RESUMEN
Corrosion-control coatings which can enhance bone formation and be completely replaced by bone are attractive for biodegradable Mg alloys. Carbonate apatite (CAp) and hydroxyapatite (HAp) coatings were formed on Mg-4 wt% Y-3 wt% rare earth (WE43) alloy as a corrosion-control and bioabsorbable coating in the coating solution with various concentrations of NaHCO3. The incorporation of carbonate group in apatite structure was examined using X-ray diffraction and Fourier transform infrared spectroscopy. Rat osteoclast precursor and MC3T3-E1 osteoblast cells were cultured on the CAp- and HAp-coated WE43 to examine the osteoclastic resorption and the alkaline phosphatase (ALP) activity, respectively. Mg ions in the used medium were quantified to examine the corrosion-control ability. The NaHCO3 addition in the solution resulted in the formation of B-type CAp in which the phosphate group of apatite structure was substituted with the carbonate group. The osteoclastic resorption was observed only for the CAp coatings as the cracking of the coatings and the corrosion of substrate WE43 strongly localized under osteoclast cell bodies. The CAp and HAp coatings significantly enhanced the ALP activity of osteoblasts. The CAp-coated WE43 specimens showed 1/5 smaller amount of Mg ion release than the uncoated WE43 on the first day of culturing osteoblasts. For the subsequent 22 days, the Mg ion release was reduced to 1/2 by the CAp coatings. In the presence of osteoclasts, the CAp coatings showed slightly lower corrosion protectiveness than the HAp coating. It was demonstrated that the CAp coatings can be a bioabsorbable and corrosion-control coating for biodegradable Mg alloys.
RESUMEN
Two strains of Pseudomonas sp., Os17 and St29, were newly isolated from the rhizosphere of rice and potato, respectively, by screening for 2,4-diacetylphloroglucinol producers. These strains were found to be the same species and were the closest to but different from Pseudomonas protegens among the sequenced pseudomonads, based on 16S ribosomal RNA gene and whole-genome analyses. Strain Os17 was as effective a biocontrol agent as reported for P. protegens Cab57, whereas strain St29 was less effective. The whole-genome sequences of these strains were obtained: the genomes are organized into a single circular chromosome with 6,885,464 bp, 63.5% G+C content, and 6,195 coding sequences for strain Os17; and with 6,833,117 bp, 63.3% G+C content, and 6,217 coding sequences for strain St29. Comparative genome analysis of these strains revealed that the complete rhizoxin analog biosynthesis gene cluster (approximately 79 kb) found in the Os17 genome was absent from the St29 genome. In an rzxB mutant, which lacks the polyketide synthase essential for the production of rhizoxin analogs, the growth inhibition activity against fungal and oomycete pathogens and the plant protection efficacy were attenuated compared with those of wild-type Os17. These findings suggest that rhizoxin analogs are important biocontrol factors of this strain.
Asunto(s)
Cucumis sativus/microbiología , Genoma Bacteriano/genética , Macrólidos/metabolismo , Enfermedades de las Plantas/prevención & control , Pseudomonas/genética , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antibiosis , Bacillus/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Agentes de Control Biológico , Fusarium/efectos de los fármacos , Genes Reporteros , Datos de Secuencia Molecular , Familia de Multigenes , Floroglucinol/análogos & derivados , Floroglucinol/metabolismo , Floroglucinol/farmacología , Enfermedades de las Plantas/microbiología , Raíces de Plantas/microbiología , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Pseudomonas/química , Pseudomonas/metabolismo , Pythium/efectos de los fármacos , Proteínas Recombinantes de Fusión , Rizosfera , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
The biocontrol strain Pseudomonas sp. Cab57 was isolated from the rhizosphere of shepherd's purse growing in a field in Hokkaido by screening the antibiotic producers. The whole genome sequence of this strain was obtained by paired-end and whole-genome shotgun sequencing, and the gaps between the contigs were closed using gap-spanning PCR products. The P. sp. Cab57 genome is organized into a single circular chromosome with 6,827,892 bp, 63.3% G+C content, and 6,186 predicted protein-coding sequences. Based on 16S rRNA gene analysis and whole genome analysis, strain Cab57 was identified as P. protegens. As reported in P. protegens CHA0 and Pf-5, four gene clusters (phl, prn, plt, and hcn) encoding the typical antibiotic metabolites and the reported genes associated with Gac/Rsm signal transduction pathway of these strains are fully conserved in the Cab57 genome. Actually strain Cab57 exhibited typical Gac/Rsm activities and antibiotic production, and these activities were enhanced by knocking out the retS gene (for a sensor kinase acting as an antagonist of GacS). Two large segments (79 and 115 kb) lacking in the Cab57 genome, as compared with the Pf-5 genome, accounted for the majority of the difference (247 kb) between these genomes. One of these segments was the complete rhizoxin analog biosynthesis gene cluster (ca. 79 kb) and another one was the 115-kb mobile genomic island. A whole genome comparison of those relative strains revealed that each strain has unique gene clusters involved in metabolism such as nitrite/nitrate assimilation, which was identified in the Cab57 genome. These findings suggest that P. protegens is a ubiquitous bacterium that controls its biocontrol traits while building up strain-specific genomic repertoires for the biosynthesis of secondary metabolites and niche adaptation.
