Does BCR-ABL transcript type influence the prognosis of patients in chronic myelogenous leukemia chronic phase?

ABSTRACT Introduction and objective: In this study, we evaluated the influence of the transcript type on hematological and clinical parameters, as well as the event-free survival of 50 patients in the Chronic myeloid leukemia chronic phase. Methods: We reviewed the medical records of 55 patients with Chronic myeloid leukemia. The eligibility criteria were based on the availability of hematological and clinical baseline data in the medical records. Data on BCR-ABL transcripts were obtained from medical records. Results: Eighteen patients (36%) had the b2a2 transcript, 24 (48%) had b3a2 and 8 (16%) had b2a2/b3a2. The median platelet count for transcripts b2a2, b3a2 and b2a2/b3a2 was 320.65 × 103/L, 396 × 103/L, and 327.05 × 103/L, respectively (p = 0.896). We could not find any differences in relation to the other hematological parameters, when compared to the transcript type. Comparison between spleen and liver size and type of transcript did not differ inside the groups (p = 0.395 and p = 0.647, respectively) and the association between risk scores and transcript type did not show statistical significance (p > 0.05). The 21-month probability for event-free survival was 21%, 48% and 66% for the transcripts b2a2, b3a2 and b2a2/b3a2 respectively (p = 0.226) Conclusion: We conclude that the expression BCR-ABL transcripts have no influence on hematological, clinical and event-free survival parameters of patients in the Chronic myeloid leukemia chronic phase.

The Inhibitory Mechanism of Gentamicin on Electrical Field Stimulation Response in Rat Bladder Smooth Muscle

To see the inhibitory mechanism of gentamicin in response to electrical field stimulation (EFS) using the rat bladder smooth muscle, atropine or guanethidine was treated but had no effect. Methylsergide, a non-selective 5-HT1, 5-HT2 receptor antagonist was also treated but had on effect. Kinase inhibitors, such as chelerythrine (PKC inhibitor), ML-9 (MLCK inhibitor), or Y27632 (rho kinase inhibitor) were pretreated before gentamicin treatment, but did not have effect. For U73122, a phospholipase C (PLC) inhibitor however, the inhibitory effect to gentamicin was significantly attenuated in all frequencies given by the EFS. Therefore gentamicin induced inhibitory effect on EFS response in rat bladder smooth muscle was not mediated by the activation of adrenergic, cholinergic, or serotonergic receptor. The inhibition of gentamicin might be mediated through the PLC dependent pathway, but not through the PKC, MLCK or rho kinase dependent pathway.

Desenvolvimento e validação de um método por CLAE-EM/EM para determinação de PGE2 e PGD2 em meio de cultivo celular e avaliação da recuperação das prostaglandinas utilizando diferentes condições na extração em fase sólida / Development and validation of HPLC-MS/MS method to determine PGE2 and PGD2 in cell culture medium and assessment of recovery of the prostaglandins by solid phase extraction under various conditions

Tradicionalmente, estas prostaglandinas são quantificadas por técnicas de imuno-ensaio, que apresentam diversas desvantagens. Estes metabólitos são isômeros estruturais, e dessa forma é necessário o uso de técnicas de detecção seletivas, como cromatografia líquida acoplada à espectrometria de massas sequencial (CLAE-EM/EM). Para a extração de prostaglandinas de matrizes complexas, destaca-se a extração em fase sólida (EFS), que otimizada, fornece excelentes taxas de recuperação. O objetivo deste trabalho foi desenvolver e validar um método rápido por CLAE-EM/EM, para análise simultânea de PGE2 e PGD2 de meio de cultivo celular e avaliar a eficiência de extração em diferentes condições de EFS, em relação ao método proposto pelo fabricante dos cartuchos. A separação ocorreu com coluna de fase reversa (C18, 150mm x 2.1mm, 5μm) eluída no modo gradiente com acetonitrila e água (0,1% AFO). Dez condições diferentes de EFS foram testadas. O método desenvolvido foi adequado para a análise simultânea de PGE2 e PGD2 , apresentando resolução de ~1,5 entre os picos e corrida de 11 minutos. LD da ordem de 0,5 ng/mL e LQ de 1,0 ng/mL foram obtidos para ambos os analitos. A linearidade de PGE2 e PGD2 apresentou r>0,99. Variações inferiores a 6,51% e 5,93% foram encontradas para repetibilidade e precisão intermediária, respectivamente. Foi possível diminuir perdas durante a EFS e aumentar a recuperação dos analitos. A condição que ofereceu melhor eficiência de extração aumentou o rendimento em 181% para PGE2 e 323% para PGD2 , em relação ao método proposto pelo fabricante.

