Monoclonal antibodies generated against recombinant ATM support kinase activity

We report on the rapid generation of two monoclonal antibodies, ATM A16.35 and ATM D16.11, that bind to the kinase domain of mutated ataxia telangiectasia (ATM). These antibodies were generated against E. coli-expressed recombinant protein using the RIMMS strategy. We show that ATM A16.35 binds ATM by Western blot analysis, and ATM D16.11 forms immune complexes with native ATM in immunoprecipitations without neutralizing kinase activity.     

Changes in dielectric properties at 460 kHz of kidney and fat during heating: importance for radio-frequency thermal therapy

We have developed a system to measure the changes due to heating to high temperatures in the dielectric properties of tissues in the radio-frequency range. A two-electrode arrangement was connected to a low-frequency impedance analyser and used to measure the dielectric properties of ex vivo porcine kidney and fat at 460 kHz. This frequency was selected as it is the most commonly used for radio-frequency thermal therapy of renal tumours. Tissue samples were heated to target temperatures between 48 and 78 degrees C in a hot water bath and changes in dielectric properties were measured during 30 min of heating and 15 min of cooling. Results suggest a time-temperature dependence of dielectric properties, with two separate components: one a reversible, temperature-dependent effect and the other a permanent effect due to structural events (e.g. protein coagulation, fat melting) that occur in tissues during heating. We calculated temperature coefficients of 1.3 +/- 0.1% degrees C(-1) for kidney permittivity and 1.6% degrees C(-1) for kidney conductivity, 0.9 +/- 0.1% degrees C(-1) for fat permittivity and 1.7 +/- 0.1% degrees C(-1) for fat conductivity. An Arrhenius model was employed to determine the first-order kinetic rates for the irreversible changes in dielectric properties. The following Arrhenius parameters were determined: an activation energy of 57 +/- 5 kcal mol(-1) and a frequency factor of (6 +/- 1) x 10(34) s(-1) for conductivity of kidney, an activation energy of 48 +/- 2 kcal mol(-1) and a frequency factor of 6 x 10(28) s(-1) for permittivity of kidney. A similar analysis led to an activation energy of 31 +/- 4 kcal mol(-1) and a frequency factor of (4.43 +/- 1) x 10(16) s(-1) for conductivity of fat, and an activation energy of 40 +/- 4 kcal mol(-1) and a frequency factor of 4 x 10(22) s(-1) for permittivity of fat. Structural events occurring during heating at different target temperatures as determined by histological analyses were correlated with the changes in the measured dielectric properties.     

Additive effect of TRAIL and p53 gene transfer on apoptosis of human lung cancer cell lines

TRAIL is a cytokine that can induce tumor-specific apoptosis through its specific death receptors (DR4 and DR5) and p53 has been proven to increase the expression of death receptors. To examine their interaction in tumor suppression, p53 and TRAIL genes were inserted in recombinant adenovirus vectors and transferred simultaneously into non-small cell lung cancer cell lines (NCI-H157, NCI-H358, NCI-H460 and A549). Western blot assay demonstrated production of TRAIL protein in NCI-H157 and A549 cell lines. Increased expressions of DR4 and DR5 of NCI-H157 and DR4 of A549 after p53 overexpression were confirmed by flow cytometry. p53 or TRAIL gene transfer increased sub-G1 fraction in cell cycle analysis and inhibited the tumor growth dose-dependently and the degree was potentiated by co-transfer. But isobologram analysis indicated an additive effect. Together, these data indicate that p53 and TRAIL interact additively on tumor apoptosis despite theoretical synergism.     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.     

Heart-lung transplantation in Korea

Heart-lung transplantation is an effective treatment for patients with various forms of congenital heart disease or pulmonary hypertension. Since the first heart-lung transplantation in 1997, five transplants have been performed in Korea. Three cases were performed in 1997, one in 1998, and the latest one in 2002. The preoperative diagnoses were complex congenital heart disease (CHD) in 2, and CHD with Eisenmenger's syndrome in 3. In this paper, we report five cases of heart-lung transplantation performed in Korea, and include a review of the relevant literature.     

