas/farmacología , Neuronas/fisiología , Péptidos Cíclicos/farmacología , Canales de Sodio/fisiología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Cerebelo/fisiología , Cristalografía por Rayos X , Carpa Dorada/metabolismo , Lipopéptidos , Ratones , Modelos Moleculares , Estructura Molecular , Neuronas/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Canales de Sodio/efectos de los fármacos , EstereoisomerismoRESUMEN
Extracts or supernatants from cultures of Lactobacilli are used for their medicinal effects, including wound healing and immune system stimulating activity. We have studied the in vivo and in vitro effects of supernatants from bacterial cultures of two strains of Lactobacillus (LS) on tissue repair and angiogenesis. Subcutaneous injection of LS into rodent ears led to proliferation of blood vessels that also exhibited strong immunostaining for Flk-1 receptor. Some inflammatory cells were scattered among the blood vessels. The continuous influx of polymorphonuclear leukocytes (PMNs) and macrophages into transcutaneous wounds in mice treated with LS resulted in prolonged inflammatory phase of wound healing and delayed wound closure, including reepithelialization. Subcutaneous injection of Matrigel impregnated with LS into the abdominal wall led to rapid and transient influx of PMNs in the vicinity of the gel. LS stimulated the proliferation of murine macrophage J774.A1 cell line and porcine lymphocytes but not that of murine fibroblast AKR-2B cells. LS also induced production of TNF-alpha by J774.A1 cells and by porcine kidney epithelial LLC-PK1 cells. LS did not appear to have an effect on collagen production. In conclusion, our study demonstrates the potential of LS to function as a stimulator of the inflammatory stage of tissue repair, TNF-alpha production, and of angiogenesis.
Asunto(s)
Lactobacillus/química , Neovascularización Fisiológica/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Animales , Secuencia de Bases , División Celular/efectos de los fármacos , Línea Celular , Quimiotaxis de Leucocito/efectos de los fármacos , Femenino , Técnicas In Vitro , Inflamación/etiología , Inflamación/patología , Células LLC-PK1 , Activación de Linfocitos/efectos de los fármacos , Ratones , Neutrófilos/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Porcinos , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Antillatoxin (ATX) is a lipopeptide derived from the pantropical marine cyanobacterium Lyngbya majuscula. ATX is neurotoxic in primary cultures of rat cerebellar granule cells, and this neuronal death is prevented by either N-methyl-d-aspartate (NMDA) receptor antagonists or tetrodotoxin. To further explore the potential interaction of ATX with voltage-gated sodium channels, we assessed the influence of tetrodotoxin on ATX-induced Ca2+ influx in cerebellar granule cells. The rapid increase in intracellular Ca2+ produced by ATX (100 nM) was antagonized in a concentration-dependent manner by tetrodotoxin. Additional, more direct, evidence for an interaction with voltage-gated sodium channels was derived from the ATX-induced allosteric enhancement of [3H]batrachotoxin binding to neurotoxin site 2 of the alpha subunit of the sodium channel. ATX, moreover, produced a strong synergistic stimulation of [3H]batrachotoxin binding in combination with brevetoxin, which is a ligand for neurotoxin site 5 on the voltage-gated sodium channel. Positive allosteric interactions were not observed between ATX and either alpha-scorpion toxin or the pyrethroid deltamethrin. That ATX interaction with voltage-gated sodium channels produces a gain of function was demonstrated by the concentration-dependent and tetrodotoxin-sensitive stimulation of 22Na+ influx in cerebellar granule cells exposed to ATX. Together these results demonstrate that the lipopeptide ATX is an activator of voltage-gated sodium channels. The neurotoxic actions of ATX therefore resemble those of brevetoxins that produce neural insult through depolarization-evoked Na+ load, glutamate release, relief of Mg2+ block of NMDA receptors, and Ca2+ influx.