PGE2 and PGD2 are very important pro-inflammatory mediators. Traditionally, these prostaglandins are estimated by immunoassay techniques, which have several disadvantages. Since these metabolites are structural isomers, it is necessary to use selective detection techniques, such as liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). For the extraction of prostaglandins from complex matrices, solid phase extraction (SPE) is an outstanding method that can be optimized to provide excellent recovery. The aim of this study was to develop and validate a rapid method for the simultaneous analysis of PGE2 and PGD2 in cell culture medium by HPLC-MS/MS and to assess the extraction efficiency of SPE under various conditions, compared to the generic method proposed by the manufacturer of the cartridges. The analytes were separated on a reversed-phase column (C18, 150mm x 2.1mm, 5μm), eluted in a gradient of acetonitrile and water (0.1% formic acid). Ten different conditions for SPE were tested. The method was suitable for the simultaneous analysis of PGE2 and PGD2 , showing a resolution of ~1.5 between the peaks and a run time of 11 minutes. LOD of 0.5 ng/mL and LOQ of 1.0 ng/mL were recorded for both analytes. The linearity of the analytical curves for both PGE2 and PGD2 showed r>0.99. Variations of less than 6.51% and 5.93% were found for repeatability and intermediate precision, respectively. It was possible to reduce the losses during SPE and enhance the recovery of the analytes. The condition affording the best extraction efficiency increased the yield by 181% for PGE2 and 323% for PGD2 , relative to the method proposed by the manufacturer.

Validación de uaa metodología para la determinaión de carbofuran en suelos meddiante cromatografía líquida de alta eficiencia con detección ultravioleta (clar-uv) / Validation ofan analytical method for carbofuran determinatin on soils by hhigh performance liquid chromatography with uv detection (hplc-uv) / Validação de um metodologia paa determinação do carbofurano em solos por crommatografia líquida da alta eficiência com detecção ultravioleta (clar-vv)

Se validó una metodología analítica que permite cuantificar residuos de carbofuran en muestras de suelo. La extracción del plaguicida desde la matriz se realizó mediante agitación mecánica empleando acetato de etilo como solvente, los extractos obtenidos se sometieron a extracción en fase sólida (EFS) utilizando cartuchos C18, finalmente, la determinación y cuantificación del carbofuran se llevó a cabo mediante cromatografía líquida de alta eficiencia con detección ultravioleta (CLAR-UV) a una longitud de onda de 205 nm. La metodología validada es específica y selectiva para el carbofuran, lineal en el rango desde 0,47 hasta 2,36 mgKg-1, precisa con un coeficiente de variación típico (CVtip) de 10,78%; exacta brindando un porcentaje de recuperación para la metodología global (porcentaje de R) equivalente a 98,25±3,97% y sensible con límites de detección y cuantifica-ción de 0,045 y 0,149 mgKg-1, respectivamente. También se verificó la robustez del método. Se analizaron dos muestras de suelo dedicados al cultivo de café, y se encontraron residuos de carbofuran durante los primeros 30 días después de su aplicación.

An analytic method was validated to quantify Carbofuran residues in soil samples. The pesticides were extracted from the matrix by mechanic stirring, using ethyl acetate as a solvent. These extracts were cleaned by using cartridges C18. The determination and quantification of Carbofuran was made by high performance liquid chromatography with ultraviolet detection (HPLC-UV). The wavelength was 205 nm. The validated method is specific and selective for Carbofuran, is linear in the range from 0.47 to 2.36 mg kg-1, is accurate with a typical variation coefficient of 10.78%, is exact with recovering percentage (% R) equivalent to 98.25±3.97% and sensitive with detection and quantification limits since 0.045 and 0.149 mg kg-1 respectively. The robustness of the method was recognized. Two samples from soil of coffee cultivation were analyzed. Residues ofCarbofuranwerefoundduringthe first thirty days after application. Two samples of soil coffee were analyzed finding residues of carbofuran during the first thirty days after application.

Neste artigo validou-se uma metodologia analítica que quantifica resíduos de carbo-furano em amostras de solo. Por agitação mecânica se extraíram pesticidas da matriz, utilizando acetato de etilo como solvente. Os extratos obtidos foram submetidos à extração em fase sólida (EFS) com cartuchos de C18. Em seguida, a identificação e quantificação de Carbofuran foi realizada por cromatografia líquida de alta eficiência com detecção ultravioleta (CLAE-UV). A longitude de onda foi 205 nm. A metodologia validada é específica e seletiva para car-bofuran; linear no intervalo de 0,47-2,36 mgkg-1, cocomumcoeficientedevariação (C. Vtip) típico de 10,78%, fornecendo uma taxa de recuperação precisa da meto-dologiaglobal(R%)equivalentea98,25± 3,97%. Os limites de sensibilidade de detecção e quantificação são 0,045 e 0,149 mg kg-1, respectivamente. Verificou-se também a robustez do método. Analisadas duas amostras de terra dedicada ao cultivo do café, se encontraram resíduos de carbofuran nos trinta primeiros dias após aplicá-lo.