Applications of the pulsatile flow versatile ECLS: in vivo studies

INTRODUCTION: T-PLS (Twin-Pulse Life Support) is the first commercial pulsatile ECLS (Extra Corporeal Life Support) device (1). The dual sac structure of T-PLS can effectively reduce high membrane oxygenator inlet pressure and hemolysis. To verify both the use of T-PLS for ECLS and the advantages of T-PLS, we tested various models. METHOD AND RESULTS: In the partial CPB (cardio pulmonary bypass) model (swine), T-PLS (N = 6), and Biopump (N = 2), a single pulsatile pump (N = 2), were compared. In the case of single pulsatile flow, during pump systole, pressure increased to 700 - 800 mmHg at the inlet port of the membrane oxygenator. fHb, a hemolysis measurement value, was about 80 mg/dL at 3 hours. On the contrary, because of T-PLS's dual sac system, the pressure of T-PLS had a maximum value of about 250 mmHg and fHb was similar to that of the commercial centrifugal pumps. In the total CPB model (bovine, N = 6), the heart was stopped via cardioplegia (Kcl). T-PLS flow was maintained at 3.0-4.5 L/min. T-PLS functioned like a natural heart, having a pulse pressure of 26-43 mmHg and a pulse rate of 40-60 bpm (beats per minute). In the emergency case model (canine, N = 6), T-PLS was started 10 minutes after cardiac arrest from electronic shock. In spite of cardiac arrest for a period of 40 minutes, the heart was recovered after defibrillation. In the ARDS (Acute Respiratory Distress Syndrome) model (canine, N = 6), minimal ventilator parameters were set: tidal volume 130 ml, respiration rate = bpm, FiO2 = 10%. Three hours after starting T-PLS, PO2 of the carotid artery blood (after 2 hours: 195 +/- 89.4; after 3 hours: 258 +/- 99.3 mmHg) was above half the value of the femoral artery but was within normal range. CONCLUSION: It is suggested that a portable pulsatile ECLS like T-PLS may be used as a CPB device and as an alternative CPR (cardiopulmonary resuscitation) device in the case of cardiac arrest. Due to the pulsatile flow, oxygenated blood is delivered to the patient without overloading the ARDS patients heart.     

Microsatellite alterations in hepatocellular carcinoma and intrahepatic cholangiocarcinoma

A series of 20 hepatocellular carcinomas and 8 intrahepatic cholangiocarcinomas was screened from the Korean population for microsatellite alterations, including a loss of heterozygosity and replication errors using nine microsatellite markers containing several genes. The microsatellite results and our previous comparative genomic hybridization results of two tumors were compared at each locus, and the correlations between these and clinicopathologic variables were examined. The most characteristic findings were found at 13q. Replication errors were prevalent at D13S160 (13q21.2 approximately q31) and D13S292(13q12). The incidence of loss of heterozygosity, however, was higher at D13S153 (13q14.1 approximately q14.3) and D13S265(13q31 approximately q32). In contrast, there were higher deletion frequencies observed in hepatocellular carcinoma (HCC) and higher amplification frequencies observed in intrahepatic cholangiocarcinoma at 13q in our previous comparative genomic hybridization (CGH) study. Higher frequencies of replication errors were observed at D16S408 (13q12 approximately q21) and D16S504(13q23 approximately q24) in the HCC. This study found that significant differences in the patterns of genetic instability of microsatellites were dependent on the chromosomal loci. It is believed that certain genes at altered CGH regions, which are relevant to the development and/or progression of these cancers, are activated by different mutation mechanisms.     

Molecular therapies for viral hepatitis

Current treatment modalities available for hepatitis B virus (HBV) or hepatitis C virus (HCV) infections are not efficient. The enormous disease burden caused by these two infections makes the development of novel therapies critical. For HCV, the development of an effective vaccine is urgent in view of the escalating number of infected individuals. Molecular therapies for HBV and HCV infection can be directed at reducing viral load by interfering with the life cycle of the viruses or at generating immune response against viral epitopes. The antiviral approaches consist of the delivery or expression of antisense RNAs, ribozymes or dominant negative proteins. Viral biology can be interrupted by attacking various potential targets within the two viruses. DNA-based vaccination strategies are being explored for both prevention and treatment of these diseases. Both non-viral and recombinant viral vectors are being developed for safe, effective and long-term gene transfer to the liver. Although no "ideal" vector is available at this time, the ingenuity of numerous investigators is leading to the improvement of the vector systems, promising successful application of gene therapy to the prevention and treatment of viral hepatitis in the foreseeable future.