Asunto(s)
Lipoproteínas/farmacología , Toxinas Marinas/farmacología , Neuronas/fisiología , Péptidos Cíclicos , Canales de Sodio/fisiología , Animales , Batracotoxinas/farmacocinética , Unión Competitiva , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Cerebelo/fisiología , Cianobacterias , Cinética , Lipopéptidos , Neuronas/citología , Neuronas/efectos de los fármacos , Nitrilos , Piretrinas/farmacología , Ratas , Ratas Sprague-Dawley , Anémonas de Mar , Canales de Sodio/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
To develop theoretical deposition models, assumptions are introduced to make the models computationally affordable. For this reason, experimental (in vivo) validation of such models is needed to give confidence to the assumptions being made. However, for an in vivo deposition experiment to be considered useful for validation of a model, a number of parameters must be measured in the experiment for input to the model. Ideally, these parameters would include time-dependent breathing flow rates during aerosol exposure, properties of the inhaled aerosol as a function of time during the breath (including particle size distribution, aerosol mass fraction, as well as hygroscopic properties, inhaled temperature and humidity if hygroscopicity is important), in addition to anatomical regional deposition data and detailed lung geometry measurements. Furthermore, because of the dependence of extrathoracic filtering on the inlet conditions at the mouth and the complexity of modeling deposition in this region, experimental data on the filtering properties of the mouth-throat are needed. Although some of the above parameters are impractical to measure with current experimental techniques, it would greatly aid the development of deposition models if as many of these parameters as possible were measured in future in vivo deposition experiments. Data exemplifying the importance of measuring the above parameters is discussed.
Asunto(s)
Aerosoles/metabolismo , Pulmón/metabolismo , Modelos Biológicos , Humanos , Pulmón/diagnóstico por imagen , Matemática , Tamaño de la Partícula , Cintigrafía , Valores de ReferenciaRESUMEN
The porcine seminal plasma protein (PSP) accounts for much more than 50% of the total proteins in seminal plasma. PSP has been previously purified and its biochemical properties characterized. However, the biological functions of PSP remain to be elucidated. We hypothesize that PSP is involved in the regulation of uterine immune activity. In the current study, effects of PSP on in vitro lymphocyte activities and the presence of PSP binding sites on lymphocytes were examined. In mitogen-induced proliferation assay, lymphocytes from peripheral blood of gilts were cultured with pokeweed mitogen (PWM), phytohemagglutinin (PHA), or concanavalin A (Con A) in the presence or absence of PSP. PSP at 50, 125, and 250 ng/well augmented PWM-induced [3H]thymidine uptake in a dose-responsive manner by 152.8 +/- 8.1%, 225.9 +/- 35.2%, and 274.8 +/- 53.6%, respectively, compared with that of control. PSP did not alter lymphocyte proliferation in the absence of PWM. Similarly, PSP had little or no effect on PHA- or Con A-induced lymphocyte proliferation. In one-way mixed lymphocyte reactions, PSP at 50, 125, and 250 ng/well enhanced [3H]thymidine uptake in a dose-responsive manner by 181.5 +/- 16.5%, 339.9 +/- 48.2%, and 600.1 +/- 84.8% of control, respectively. Using biotinylated PSP-I, PSP binding sites were localized on approximately 3-5% of the lymphocyte population. In summary, we have demonstrated that PSP itself is not a mitogen/antigen to porcine lymphocytes but that it has a stimulatory effect on lymphocyte activities initiated by PWM or surface antigens of lymphocytes. PSP may exert its functions by interacting with PSP binding sites on a subpopulation of porcine lymphocytes. The high potency of PSP on lymphocyte activities and the abundance of PSP in seminal plasma have suggested that PSP may play an important role in regulating immune responses in the porcine uterine environment.