The Influences of G Proteins, Ca2+, and K+ Channels on Electrical Field Stimulation in Cat Esophageal Smooth Muscle

NO released by myenteric neurons controls the off contraction induced by electrical field stimulation (EFS) in distal esophageal smooth muscle, but in the presence of nitric oxide synthase (NOS) inhibitor, L-NAME, contraction by EFS occurs at the same time. The authors investigated the intracellular signaling pathways related with G protein and ionic channel EFS-induced contraction using cat esophageal muscles. EFS-induced contractions were significantly suppressed by tetrodotoxin (1 micrometer) and atropine (1 micrometer). Furthermore, nimodipine inhibited both on and off contractions by EFS in a concentration dependent meaner. The characteristics of 'on' and 'off' contraction and the effects of G-proteins, phospholipase, and K+ channel on EFS-induced contraction in smooth muscle were also investigated. Pertussis toxin (PTX, a Gi inactivator) attenuated both EFS-induced contractions. Cholera toxin (CTX, Gs inactivator) also decreased the amplitudes of EFS-induced off and on contractions. However, phospholipase inhibitors did not affect these contractions. Pinacidil (a K+ channel opener) decreased these contractions, and tetraethylammonium (TEA, K+ Ca channel blocker) increased them. These results suggest that EFS-induced on and off contractions can be mediated by the activations Gi or Gs proteins, and that L-type Ca2+ channel may be activated by G-protein alpha subunits. Furthermore, K+ Ca-channel involve in the depolarization of esophageal smooth muscle. Further studies are required to characterize the physiological regulation of Ca2+ channel and to investigate the effects of other K+ channels on EFS-induced on and off contractions.

Vitrification of Mouse Embryos in Ethylene Glycol-based Solutions / 대한불임학회지

OBJECTIVE: This study was conducted to find an optimal condition for the vitrification of mouse morulae and expanded blastocysts. MATERIALS AND METHODS: Mouse embryos were obtained at 2-cell stage and cultured to morula and expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% Serum Substitute Supplement (SSS). The vitrification solutions used were EFS30, EFS35 and EFS40 that contains 30%, 35% and 40% ethylene glycol, respectively, with 18% ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% SSS. The vitrification procedure was performed in EFS solution with three steps, followed by thawing in 6 steps with 0.5 M sucrose, and then survival and hatching-hatched rate per embryos recovered were compared among six groups. RESULTS: After 24 h culture in different vitrification and thawing solution, the survival rate of morula embryos was 94.1%, 85.4% and 59.7% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of morula embryos after 72 h culture was 30.6%, 25% and 11.3% for EFS30, EFS35 and EFS40 group, respectively. The survival rate of expanded blastocyst embryos after 24 h culture was 90.4%, 98.5% and 100% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of expanded blastocyst embryos after 48 h culture was 46.2%, 57.6% and 64.3% for EFS30, EFS35 and EFS40 group, respectively. CONCLUSION: The EFS30 solution was the best for vitrification of mouse morulae. The EFS40 solution was the best for vitrification of mouse expanded blastocysts. The mouse expanded blastocyst was better than mouse morula for vitrification of mouse embryos.

Vitrification of Mouse Blastocyst / 대한산부인과학회잡지

OBJECTIVE: The aim of this study was to compare the survival and developmental rate of two vitrification solutions for the vitrification of mouse expanded blastocysts. METHODS: Mouse embryos were obtained at 2 cell stage and cultured to expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% serum substitute supplement (SSS). The vitrification solutions used were EFS40 and VS. EFS40 consisted of 40% ethylene glycol, 18% ficoll, and 0.5 M sucrose while VS consisted of 20% ethylene glycol, 20% DMSO, and 10% 1,3-butanediol diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% calf serum (CS). Toxicity was tested by exposing expanded blastocysts to vitrification solution. The vitrification procedure used for EFS40 was performed in three steps, after which they were warmed in 5 steps with 0.5 M sucrose. VS was performed in two steps, after which they were warmed with 1.0 M trehalose. Recovery, survival and hatching rate per expanded blastocysts recovered were compared between two groups. RESULTS: In toxicity test, survival and hatching rate of EFS40 group were 95% and 100%, respectively. In contrast, survival and hatching rate of VS group were 100% and 87.5%, respectively. After vitrification and warming in solution, recovery rate for EFS40 group was 73.7% whereas recovery rate for VS group was 66.5%. After 24 h culture, survival and hatching rate were 80.5% and 20.7% for EFS40 and 66% and 0% for VS group, respectively. After 48 h culture, survival and hatching rate were 69% and 33.3% for EFS40 and 58.3% and 1.9% for VS group, respectively. Survival and hatching rate in EFS40 group were significantly higher than those found in VS group. CONCLUSION: The EFS40 solution was better than VS solution for vitrification of mouse expanded blastocysts.