Asunto(s)
Genoma Bacteriano , Pseudomonas/genética , Secuencia de Bases , Elementos Transponibles de ADN , Japón , Datos de Secuencia Molecular , Operón , Pseudomonas/clasificación , Especificidad de la EspecieRESUMEN
In Pseudomonas protegensâ CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway controls secondary metabolism and suppression of fungal root pathogens via the expression of regulatory small RNAs (sRNAs). Because of its high cost, this pathway needs to be protected from overexpression and to be turned off in response to environmental stress such as the lack of nutrients. However, little is known about its underlying molecular mechanisms. In this study, we demonstrated that Lon protease, a member of the ATP-dependent protease family, negatively regulated the Gac/Rsm cascade. In a lon mutant, the steady-state levels and the stability of the GacA protein were significantly elevated at the end of exponential growth. As a consequence, the expression of the sRNAs RsmY and RsmZ and that of dependent physiological functions such as antibiotic production were significantly enhanced. Biocontrol of Pythium ultimum on cucumber roots required fewer lon mutant cells than wild-type cells. In starved cells, the loss of Lon function prolonged the half-life of the GacA protein. Thus, Lon protease is an important negative regulator of the Gac/Rsm signal transduction pathway in P. protegens.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Proteasa La/genética , Pseudomonas/genética , ARN Nuclear Pequeño/genética , Antibacterianos/metabolismo , Antibiosis , Proteínas Bacterianas/metabolismo , Cucumis sativus/microbiología , Mutación , Raíces de Plantas/microbiología , Proteasa La/metabolismo , Estabilidad Proteica , Pseudomonas/metabolismo , Pythium/patogenicidad , Pythium/fisiología , ARN Nuclear Pequeño/metabolismo , Transducción de SeñalRESUMEN
Genes involved in lipid accumulation were identified in Saccharomyces cerevisiae using transposon insertion mutagenesis. Five ORFs, such as SNF2, IRA2, PRE9, PHO90, and SPT21 were found from the analysis of the insertion sites in transposon insertion mutants with higher lipid content. Since these ORFs are not directly involved in storage lipid biosynthesis, we speculate that they are involved in carbon fluxes into storage lipids in response to nutrient conditions. Lipid analysis of disruptants of these ORFs indicated that the Deltasnf2, and Deltaira2 disruptants had significantly higher lipid content. Cultivation in a nitrogen-limited medium increased the lipid content in all disruptants, among which the Deltapre9 disruptant was the most sensitive to nitrogen limitation. We then focused on the Deltasnf2 disruptant due to its higher lipid content and its function as a regulator of phospholipid synthesis. Lipid class analysis indicated that triacylglycerol and free fatty acids contributed to the increase in total lipids of the Deltasnf2 disruptant. The addition of exogenous fatty acids was not so effective at increasing the lipid content in the Deltasnf2 disruptant as it was in the wild type. It should be noticed that exogenous free linoleic acid was much higher in the Deltasnf2 disruptant than in the wild type, as in the case of endogenous free fatty acids. In addition, the incorporation of exogenous fatty acids into cells increased in the disruptant, suggesting that fatty acid transporters were regulated by SNF2. The results suggest that metabolic fluxes into storage lipids, which are activated in the Deltasnf2 disruptant, is repressed by the incorporation of exogenous fatty acids. They provide new insight into the biosynthesis of storage lipids in yeast.