Asunto(s)
Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Proteínas de Secreción Prostática , Proteínas/farmacología , Animales , Sitios de Unión , Biotinilación , Células Cultivadas , Concanavalina A/farmacología , ADN/biosíntesis , Femenino , Activación de Linfocitos/efectos de los fármacos , Masculino , Mitosis , Fitohemaglutininas/farmacología , Mitógenos de Phytolacca americana/farmacología , Proteínas/aislamiento & purificación , Proteínas de Plasma Seminal , PorcinosRESUMEN
Pig seminal proteins PSP-I and PSP-II are major protein components of boars' ejaculate and are present as heterodimers (PSP-dimer) in seminal plasma. These proteins were examined for their ability to modulate pig lymphocyte activity in vitro in mitogen-induced lymphocyte proliferation assays and in one-way mixed lymphocyte reactions. Pig lymphocytes were cultured with phytohaemagglutinin, concanavalin A, or pokeweed mitogen (PWM) in the presence or absence of pig seminal proteins and the amount of cellular [3H]thymidine was used as an indication of proliferation. In the absence of mitogens, none of the three pig seminal proteins affected lymphocyte proliferation suggesting that these proteins are not antigenic or mitogenic. PSP-dimer enhanced lymphocyte proliferation induced by PWM (156-227%, P < 0.05) in a concentration-dependent manner, but had no effect on phytohaemagglutinin- or concanavalin A-induced proliferation. PSP-I enhanced (127-185%, P < 0.05) phytohaemagglutinin-induced proliferation. PSP-II augmented (130-240%, P < 0.05) lymphocyte proliferation induced by concanavalin A and PWM. Lymphocytes from gilts were significantly more responsive to concanavalin A- and PWM-induced lymphocyte proliferation in the presence of PSP-I compared with boars (concanavalin A: gilts 131%, boars 91%; PWM: gilts 188%, boars 134%; P < 0.05). In the mixed lymphocyte reaction, pretreating stimulating cells with increasing concentrations of PSP-I or PSP-II elicited a 400% concentration-dependent increase (P < 0.01) in lymphocyte proliferation. The abundance of pig seminal proteins in boar seminal plasma, their ability to enhance lymphocyte proliferation, and their previously reported ability to bind to lymphocytes suggest that these proteins are immunostimulatory and supports the hypothesis that they modulate uterine immune activity to ensure reproductive success.
Asunto(s)
Glicoproteínas/farmacología , Linfocitos/efectos de los fármacos , Proteínas de Secreción de la Vesícula Seminal , Porcinos/inmunología , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Concanavalina A/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Fitohemaglutininas/farmacología , Mitógenos de Phytolacca americana/farmacologíaRESUMEN
During its asexual life cycle, the human malaria parasite Plasmodium falciparum exports numerous proteins beyond its surface to its host erythrocyte. We have studied the biosynthesis, processing and export of a 45 kDa parasite protein resident in membrane clefts in the erythrocyte cytoplasm. Our results indicate that this cleft protein is made as a single tightly membrane-bound 45 kDa polypeptide in ring- and trophozoite-infected erythrocytes (0-36 h in the life cycle). Using ring/trophozoite parasites released from erythrocytes, the 45 kDa protein is shown to be efficiently transported to the cell surface. This export is specifically blocked by the drug brefeldin A, and at 15 and 20 degrees C. These results indicate that transport blocks seen in the Golgi of mammalian cells are conserved in P. falciparum. Further, the newly synthesized 45 kDa protein passes through parasite Golgi compartments before its export to clefts in the erythrocyte. In mid-to-late-ring-infected erythrocytes, a fraction of the newly synthesized 45 kDa protein is processed to a second membrane-bound phosphorylated 47 kDa protein. The t1/2 of this processing step is about 4 h, suggesting that it occurs subsequent to protein export from the parasite. Evidence is presented that, in later trophozoite stages (24-36 h), the exported 45 and 47 kDa proteins are partially converted into soluble molecules in the intraerythrocytic space. Taken together, the results indicate that the lower eukaryote P. falciparum modulates a classical secretory pathway to support membrane export beyond its plasma membrane to clefts in the erythrocyte. Subsequent to export, phosphorylation and/or conversion into a soluble form may regulate the interactions of the 45 kDa protein with the clefts during parasite development.