The Study on Vitrification and Ultrarapid Thawing of Human Embryonic Stem Cells / 대한불임학회지

OBJECTIVE: This study was carried out to establish the effectiveness of the vitrification method and the optimal cryoprotectants in the cryopreservation of human embryonic stem cells (ESC). MATERIALS AND METHODS: Human ESC clumps established at Seoul National University Hospital (SNUhES 1) were cryopreserved with the vitrification method using the EM grid. EDS and EFS40 were used as vitrification solutions. RESULTS: Between the EDS and EFS40 groups, there was no significant difference in the recovery rate after cryopreservation of human ESC. The formation rates of ESC colonies in the vitrified groups were significantly lower than those in the control ESC group (p<0.05, p<0.05). In addition, the formation rate of ESC colonies in the EDS group was significantly higher than that in the EFS40 group (p<0.05). The ESC colonies in the vitrified groups were significantly smaller after culture duration of 2 and 4 days, respectively, compared with the control ESC group (p<0.01, p<0.05). However, these effects could be reduced to nonsignificant level by the additional culture of ESC colonies. The vitrified human ESC retained the properties of pluripotent cells, including the expression of cell surface markers for the undifferentiated cells such as alkaline phosphatase and SSEA-4 (stage-specific embryonic antigen-4), and the expression of transcription factor Oct-4 (octamer-binding transcription factor-4), and the normal karyotype. CONCLUSION: The vitrification method using the EM grid and EDS solution was confirmed to be very effective for the cryopreservation of human ESC.

Effects of Electrical Field Stimulation of the Isolated Corpus Cavernosum from Hypertensive Rats / 대한비뇨기과학회지

The present study was aimed to investigate whether and to what extent hypertension affects the relaxation of the corpus cavernosum. The corpus cavernosum was isolated from 12-week 2- kidney, 1-clip hypertensive rats. The corporal strips were isolated and suspended longitudinally in an organ bath. They were precontracted with phenylephrine, and their responses to electrical field stimulation (EFS) were examined. EFS caused a frequency-dependent contraction (60%) or relaxation (40%) of the corpus cavernosum precontracted with phenylephrine. The contraction response was inhibited or abolished and only frequency-dependent relaxation appeared in the presence of atropine (0.00001mol/L) and guanethidine (0.00001mol/L). The relaxation response to EFS of the corporal preparation precontracted with phenylephrine was attenuated or abolished in the presence of L-NAME (0.0001mol/L). The corporal preparation from the hypertensive rats also showed a frequency-dependent relaxation, however, the degree of which was lower at a low frequency of stimulation than that from the normotensive control. These results suggest that endothelium-derived nitric oxide released upon neural stimulation partly mediate the relaxation of the corpus cavernosum. It is also suggested that hypertension is associated with a partly attenuated relaxation response to EFS.

Effects of octreotide on the contractility of isolated rat vas deferens / 영남의대학술지

This study was performed to investigate the effect of octreotide on the contractility of rat vas deferens. The -smooth muscle strips isolated from the prostatic portion were myographied in isolated organ bath. Electric -field stimulation (monophasic square wave, duration : 1. mSec, voltage : 50 V, frequency : 5 Hz or 30 Hz, train : 10 Sec) produced reproducible contraction. The contraction was composed of two component, first phasic component (FPC) and second tonicc component (STC).. These contractions were abolished by -tetrodotoxin (1 microM). Octreotide inhibited the field stimulation induced contractions both FPC and STC concentration- dependently. The FPC was decreased by a desentization of purinergic receptor by pretreatment of mATP, and the STC was decreased by pr,,creatment of reserpine (3 mg/kg, EP) 24 hours before experiments. Octreotide reduced the field stimulation induced contraction in the presence of mATP and of reserpinized muscle strips. The inhibitory effect of octreotide was more potent at 5 Hz than at 30 Hz. Octreotide did not affect basal ton and exogenous norepinephrine- or ATP-induced contraction. These results suggest that octreotide inhibit the contractility of the isolated rat vas deferens by inhibition of the release of neurotransmitters, both ATP and norepinephrine from adrenergic nerve terminal.