Asunto(s)
Elementos Transponibles de ADN/genética , Metabolismo de los Lípidos , Mutagénesis/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas , Forma de la Célula , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ácidos Grasos/farmacología , Eliminación de Gen , Sistemas de Lectura Abierta , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
During the peri-implantation period, the endometrium undergoes tissue remodeling and cellular rearrangement. To clarify the involvement of matrix metalloproteinases (MMPs) in endometrial remodeling, we isolated total RNAs from the endometrium of non-pregnant and pregnant mice on days 3 to 5 and evaluated mRNA expression of MMP-2, -3, -9, -11 and -13 using reverse transcription-polymerase chain reaction (PCR). Prompt increases in MMP-3 and -13 mRNA were found on day 4 of pregnancy. Quantitative real-time PCR showed that expression of MMP-3 and -13 increased significantly on day 4, up to 8.4 +/- 2.7 times and 3.4 +/- 1.5 times, respectively, the level in non-pregnant endometrium (p < 0.05). On day 4, immunohistochemistry demonstrated MMP-3-positive endometrial stromal cells. At the same time, tenascin-C (TN-C) mRNA increased 11.1 +/- 4.0 times from the level in non-pregnant endometrium (p < 0.004). To clarify regulation of MMP-3 expression, we examined the effects of interleukin-1alpha (IL-1alpha) and TN-C on MMP-3 mRNA in cultured mouse endometrial stromal cells. Both substances resulted in a dose-dependent increase in MMP-3 mRNA (6.1 +/- 1.8-fold at 1 ng/ml of IL-1alpha and 3.9 +/- 1.8-fold at 10 mug/ml of TN-C). This study shows that MMP-3 expression is upregulated in endometrial stromal cells of the peri-implantation period and may be controlled by IL-1alpha and TN-C.
Asunto(s)
Colagenasas/biosíntesis , Colagenasas/efectos de los fármacos , Interleucina-1/farmacología , Tenascina/farmacología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Implantación del Embrión/fisiología , Endometrio/citología , Endometrio/metabolismo , Femenino , Inmunohistoquímica , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 3 de la Matriz , Ratones , Ratones Endogámicos C3H , Embarazo , ARN Mensajero/metabolismo , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Regulación hacia ArribaRESUMEN
Oligopeptide hormones are involved in cell-cell interaction during embryonal implantation and neuropeptide Y (NPY) is expressed in the human placenta and decidual cells in the third trimester of pregnancy. However, there is no report regarding the intrauterine localisation and the functions of NPY during the peri-implantation period. In the present study, the spatiotemporal changes in NPY expression in the murine uterus during the peri-implantation period were investigated using reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR and immunohistochemical techniques, as were the effects of sex steroids on NPY mRNA expression in primary cultured murine uterine epithelial cells. Neuropeptide Y mRNA was increased in the pregnant murine uterus, as well as in the pseudopregnant murine uterus, during the peri-implantation period. Immunohistochemical analysis revealed increases in NPY expression in luminal and glandular epithelial cells and decidualised stromal cells. Neuropeptide Y mRNA expression was strongly induced in cultured epithelial cells in response to sex steroids. The data suggest that NPY is involved in cell-cell interactions during embryonic implantation.
Asunto(s)
Comunicación Celular , Implantación del Embrión , Endometrio/metabolismo , Neuropéptido Y/metabolismo , Animales , Comunicación Celular/efectos de los fármacos , Comunicación Celular/genética , Endometrio/química , Endometrio/efectos de los fármacos , Células Epiteliales/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Epitelio/química , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Estradiol/farmacología , Femenino , Expresión Génica , Inmunohistoquímica , Ratones , Neuropéptido Y/análisis , Neuropéptido Y/genética , Embarazo , Progesterona/farmacología , ARN Mensajero/análisis , ARN Mensajero/metabolismoRESUMEN
We studied the regulation of lipid body biogenesis in the oleaginous fungus Mortierella ramanniana var. angulispora by investigating culture conditions to modulate lipid body size, which we found was affected by the carbon-to-nitrogen ratio (C/N ratio) in the culture medium. Increasing the nitrogen source or decreasing the C/N ratio from 38 to 9 induced the appearance of lipid bodies with diameters less than 2-3 micro m, which are usually found at a C/N ratio of 38 in this fungus. To determine factors regulating lipid body size, we compared lipid body fractions from fungal cells cultured at different C/N ratios. We found some differences in polypeptide profiles between lipid body fractions from fungal cells cultured at different C/N ratios for 2 days when the lipid bodies were enlarged at a C/N ratio of 38. We then compared the phosphorylation of lipid body proteins, since protein phosphorylation plays a pivotal role in various aspects of signal transduction. In vitro phosphorylation in the lipid body fraction indicated that protein kinase activity toward endogenous and exogenous substrates such as histone IIIS, VIIS, and myelin basic protein increased in the lipid body fraction at a C/N ratio of 9. Further analysis by in-gel protein kinase assay indicated the presence of at least three activated protein kinases with molecular masses of 75, 72, and 42 kDa, which were also autophosphorylated. These results indicate the presence of nutrient-regulated protein kinases and increased phosphorylation in lipid bodies, which correlate with the appearance of smaller lipid bodies in this fungus. Further studies to characterize these protein kinases at the molecular level should provide new insights into the link between nutrient sensing and lipid storage.