Asunto(s)
Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Plasmodium falciparum/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/metabolismo , Animales , Transporte Biológico , Medios de Cultivo , Eritrocitos/parasitología , Técnica del Anticuerpo Fluorescente , Aparato de Golgi/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Peso Molecular , Fosforilación , Plasmodium falciparum/crecimiento & desarrollo , Pruebas de Precipitina , Proteínas Protozoarias/biosíntesisRESUMEN
Incubation of a rabbit endometrial cell line (HRE-H9 cells) with KCl (5-60 mM) for 30 min enhanced IR-GnRH secretion, with 30 and 60 mM producing the greatest stimulatory effect (280 +/- 19% and 298 +/- 49% of control group, respectively). By adding 30 mM KCl into HRE-H9 culture and increasing the incubation time to 90 min, there was a stepwise increase in IR-GnRH secretion. In the third experiment, treatment of HRE-H9 cells with estradiol (E2, 10(-9)-10(-8) M) for 48 h stimulated IR-GnRH secretion (215 +/- 17%, 168 +/- 19%, respectively), whereas P4 treatment did not produce any significant change. Treatment with E2 + P4 at all doses tested (10(-10)-10(-6) M) augmented the secretion of IR-GnRH (140 +/- 16%, 153 +/- 14%, 276 +/- 23%, 259 +/- 26%, 198 +/- 16%, respectively). Increased IR-GnRH secretion by E2 (10(-9) M) and E2 + P4 (10(-8)-10(-7) M) resulted in a reduction in cell content of IR-GnRH (p < 0.05). In conclusion, secretion of IR-GnRH by HRE-H9 cells can be induced by KCl depolarization. Treatment of HRE-H9 cells with E2 and E2 + P4 enhanced their secretion of IR-GnRH. Under such conditions, secreted IR-GnRH appears to be derived primarily from intracellular IR-GnRH pools.
Asunto(s)
Endometrio/efectos de los fármacos , Estradiol/farmacología , Hormona Liberadora de Gonadotropina/metabolismo , Progesterona/farmacología , Análisis de Varianza , Animales , Línea Celular , Medios de Cultivo , Endometrio/citología , Endometrio/metabolismo , Femenino , Potenciales de la Membrana/efectos de los fármacos , Cloruro de Potasio/farmacología , Conejos , RadioinmunoensayoRESUMEN
Immunoreactive beta-endorphin (ir-BEND) and GnRH (ir-GnRH) have been identified in the pig uterus. The study reported here examined (i) possible biochemical differences between ir-BEND and ir-GnRH present in pig uterine fluids and standard synthetic peptides, and (ii) the secretory profiles of uterine ir-BEND and ir-GnRH during the oestrous cycle and early pregnancy for two breeds of pig, Large White and the highly prolific Chinese Meishan. Reverse phase (RP)-HPLC analysis of concentrated uterine fluids indicated that the majority of ir-BEND eluted with a hydrophobicity similar to that of synthetic BEND 1-31 (BEND) and alpha-N-acetylated BEND 1-31 (Nac-BEND), with a ratio of BEND to Nac-BEND of approximately 1.8:1. The RP-HPLC profiles of ir-GnRH demonstrated a major peak coeluting with synthetic GnRH, along with a minor peak eluting at the void volume. Total content of ir-BEND (pg per uterus) was affected by the interaction of breed with day (P < 0.001), but was independent of reproductive status. In Large White gilts, uterine fluid content of ir-BEND was higher (P < 0.05) on days 10 and 11 than on days 8, 12 and 14; however, in Meishan gilts, ir-BEND decreased from day 8 to days 10 and 11, and remained low on days 12 and 14. Compared to Meishan gilts, Large White gilts had higher ir-BEND concentrations on day 10 (P < 0.01), day 11 (P < 0.0001), day 12 (P < 0.01) and day 14 (P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hormona Liberadora de Gonadotropina/metabolismo , Reproducción/fisiología , Porcinos/fisiología , Útero/metabolismo , betaendorfina/metabolismo , Animales , Líquidos Corporales/química , Cromatografía Líquida de Alta Presión , Estro/sangre , Femenino , Hormona Liberadora de Gonadotropina/química , Embarazo , Porcinos/genética , betaendorfina/químicaRESUMEN
Studies were aimed to identify and characterize IR-GnRH in the porcine endometrium, ovary, oviduct, and compare that with GnRH of hypothalamic and placental origin. RP-HPLC profiles revealed that all tissue extracts contained three peaks of IR-GnRH. Extraction with radioiodinated 125I-GnRH also resulted in three similar peaks, indicating these two extra peaks as an extraction artifact. Radioreceptor assay showed endometrial and ovarian ultrafiltrates displaced binding of 125I-GnRH analog to pituitary membrane. Secretion of uterine IR-GnRH was increased in ovariectomized, progesterone-treated gilts, as compared to the control. Treatment with estradiol + progesterone in ovariectomized gilts further increase uterine secretion of IR-GnRH. These results demonstrate that IR-GnRH identified in the porcine reproductive tissues possesses hydrophobicity similar to those in hypothalamus and placenta; endometrial IR-GnRH binds to pituitary GnRH receptors; and uterine secretion of IR-GnRH is modulated by ovarian steroids.
Asunto(s)
Endometrio/química , Trompas Uterinas/química , Hormona Liberadora de Gonadotropina/análisis , Ovario/química , Animales , Cromatografía Líquida de Alta Presión , Femenino , Hormonas Esteroides Gonadales/fisiología , Hormona Liberadora de Gonadotropina/metabolismo , Hipotálamo/química , Placenta/química , Embarazo , Radioinmunoensayo , Ensayo de Unión Radioligante , Receptores LHRH/metabolismo , Porcinos , Útero/metabolismoRESUMEN
The effects of porcine uterine fluid filtrate (PUF) on lymphocyte activity were examined. PUF was added to porcine lymphocyte cultures in the presence or absence of phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM). Lymphocyte response was determined by 3H-thymidine incorporation. Results indicated that PUF enhanced PHA-, Con A-, and PWM-induced lymphocyte mitogenesis by 222%, 207%, and 261%, respectively, as compared to control. The stimulatory effect of PUF was unlikely to be due to the presence of interleukin-2 in PUF, by evidence that PUF alone did not support cytotoxic T-lymphocyte proliferation, nor was the stimulatory effect due to microbial contamination, because treatment of PUF through PyroBind columns to remove endotoxin did not affect its activity. Incubation of PUF at 56C for 40 min did not alter the enhancement effect. Treatment of PUF with trypsin or pronase for up to 24 hr did not change its activity. In mixed lymphocytes reaction, PUF augmented 3H-thymidine uptake by 242%. The molecular (mol) mass of the immunostimulatory activity in PUF was examined by size-exclusion HPLC, Sephadex G-50, and G-15 gel filtration, and it was found to be 0.8 kDa and < 0.5 kDa. Extracts of porcine endometrium also enhanced PHA-induced lymphocyte mitogenesis, suggesting that the factor(s) may be of uterine origin. In conclusion, we have identified an factor(s) in PUF capable of stimulating lymphocyte mitogenesis and mixed lymphocyte reaction.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Útero/inmunología , Adyuvantes Inmunológicos/análisis , Animales , Líquidos Corporales/inmunología , Cromatografía Liquida , Femenino , Técnicas In Vitro , Interleucina-2/análisis , Lectinas/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Prueba de Cultivo Mixto de Linfocitos , PorcinosRESUMEN
Immunoreactive (ir) beta-endorphin (BEND) was recently identified in porcine uterine fluids. In the study reported here, we examined the hypothesis that porcine endometrium serves as a source of uterine fluid ir-BEND during the estrous cycle and early pregnancy. Endometrial ir-BEND was chromatographically characterized, sites of ir-BEND synthesis were immunocytochemically localized, and concentrations of endometrial ir-BEND during the estrous cycle and early pregnancy were measured. Sephadex G-50 chromatographic profiles of endometrial extracts from Day 15 of the estrous cycle revealed three distinct peaks of ir-BEND, with the first peak occurring near void volume and the second and third peaks coinciding with standard porcine beta-lipotropin and standard porcine BEND, respectively. Reverse-phase HPLC C18 chromatographic profiles indicated that endometrial ir-BEND contained both standard BEND and alpha-N-acetylated BEND. Immunocytochemical studies demonstrated ir-BEND in the surface and glandular epithelial cells of the endometrium, with immunostaining most prominent in the apical portion of epithelial cells. Concentrations of ir-BEND in endometrial tissues were higher on Days 14-15 than on Days 8-12 during the estrous cycle and pregnancy (p less than 0.05); however, values were not different in pregnant and cyclic gilts. Biochemical and immunocytochemical evidence supports our hypothesis that ir-BEND present in uterine fluids is derived from the endometrium. The increase in endometrial ir-BEND concentration during Days 14-15 in cyclic and pregnant gilts indicates that ovarian steroids may influence the synthesis of endometrial ir-BEND.