Asunto(s)
Cuerpo Adiposo/metabolismo , Metabolismo de los Lípidos , Mortierella/metabolismo , Nitrógeno/farmacología , Proteínas Quinasas/metabolismo , Animales , Carbono/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Medios de Cultivo , Electroforesis , Cuerpo Adiposo/química , Lípidos/química , Peso Molecular , Mortierella/citología , Mortierella/efectos de los fármacos , Mortierella/ultraestructura , Nitrógeno/metabolismo , Fosforilación , Transducción de Señal , Factores de TiempoRESUMEN
OBJECTIVE: To evaluate the endocrinologic profile and reproductive outcome after laparoscopic drilling using a harmonic scalpel for polycystic ovarian syndrome (PCOS) in clomiphene-resistant infertile women. STUDY DESIGN: We performed a prospective, randomized study of 34 infertile women with PCOS. Group A (17 women) underwent laparoscopic ovarian drilling using a harmonic scalpel laser. Group B (control group, 17 women) underwent laparoscopic ovarian drilling using a neodymium-yttrium-aluminum-garnet laser. Change in the hormonal profile after surgery, ovulation rate and pregnancy rate were compared between groups A and B. RESULTS: LH and testosterone serum levels and the LH-FSH ratio showed a statistically significant reduction after surgery, and the spontaneous ovulation rate was 94% in both groups. The cumulative pregnancy rates within two years of follow-up were 77% in group A and 60% in group B. CONCLUSION: Laparoscopic ovarian drilling using a harmonic scalpel is an effective treatment for PCOS in clomiphene-resistant, anovulatory women: it results in ovulation and conception without major complications.
Asunto(s)
Anovulación/etiología , Infertilidad Femenina/etiología , Laparoscopía/métodos , Síndrome del Ovario Poliquístico/cirugía , Vibración/uso terapéutico , Adulto , Índice de Masa Corporal , Sulfato de Deshidroepiandrosterona/sangre , Femenino , Hormona Folículo Estimulante/sangre , Humanos , Hormona Luteinizante/sangre , Ovulación , Síndrome del Ovario Poliquístico/sangre , Síndrome del Ovario Poliquístico/complicaciones , Síndrome del Ovario Poliquístico/diagnóstico , Embarazo , Resultado del Embarazo , Índice de Embarazo , Estudios Prospectivos , Testosterona/sangre , Factores de Tiempo , Resultado del TratamientoRESUMEN
Tenascin-C (TNC) is an extracellular matrix protein which appears at active sites of tissue remodelling during embryogenesis or cancer invasion. In normal heart, TNC is only present during the early stages of development but reappears in pathological states. This study examined the diagnostic value of TNC for assessing disease activity of myocarditis. Expression of TNC was examined in myosin-induced autoimmune myocarditis mouse models. Sequential changes in amount, localization and the producing cells were analysed by reverse transcriptase-polymerase chain reaction, western blotting, immunohistochemistry and in situ hybridization and compared with the histological picture. The expression of TNC was upregulated at a very early stage of myocarditis. Immunostaining was detectable before cell infiltration and myocytolysis became histologically apparent, remained during the active stage while cell infiltration and necrosis continued, and disappeared in scar tissue with healing. TNC immunostaining was always observed at the periphery of necrotic or degenerating cardiomyocytes in foci of inflammation, the expression level correlating with histological evidence of inflammatory activity. Interstitial fibroblasts were the major source of TNC, expressing the large isoform containing alternative splicing sites. These data demonstrate that TNC is a useful marker for evaluation of disease activity in myocarditis.