Asunto(s)
Endometrio/metabolismo , Estro/metabolismo , Preñez/metabolismo , Porcinos/metabolismo , betaendorfina/metabolismo , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Endometrio/química , Femenino , Inmunohistoquímica , Embarazo , Radioinmunoensayo , Factores de Tiempo , betaendorfina/análisisRESUMEN
The purpose of this study was to immortalize porcine endometrial cells and to characterize the transformed cells. Primary porcine endometrial cells were transfected with the plasmid vector (pmk16) containing SV40 DNA using a liposome-mediated method. The viral DNA was from a replication-defective, origin-minus, temperature-sensitive mutant strain (A58). One clone, designated PE-1, has been propagated for over 120 passages. PE-1 cells grown at 33C (33C cells) exhibit spindle-shaped morphology; when cultured at 40C (40C cells), they took on a polygonal or spherical shape. Morphology of 40C cells returned to the spindle shape after culture flasks were shifted back to 33C. During a 2-week period, 33C cells propagated approximately 30-fold faster than 40C cells, whereas protein concentration was higher in 40C cells. Southern blot analysis of PE-1 cells demonstrated successful integration of the ts-SV40 DNA sequence into the porcine endometrial cells, possibly at multiple sites. The presence of cytokeratin on PE-1 cell membranes was shown by immunocytochemical studies, suggesting that the PE-1 cell clone was of epithelial origin. Reverse phase (RP)-HPLC analysis of PE-1 cell extract indicated that the majority of immunoreactive beta-endorphin (ir-BEND) eluted with a hydrophobicity similar to that of synthetic BEND and alpha-N-acetylated BEND (Nac-BEND). These results demonstrate that a porcine endometrial cell line has been established, and that this cell line possesses characteristics of temperature sensitivity in cell morphology, growth rate, and protein synthesis.
Asunto(s)
ADN Viral/genética , Endometrio/citología , Virus 40 de los Simios/genética , Transfección , Animales , Southern Blotting , División Celular , Línea Celular Transformada , Transformación Celular Viral , Cromatografía Líquida de Alta Presión , Endometrio/metabolismo , Células Epiteliales , Epitelio/metabolismo , Femenino , Queratinas/metabolismo , Fenotipo , Porcinos , Temperatura , betaendorfina/metabolismoRESUMEN
Immunoreactive methionine-enkephalin (ir-MENK) has been identified in the porcine uterine fluid and endometrium. Previously, we have established a porcine endometrial cell line of epithelial origin (PE-1) by transfecting primary endometrial cells with temperature sensitive SV40 DNA. The current study was conducted to identify and characterize ir-MENK present in PE-1 cells, and to investigate the effect of KCl depolarization on the kinetics of ir-MENK secretion. PE-1 cells were cultured at 33C until confluency was reached (33C cells), after which they were incubated at 40C for 2 days (40C cells). Ir-MENK in PE-1 cells was analyzed by Sephadex G-15 gel filtration and reverse phase (RP)-HPLC. Analysis of 40C cell extract by Sephadex G-15 and RP-HPLC indicated that the major portion of ir-MENK present in PE-1 cells was eluted at a position similar to that of synthetic MENK. The effect of temperature on ir-MENK synthesis in PE-1 cells was examined by measuring ir-MENK content in 33C and 40C cells over a 14-day culture period. Compared to 33C cells, 40C cells maintained higher and steadier levels of ir-MENK, suggesting that synthesis of ir-MENK is temperature sensitive. KCl stimulated ir-MENK secretion at all concentrations tested (5-60 mM for 60 min), with 30 mM being the optimal concentration. Temporal analysis of ir-MENK secretion showed that incubation for 60 min with 30 mM KCl allowed maximal secretion. Secretion of ir-MENK from PE-1 cells resulted in depletion of ir-MENK in cell content. These results demonstrate that PE-1 cells contain ir-MENK which is biochemically similar to synthetic MENK, PE-1 cells synthesize ir-MENK in a temperature sensitive manner, and these cells secrete ir-MENK upon KCl stimulation.
Asunto(s)
Endometrio/efectos de los fármacos , Encefalina Metionina/metabolismo , Cloruro de Potasio/farmacología , Animales , Línea Celular Transformada , Transformación Celular Viral , Células Cultivadas , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , ADN Viral/genética , Endometrio/metabolismo , Epitelio/efectos de los fármacos , Femenino , Calor , Cinética , Radioinmunoensayo , Virus 40 de los Simios/genética , Porcinos , TransfecciónRESUMEN
The expression of the proenkephalin gene has been demonstrated in the reproductive tissues of several animal species. The objectives of the experiments reported here were to (a) examine the presence of immunoreactive methionine-enkephalin (ir-MENK) in rabbit ovary, oviduct, and uterus and in a rabbit endometrial cell line (HRE-H9), (b) characterize ir-MENK biochemically, (c) investigate the effect of eCG + hCG treatment on the synthesis and secretion of ir-MENK in vivo, and (d) study the effect of K+ depolarization on the secretion of ir-MENK from HRE-H9 cells. Uterine fluid was collected by flushing the uterine lumen with saline. Reproductive tissues and HRE-H9 cells were extracted with 0.1 N acetic acid. Both the uterine fluid and extracts of uterus, ovary, oviduct, and HRE-H9 cells exhibited inhibition curves parallel to that of authentic MENK in the MENK RIA system. Sephadex G-15 gel filtration profiles indicated that in the extracts of rabbit uterus and HRE-H9 cells, most ir-MENK co-eluted with standard MENK, with a minor portion eluting near the void volume (Vo). Reverse-phase-HPLC (RP-HPLC) profiles showed a major peak coinciding with standard MENK, plus a minor peak of highly hydrophilic ir-MENK. The effect of eCG + hCG treatment was studied by i.m. injection of eCG (150 IU), followed by i.v. injection of hCG (75 IU) 4 days later. Ir-MENK concentration in the uteri and ovaries was significantly (p less than 0.05) increased (9.06 +/- 1.89 and 2.05 +/- 0.32 ng/mg protein, respectively), compared to control levels (2.31 +/- 0.86 and 0.24 +/- 0.77).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Encefalina Metionina/biosíntesis , Genitales Femeninos/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Gonadotropina Coriónica/farmacología , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Endometrio/efectos de los fármacos , Endometrio/metabolismo , Encefalina Metionina/metabolismo , Trompas Uterinas/efectos de los fármacos , Trompas Uterinas/metabolismo , Femenino , Genitales Femeninos/efectos de los fármacos , Datos de Secuencia Molecular , Ovario/efectos de los fármacos , Ovario/metabolismo , Cloruro de Potasio/farmacología , Conejos , Útero/efectos de los fármacos , Útero/metabolismoRESUMEN
A near-infrared (IR) spectrophotometer, integrating optics, and parallel-vector supercomputer are employed to develop a mathematical model that predicts the dissolution rate of individual intact tablets from near-IR spectra (r2 = 0.985). Each tablet can be analyzed nondestructively by the spectrophotometer in less than 1 min. The model permits hundreds of near-IR wavelengths to be used in the determination of dissolution rate, leading to increased accuracy.
Asunto(s)
Carbamazepina/química , Solubilidad , Espectrofotometría Infrarroja , Espectrofotometría Ultravioleta , ComprimidosRESUMEN
The micropipet aspiration technique and the parallel-plate flow chamber were used to investigate the deformation and detachment properties, respectively, of normal and transformed rat fibroblasts. The normal Cloned Rat Embryo Fibroblasts (CREF) cell line was transfected with the T24 ras oncogene to produce the transformed cell line CREF T24. The CREF T24 cell line was transfected with a Kirsten ras revertant gene (K-rev 1a suppressor) to produce the CT24HKB1 cells, which have the same morphological characteristics as the cells in the CREF line. The cells utilized in this investigation were derived from the parent cell line CREF, the only differences being the presence or absence of the T24 ras oncogene and the Kirsten ras revertant gene. The detachment and deformation properties, therefore, could be related to the metastatic phenotype of the cell rather than inherent differences between disparate cell lines. Results indicated that transfecting the CREF cell line with the ras oncogene greatly modified the detachment and deformation properties. The CREF T24 cells were more easily detached from normal cells and were 50% more deformable. Both CREF and CT24HKB1 showed similar detachment properties. Based on these results, it is speculated that K-rev 1a reversed ras-induced membrane alterations in these cells. Preliminary investigations have demonstrated that both CREF and CREF T24 cells in different phases of the cell cycle differed in morphological characteristics. However, the majority of the cells within a given cell line showed similar deformation characteristics. Current investigations are focusing on characterization of both detachment and deformation properties of these cells as a function of the cell cycle using synchronization techniques.
Asunto(s)
Fibroblastos/citología , Animales , Adhesión Celular/fisiología , Ciclo Celular/fisiología , Línea Celular Transformada , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/fisiología , Genes ras/genética , Metástasis de la Neoplasia/patología , Fenotipo , Ratas , Reología , Estrés Mecánico , TransfecciónRESUMEN
The current study examined the presence of immunoreactive methionine-enkephalin (ir-MENK) in porcine uterine fluid and endometrial extracts, characterized ir-MENK biochemically, and investigated the effect of ovarian steroids on uterine secretion of ir-MENK. Porcine uterine fluid was collected by flushing the uterine lumen with saline. Endometrial tissues were extracted with acetic acid. Both uterine fluid and endometrial extracts exhibited inhibition curves parallel to that of authentic MENK in the MENK RIA system. Sephadex G-15 gel filtration chromatographic profiles indicated that both concentrated uterine fluid and endometrial extracts contained two peaks of ir-MENK, a major peak which coeluted with standard MENK, and a minor peak eluting near the void volume (Vo). Reverse phase-HPLC chromatographic profiles also demonstrated two peaks of ir-MENK for concentrated uterine fluid and endometrial extracts, a major peak which coincided with standard MENK, plus a highly hydrophilic peak. The effect of ovarian steroids on the uterine secretion of ir-MENK was examined by measuring ir-MENK in uterine fluids from cyclic and pregnant gilts as well as ovariectomized, ovarian steroid-treated gilts. Day effects (P less than 0.01) were detected for cyclic and pregnant gilts, since values for ir-MENK increased between days 8 and 14 after onset of estrus. In ovariectomized gilts, treatment with progesterone (P4) increased the uterine secretion of ir-MENK (202 +/- 9 vs. 65 +/- 4 pg/ml for control, P less than 0.05). The combined treatment of P4 and estradiol did not further enhance secretion of ir-MENK, while treatment with estradiol did not alter ir-MENK levels relative to values for control gilts. These results indicate the presence of ir-MENK in porcine uterine fluid and endometrium, and suggest that uterine secretion of ir-MENK is regulated primarily by P4.
Asunto(s)
Encefalina Metionina/metabolismo , Útero/metabolismo , Animales , Cromatografía en Gel , Endometrio/metabolismo , Encefalina Metionina/aislamiento & purificación , Estradiol/farmacología , Femenino , Embarazo , Progesterona/farmacología , Radioinmunoensayo , Porcinos , Útero/efectos de los fármacosRESUMEN
The micropipette aspiration technique was used to investigate the deformation properties of a panel of nontransformed and transformed rat fibroblasts derived from the same normal cell line. In this method, a step negative pressure is applied to the cell via a micropipette and the aspiration distance into the pipette as a function of time is determined using video techniques. A standard solid viscoelastic model was then used to analyze the viscoelastic properties of the cell. From these results, it is concluded that a direct correlation exists between an increase in deformability and progression of the transformed phenotype from a nontumorigenic cell line into a tumorigenic, metastatic cell line.